**Meet the editor**

Dr. Nancy Dumais is a Professor of Virology at Université de Sherbrooke, Canada. She received her Diploma (M.Sc.) in Cellular and Molecular Biology and her Doctorate (Ph.D.) in Virology, in 1996 and 2001, respectively, both from Université Laval in Canada. Then, she was a Postdoctoral Researcher at McMaster University where she studied mucosal immunization against HIV. Her

research interests include chemokines and chemokines receptors in HIV-1 pathogenesis. Her laboratory also investigates the roles of prostaglandins in HIV transcription and replication. In addition, she is interested in scientific and health education.

Contents

**Preface IX** 

**Part 1 From the Laboratory to the Clinic: HIV and Cellular Interactions 1** 

Chapter 2 **The Role of Human Immunodeficiency** 

Chapter 4 **HIV Recombination and Pathogenesis –** 

Nitin K. Saksena, Dominic E. Dwyer and

Monalisa Swain, Harsha Balaram and

Chapter 6 **Cellular Restriction Factors: Exploiting the** 

Jenna N. Kelly, Jessica G.K. Tong,

Ryuta Sakuma and Hiroaki Takeuchi

**TRIM5, APOBEC3G and Cyclophilins 183** 

Thatiane L. Sampaio

Chapter 3 **HIV Toxins: Gp120 as an** 

Bin Wang

Hanudatta S. Atreya

Chapter 7 **Retroviral Host Cell Factors:** 

Chapter 1 **Functions of the Lentiviral Accessory Protein Nef** 

Luciana J. Costa, Luiza M. Mendonça and

**Virus Type 1 (HIV-1) Proteins and Antiretroviral Drug Therapy in HIV-1-Induced Oxidative Stress 41**  Edna Maria Vissoci Reiche and Andréa Name Colado Simão

**Independent Modulator of Cell Function 69**  Leonor Huerta and César N. Cortés Rubio

**Biological and Epidemiological Implications 97** 

Chapter 5 **Insulin-Like Growth Factor System in HIV/AIDS: A Structure** 

**Based Approach to the Design of New Therapeutics 125** 

**Body's Antiviral Proteins to Combat HIV-1/AIDS 143** 

Clayton J. Hattlmann,Matthew W. Woods and Stephen D. Barr

**During the Distinct Steps of HIV and SIV Replication Cycle 3** 

## Contents

#### **Preface XIII**


X Contents


Contents VII

Chapter 18 **AIDS and Trauma 443**

Chapter 20 **Benign and Malignant** 

Chapter 21 **Sexual Dysfunctions 503** 

Chapter 22 **AIDS Changed America with** 

Arne N. Gjorgov

Chapter 24 **Molecular Epidemiology of** 

Chapter 25 **Saliva Testing as a Practical** 

Chapter 26 **HAART and Causes of Death in** 

Chapter 28 **Small Livestock, Food Security,** 

Mª Ángeles Muñoz-Fernández

**Part 4 From the Clinic to the Patients:** 

Chapter 23 **Transmission of HIV Through Blood –** 

AB (Sebastian) van As

Chapter 19 **Cutaneous Manifestations of**

Erik Vakil, Caroline Zabiegaj-Zwick and

**HIV/AIDS in Sub-Sahara African 453**

Etienne Mahe and Monalisa Sur

**the Twin Breast Cancer Epidemic:** 

Marco de Tubino Scanavino

Innocent Ocheyana George and Dasetima Dandison Altraide

**Lymphoproliferative Disorders in HIV/AIDS 463** 

**Exploring the Consequences of Condomization 519** 

**Transmission, Diagnosis and Therapies 581** 

**How To Bridge the Knowledge Gap 583**  Smit Sibinga, Cees Th and John P. Pitman

**HIV-1 Infection in the Amazon Region 619** 

**Tool for Rapid HIV Screening 627** H. Blake, P. Leighton and S. Sharma

**Perinatally HIV-1-Infected Children 641** 

María del Palacio Tamarit, Isabel de José and

Alicja Szulakowska and Halina Milnerowicz

John Cassius Moreki and Richard Dikeme

Chapter 27 **Cannabinoids – Influence on the Immune System and Their** 

**Nutrition Security and HIV/AIDS Mitigation 681**

Marluísa de Oliveira Guimarães Ishak and Ricardo Ishak

Claudia Palladino, Jose María Bellón, Francisco J. Climent,

**Potencial Use in Supplementary Therapy of HIV/AIDS 665**

Antonio Carlos Rosário Vallinoto, Luiz Fernando Almeida Machado,

Yusuke Okuma, Naoki Yanagisawa, Yukio Hosomi, Atsushi Ajisawa and Masahiko Shibuya

Chapter 17 **Neuropsychiatric Manifestations of HIV Infection and AIDS 415**  Victor Obiajulu Olisah

Chapter 18 **AIDS and Trauma 443**  Erik Vakil, Caroline Zabiegaj-Zwick and AB (Sebastian) van As

VI Contents

**Part 2 From the Laboratory to the Clinic:** 

Chapter 9 **Immunotherapies and Vaccines 229** 

Chapter 8 **HIV Without AIDS:** 

**HIV and the Immune System 197**

Hermancia S. Eugene and Ted M. Ross

Fadi T. Khasawneh, Beatrice Saviola, Timothy Guilford and Clare Donahue

Maria Angelica Ehara Watanabe

Nancy Dumais, Sandra C. Côté and

**HIV and Clinical Manifestations 371** 

Atsushi Ajisawa and Masahiko Shibuya

**of HIV Infection and AIDS 415** 

Chapter 13 **CXCL8 Regulation and Function in HIV**

Anne-Marie Ducharme

**Part 3 From the Clinic to the Patients:** 

Alexander Dorosevich

Chapter 17 **Neuropsychiatric Manifestations** 

Victor Obiajulu Olisah

Chapter 16 **HIV and Lung Cancer 393** 

Chapter 14 **Emerging Roles of** 

**The Immunological Secrets of Natural Hosts 199**  Zachary Ende, Michelle Bonkosky and Mirko Paiardini

Chapter 10 **HIV Envelope-Specific Antibody and Vaccine Efficacy 257**

Chapter 11 **Role of Cytokines and Chemokines in HIV Infection 281**  Vishwanath Venketaraman, Devin Morris, Clare Donohou, Andrea Sipin, Steven Kung, Hyoung Oh, Mesharee Franklin, John P. Murad,

Chapter 12 **The Role of Genetic Polymorphisms in the Chemokine** 

Per-Erik Olsson, Hazem Khalaf and Jana Jass

**Prostaglandins in HIV-1 Transcription 345** 

Chapter 15 **Pathology of HIV/AIDS: Lessons from Autopsy Series 373** Andrey Bychkov, Shunichi Yamashita and

Yusuke Okuma, Naoki Yanagisawa, Yukio Hosomi,

**and their Receptors and Cytokines in the Human Immunodeficiency Virus Type 1 (HIV-1) Infection 301**  Edna Maria Vissoci Reiche, Marla Karine Amarante and

**Infections and Potential Treatment Strategies 327**

Egidio Brocca-Cofano, Peng Xiao and Marjorie Robert-Guroff

	- **Part 4 From the Clinic to the Patients: Transmission, Diagnosis and Therapies 581**

Preface

an understanding and finally, a cure of HIV.

new avenues for AIDS therapies.

The ongoing research efforts that started even before our recognition of the HIV/AIDS syndrome and identification of HIV as the causative agent have made important inroads into our knowledge and understanding of this terrible disease. Nevertheless, the continuing AIDS pandemic and profound human and socio-economic impacts remind us that despite the unrelenting quest for knowledge since the early 1980s, we have much to learn about HIV and AIDS. Moreover, the copious amount of research performed on HIV and AIDS requires comprehensive overviews on this subject in order to provide clues and opportunities for future research. With this in mind, the purpose of this book is to aid clinicians, provide a source of inspiration for researchers, and serve as a guide for graduate and medical students in their continued search for

This volume has four sections grouped in two parts. The first part, "From the laboratory to the clinic," and the second part, "From the clinic to the patients," represent the unique but intertwined mission of this work: to provide basic and clinical knowledge on HIV/AIDS. The first two sections describe how basic research generates knowledge that impacts clinical practices. The first section details the multifaceted interactions that occur between HIV and host cells. Functions of the accessory protein Nef during the lentivirus replication cycle are reviewed in Chapter 1. Chapter 2 explains how HIV-1 proteins and therapeutic antiretroviral drugs cause oxidative stress-induced cardiovascular disease and neurological disorders. Chapter 3 is a comprehensive update on the prevalence of multiple HIV-1 subtypes, and how this influences pathogenesis, evasion on the immune system, and vaccine development. Insulin-like growth factor (IGF) levels are closely monitored in HIV/AIDS patients and help track the progression of the disease and Chapter 4 provides structural information on IGF and explains how understanding the IGF system may be useful for developing potent therapeutics. The information of Chapters 5 and 6 is presented to better understand the interactions between HIV and cellular proteins and the development of intrinsic immunity. These chapters discuss how host proteins are capable of inhibiting HIV-1 replication. These host proteins, termed cellular restriction factors, are a new and important research area that may provide

## Preface

The ongoing research efforts that started even before our recognition of the HIV/AIDS syndrome and identification of HIV as the causative agent have made important inroads into our knowledge and understanding of this terrible disease. Nevertheless, the continuing AIDS pandemic and profound human and socio-economic impacts remind us that despite the unrelenting quest for knowledge since the early 1980s, we have much to learn about HIV and AIDS. Moreover, the copious amount of research performed on HIV and AIDS requires comprehensive overviews on this subject in order to provide clues and opportunities for future research. With this in mind, the purpose of this book is to aid clinicians, provide a source of inspiration for researchers, and serve as a guide for graduate and medical students in their continued search for an understanding and finally, a cure of HIV.

This volume has four sections grouped in two parts. The first part, "From the laboratory to the clinic," and the second part, "From the clinic to the patients," represent the unique but intertwined mission of this work: to provide basic and clinical knowledge on HIV/AIDS. The first two sections describe how basic research generates knowledge that impacts clinical practices. The first section details the multifaceted interactions that occur between HIV and host cells. Functions of the accessory protein Nef during the lentivirus replication cycle are reviewed in Chapter 1. Chapter 2 explains how HIV-1 proteins and therapeutic antiretroviral drugs cause oxidative stress-induced cardiovascular disease and neurological disorders. Chapter 3 is a comprehensive update on the prevalence of multiple HIV-1 subtypes, and how this influences pathogenesis, evasion on the immune system, and vaccine development. Insulin-like growth factor (IGF) levels are closely monitored in HIV/AIDS patients and help track the progression of the disease and Chapter 4 provides structural information on IGF and explains how understanding the IGF system may be useful for developing potent therapeutics. The information of Chapters 5 and 6 is presented to better understand the interactions between HIV and cellular proteins and the development of intrinsic immunity. These chapters discuss how host proteins are capable of inhibiting HIV-1 replication. These host proteins, termed cellular restriction factors, are a new and important research area that may provide new avenues for AIDS therapies.

#### X Preface

The second section focuses on the relationship between HIV and the immune system. Continued study of the persistent HIV animal reservoir may provide insight into new vaccine strategies or therapeutic approaches for the treatment of HIV-infected humans. This very important area of HIV research is reviewed in Chapter 7. Chapters 8 and 9 approach some fundamental principles behind the design and development of effective vaccines and/or immunotherapies. These chapters are followed by a series of chapters (Chapter 10 to Chapter 13) that provide information on the role of cytokines, chemokines, and prostaglandins in HIV infection and pathogenesis.

Preface XI

**Nancy Dumais** 

Canada

Département Biologie, Faculté des Sciences,

Université de Sherbrooke, Québec

We are grateful to all the authors and researchers who contribute to the writing and content of this book on HIV/AIDS research. Their willingness to participate in this endeavour was fuelled by an unprecedented enthusiasm and I am privileged to be the editor of such a meaningful book. This book captures and describes in clear detail many aspects of basic and clinical research on HIV/AIDS, which is one of the most

profound worldwide devastating diseases of our time.

The second part of this book is a compendium of chapters dedicated to AIDS and HIV epidemiology and clinical research. The third section entitled "HIV/AIDS and clinical manifestations" describes clinical manifestations of HIV/AIDS. Postmortem examinations provide important diagnostic and epidemiological data on the myriad diseases associated with HIV infection. Chapter 14 presents data from post-mortem surveillance from 1982 to 2011 showing differences in HIV epidemiology in Africa, Asia, the U.S., and Europe. Chapter 15 discusses the epidemiology, frequency, risk factors, clinical management, and treatment of HIV-infected lung cancer patients. Neuropsychiatric manifestations of HIV infection and AIDS are presented in Chapter 16. Chapter 17 presents information related to the treatment of HIV/AIDS patients in trauma units, an area of study that has been neglected. The aim of Chapter 18 is to highlight common cutaneous manifestations of HIV/AIDS in sub-Sahara Africa. Chapter 19 explores a number of the many possible HIV/AIDS associated disorders of the lymphoid system. Chapter 20 investigates the etiologic factors involved in the sexual dysfunctions of HIV/AIDS patients, proposes steps for assessment and diagnosis, and recommends therapeutic strategies for these patients. Chapter 21 explores consequences of condomization of women's sexuality on female diseases and dysfunctions. Several aspects of two devastating epidemics, breast cancer and AIDS, are discussed in this chapter.

The final set of chapters presented in the fourth section offers scientific data on epidemiology, transmission, diagnosis, and therapies for HIV. Lack of education increases the risk of HIV transmission and Chapter 22 gives strategies to better understand transmission of HIV through blood. Chapter 23 focuses on molecular epidemiology of HIV-1 infection in the Amazon region, while Chapter 24 presents data on HIV/AIDS in Nigeria. Methods for saliva testing for HIV screening are described in Chapter 25. In Chapter 26 examines paediatric HIV infection risk factors, causes of death, and the impact of HAART in HIV-1 infected children. The effect of nutritional status among persons with HIV and drug addictions is explored in Chapter 27. Chapter 28 reviews the influence of cannabinoids on the immune system and their potential use in supplementary therapy for HIV/AIDS, as well as the importance of food and nutrition security in the mitigation of HIV/AIDS in sub-Saharan Africa.

Preface XI

We are grateful to all the authors and researchers who contribute to the writing and content of this book on HIV/AIDS research. Their willingness to participate in this endeavour was fuelled by an unprecedented enthusiasm and I am privileged to be the editor of such a meaningful book. This book captures and describes in clear detail many aspects of basic and clinical research on HIV/AIDS, which is one of the most profound worldwide devastating diseases of our time.

X Preface

infection and pathogenesis.

are discussed in this chapter.

Saharan Africa.

The second section focuses on the relationship between HIV and the immune system. Continued study of the persistent HIV animal reservoir may provide insight into new vaccine strategies or therapeutic approaches for the treatment of HIV-infected humans. This very important area of HIV research is reviewed in Chapter 7. Chapters 8 and 9 approach some fundamental principles behind the design and development of effective vaccines and/or immunotherapies. These chapters are followed by a series of chapters (Chapter 10 to Chapter 13) that provide information on the role of cytokines, chemokines, and prostaglandins in HIV

The second part of this book is a compendium of chapters dedicated to AIDS and HIV epidemiology and clinical research. The third section entitled "HIV/AIDS and clinical manifestations" describes clinical manifestations of HIV/AIDS. Postmortem examinations provide important diagnostic and epidemiological data on the myriad diseases associated with HIV infection. Chapter 14 presents data from post-mortem surveillance from 1982 to 2011 showing differences in HIV epidemiology in Africa, Asia, the U.S., and Europe. Chapter 15 discusses the epidemiology, frequency, risk factors, clinical management, and treatment of HIV-infected lung cancer patients. Neuropsychiatric manifestations of HIV infection and AIDS are presented in Chapter 16. Chapter 17 presents information related to the treatment of HIV/AIDS patients in trauma units, an area of study that has been neglected. The aim of Chapter 18 is to highlight common cutaneous manifestations of HIV/AIDS in sub-Sahara Africa. Chapter 19 explores a number of the many possible HIV/AIDS associated disorders of the lymphoid system. Chapter 20 investigates the etiologic factors involved in the sexual dysfunctions of HIV/AIDS patients, proposes steps for assessment and diagnosis, and recommends therapeutic strategies for these patients. Chapter 21 explores consequences of condomization of women's sexuality on female diseases and dysfunctions. Several aspects of two devastating epidemics, breast cancer and AIDS,

The final set of chapters presented in the fourth section offers scientific data on epidemiology, transmission, diagnosis, and therapies for HIV. Lack of education increases the risk of HIV transmission and Chapter 22 gives strategies to better understand transmission of HIV through blood. Chapter 23 focuses on molecular epidemiology of HIV-1 infection in the Amazon region, while Chapter 24 presents data on HIV/AIDS in Nigeria. Methods for saliva testing for HIV screening are described in Chapter 25. In Chapter 26 examines paediatric HIV infection risk factors, causes of death, and the impact of HAART in HIV-1 infected children. The effect of nutritional status among persons with HIV and drug addictions is explored in Chapter 27. Chapter 28 reviews the influence of cannabinoids on the immune system and their potential use in supplementary therapy for HIV/AIDS, as well as the importance of food and nutrition security in the mitigation of HIV/AIDS in sub-

**Nancy Dumais**  Département Biologie, Faculté des Sciences, Université de Sherbrooke, Québec Canada

**Part 1** 

**From the Laboratory to the Clinic:** 

**HIV and Cellular Interactions** 

## **Part 1**

**From the Laboratory to the Clinic: HIV and Cellular Interactions** 

**1** 

*Brazil* 

**Replication Cycle** 

*Universidade Federal do Rio de Janeiro* 

**Functions of the Lentiviral Accessory Protein** 

Human and Simian Immunodeficiency Viruses (HIV and SIV) are the etiological agents of the Acquired Immunodeficiency Syndrome (AIDS) in humans and the Simian AIDS (SAIDS) in macaques, respectively. HIVs and SIVs are members of the *Retroviridae* family, Lentivirus genera, and are considered complex retroviruses since its genome organization predicts the presence of at least 6 open reading frames (ORFs) in addition to the main Gag, Pol and Env ORFs present in the genomes of all retroviruses. These additional ORFs code for both regulatory (Tat and Rev) and accessory (Nef, Vif, Vpr, Vpu and Vpx) viral proteins and are all organized from the 5' half of the genome in a way that overlap both with each other and with the Pol and Env ORFs and the non-coding 3' Long Terminal Repeat (LTR) region (Figure 1). To ensure its expression and to achieve an optimal production of the viral progeny, complex mechanisms have evolved in these viruses that tightly control the expression of these ORFs during the viral replication cycle. The existence of such a number of viral proteins in addition to the viral structural (Gag and Env) and enzymatic (Pol) proteins allows the virus to explore new mechanisms to control the different steps of the replication cycle and to avoid the host cell defense. In this chapter we shall review the different steps of the HIV and SIV replication cycle with emphasis in the role taken by the viral accessory protein Nef, in both subverting the host cell machinery and influencing the function and activation of viral structural and enzymatic proteins in order to optimize viral

Lentiviral accessory proteins Vif, Vpr, Vpu and Nef were classically regarded as nonessential for virus production and/or infectivity since laboratory adapted HIV strains lacking the expression of these proteins could still replicate to several levels (Adachi et al., 1991). Since then, several studies demonstrated the crucial importance of these proteins to

Vif (Aguiar and Peterlin, 2008) and Vpu (Adachi et al., 1991) have now been acknowledged as crucial viral factors that counteract the host cell innate defense. Vif interacts and prompts the degradation of a family of cytidine deaminases DNA/RNA editing enzymes, known as Apoliprotein B mRNA-editing Enzymes (APOBECs), that otherwise would inhibit HIV and SIV replication by causing hypermutation of nascent

the efficient replication, infectivity and spread of both HIV and SIV (Kirchhoff, 2010).

progeny production as well as in evading the host cell defenses.

**1. Introduction** 

**Nef During the Distinct Steps of HIV and SIV** 

Luciana J. Costa, Luiza M. Mendonça and Thatiane L. Sampaio *Departamento de Virologia, Instituto de Microbiologia Paulo de Góes,* 

## **Functions of the Lentiviral Accessory Protein Nef During the Distinct Steps of HIV and SIV Replication Cycle**

Luciana J. Costa, Luiza M. Mendonça and Thatiane L. Sampaio *Departamento de Virologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro Brazil* 

## **1. Introduction**

Human and Simian Immunodeficiency Viruses (HIV and SIV) are the etiological agents of the Acquired Immunodeficiency Syndrome (AIDS) in humans and the Simian AIDS (SAIDS) in macaques, respectively. HIVs and SIVs are members of the *Retroviridae* family, Lentivirus genera, and are considered complex retroviruses since its genome organization predicts the presence of at least 6 open reading frames (ORFs) in addition to the main Gag, Pol and Env ORFs present in the genomes of all retroviruses. These additional ORFs code for both regulatory (Tat and Rev) and accessory (Nef, Vif, Vpr, Vpu and Vpx) viral proteins and are all organized from the 5' half of the genome in a way that overlap both with each other and with the Pol and Env ORFs and the non-coding 3' Long Terminal Repeat (LTR) region (Figure 1). To ensure its expression and to achieve an optimal production of the viral progeny, complex mechanisms have evolved in these viruses that tightly control the expression of these ORFs during the viral replication cycle. The existence of such a number of viral proteins in addition to the viral structural (Gag and Env) and enzymatic (Pol) proteins allows the virus to explore new mechanisms to control the different steps of the replication cycle and to avoid the host cell defense. In this chapter we shall review the different steps of the HIV and SIV replication cycle with emphasis in the role taken by the viral accessory protein Nef, in both subverting the host cell machinery and influencing the function and activation of viral structural and enzymatic proteins in order to optimize viral progeny production as well as in evading the host cell defenses.

Lentiviral accessory proteins Vif, Vpr, Vpu and Nef were classically regarded as nonessential for virus production and/or infectivity since laboratory adapted HIV strains lacking the expression of these proteins could still replicate to several levels (Adachi et al., 1991). Since then, several studies demonstrated the crucial importance of these proteins to the efficient replication, infectivity and spread of both HIV and SIV (Kirchhoff, 2010).

Vif (Aguiar and Peterlin, 2008) and Vpu (Adachi et al., 1991) have now been acknowledged as crucial viral factors that counteract the host cell innate defense. Vif interacts and prompts the degradation of a family of cytidine deaminases DNA/RNA editing enzymes, known as Apoliprotein B mRNA-editing Enzymes (APOBECs), that otherwise would inhibit HIV and SIV replication by causing hypermutation of nascent

Functions of the Lentiviral Accessory Protein

are reviewed in the sections bellow.

**2.1 Downmodulation of CD4 and MHC-I** 

and MHC-I expression are described below.

genome.

Swigut et al., 2000).

Nef During the Distinct Steps of HIV and SIV Replication Cycle 5

As soon as it was demonstrated that Nef has an important role for HIV and SIV infections several studies were conducted to ellucidate the mechanism by which this protein influences viral replication. The several functions described for Nef in the last two decades

Fig. 1. Representative genomic organization of the provirus of the primate lentiviruses. The main (*gag, pol and env*); regulatory (*tat and rev*); and accessory (*vif, vpr, vpu and nef*) ORFs are represented by horizontal lines. The black squares represent the LTRs present at the 5'and 3' extremities of the genome. The two exons of tat and rev are conected by angled lines. Note the extensive overlap of the structural, regulatory and accessory ORFs from the 3'half of the

The Nef protein from HIV and SIV have multiple functions and achieve its biochemical effects upon interactions with cellular components. Over thirty putative Nef targets have already been described. One of the first proteins that have been found associated with Nef is the human transmembrane CD4, which is downregulated from the cell surface by Nef (Garcia and Miller, 1991). Moreover, Nef also down-regulates other cell-surface proteins, as the major histocompatibility complex class I (MHC-I) molecules (Greenberg et al., 1998b;

Observations from naturally HIV-1-infected individuals indicated that Nef functions on downmodulation of CD4 and MHC-I could be related to the pathogenesis of AIDS (Carl et al., 2001; Tobiume et al., 2002), however the real contribution of these functions still needs to be demonstrated (Crotti et al., 2006). The motifs in Nef that mediate CD4 downregulation were considered critical for SIVmac replication in rhesus macaques, however MHC-I downregulation by Nef is not sufficient for optimal virulence of SIVmac early in infection (Iafrate et al., 2000; Lang et al., 1997; Schindler et al., 2004). Moreover, the solely importance of the CD4 downmodulation for SIVmac pathogenesis in experimental models has been challenged (Jesus da Costa et al., 2009). The mechanisms by which Nef interferes with CD4

CD4 is a type I integral membrane glycoprotein expressed primarily on T cells, thymocytes, and cells of the macrophage–monocyte lineage (Littman, 1987). CD4 is required for T-cell activation by the TCR signaling pathway and serves as the primary receptor for HIV and SIV. However, its continuous presence on the surface of HIV/SIV infected cell after viral entry is problematic for several reasons. First, because of their capacity to form complexes, co-expression of CD4 and the viral envelope protein gp120 disrupts the trafficking of both proteins (Lama et al., 1999; Ross et al., 1999). In addition, the presence of CD4 on the cell membrane reduces the ability of the newly formed particles to be properly released from the

**2. The classical functions of the HIV and SIV Nef proteins** 

retroviral genomes by deamination of cytidine residues (Aguiar and Peterlin, 2008). Vpu is a type-1 membrane associated protein that was first demonstrated to be involved in the downmodulation of the Cluster of Differentiation Antigen 4 (CD4) receptor from the infected cell surface by a mechanism of inducing the proteassomal degradation of the newly synthesized CD4 molecules in the Endoplasmic Reticulum (ER) (Schubert et al., 1998; Willey et al., 1992). Expression of Vpu is restricted to HIV-1 and a subset of SIV linages related to HIV-1. In HIV-2 and the related SIV*sooty mangabey* (SIVsm) linage, which lack the *vpu* gene, the cytoplasmic domain of the viral envelope protein gp41 mimicks this function of Vpu (Bour et al., 1996). For a long time it was considered to be the mechanism by which Vpu lead to an increase in HIV infectivity. Recently, however, it was demonstrated that Vpu in HIV-1 and related SIVs increases viral particle release from the infected cells by removing an Interferon-regulated protein (BST-2 or Theterin) from the surface of the infected cells, that otherwise would function as a host restriction factor inhibiting viral release (Neil et al., 2006; Neil et al., 2007; Neil et al., 2008).

Nef, the misnamed Negative Factor, is a myristoylated 27-35 kDa protein encoded at the far 3'end of the primate lentiviral genome (Figure 1). Nef is post-translationally modified by the addition of a myristic acid at glycine residue at amino acid position 2 (G2) on the N-terminal of the protein. This modification is required for the association of Nef to cellular membranes. Another post-translational modification in Nef is its cleavage by the virally encoded Protease (PR), which will be discussed further. The terciary structure of the Nef protein predicts an anchor domain encompassing the first 57 amino acids from the Nterminal, a highly structured central core domain encompassing amino acid residues 58-147, and a unstructures flexible C-terminal domain encompassing amino acid residues 148-180 which is commonly named C-terminal flexible-loop (Geyer et al., 2001)

Nef was first described as a negative regulator of the HIV-1 replication since in the absence of its expression levels of transcription from the viral LTR and viral replication were reported to be higher than in cells infected with the Nef positive virus counterpart (Ahmad and Venkatesan, 1988; Cheng-Mayer et al., 1989). Soon it was demonstrated that in fact the absence of Nef expression has a negative effect for virus spread in HIV-1-infected CD4 (+) cell cultures (Cheng-Mayer et al., 1989; Hammes et al., 1989; Kim et al., 1989; Lama et al., 1999; Ross et al., 1999). Moreover, evidences accumulated that the lack of Nef expression even in CD4 (-) cell cultures leads to reduction in the infectivity of the viral progeny (Chowers et al., 1995; Goldsmith et al., 1995). The effect of the Nef protein in virus infectivity is direct and specific since it can be rescued by providing Nef in *trans* to the virus producer cell (Miller et al., 1995; Pandori et al., 1996). The increase in virus infectivity by Nef is wellconserved amonsgst alleles from both HIVs and SIVs and can vary from 4-40-fold increase depending on the *nef* allele and the cell system being assayed. Nowdays, the increase in viral infectivity by Nef is the only consensus phenotype recognized for this primate lentiviral accessory protein.

The definitive proof of the crucial importance of Nef for HIV and SIV infections came from studies demonstrating that the disruption or the absence of the *nef* gene in both viruses was related to a slower or non-progression to AIDS in naturally infected humans and experimentally infected rhesus macaques, respectively (Daniel et al., 1992; Deacon et al., 1995; Kirchhoff et al., 1995). It was clearly demonstrated in these studies that in the absence of Nef expression infection with these viruses failed to mantain high levels of viremia and to progress to AIDS, defining Nef as a key factor for the pathogenesis of primate lentiviruses.

retroviral genomes by deamination of cytidine residues (Aguiar and Peterlin, 2008). Vpu is a type-1 membrane associated protein that was first demonstrated to be involved in the downmodulation of the Cluster of Differentiation Antigen 4 (CD4) receptor from the infected cell surface by a mechanism of inducing the proteassomal degradation of the newly synthesized CD4 molecules in the Endoplasmic Reticulum (ER) (Schubert et al., 1998; Willey et al., 1992). Expression of Vpu is restricted to HIV-1 and a subset of SIV linages related to HIV-1. In HIV-2 and the related SIV*sooty mangabey* (SIVsm) linage, which lack the *vpu* gene, the cytoplasmic domain of the viral envelope protein gp41 mimicks this function of Vpu (Bour et al., 1996). For a long time it was considered to be the mechanism by which Vpu lead to an increase in HIV infectivity. Recently, however, it was demonstrated that Vpu in HIV-1 and related SIVs increases viral particle release from the infected cells by removing an Interferon-regulated protein (BST-2 or Theterin) from the surface of the infected cells, that otherwise would function as a host restriction factor

Nef, the misnamed Negative Factor, is a myristoylated 27-35 kDa protein encoded at the far 3'end of the primate lentiviral genome (Figure 1). Nef is post-translationally modified by the addition of a myristic acid at glycine residue at amino acid position 2 (G2) on the N-terminal of the protein. This modification is required for the association of Nef to cellular membranes. Another post-translational modification in Nef is its cleavage by the virally encoded Protease (PR), which will be discussed further. The terciary structure of the Nef protein predicts an anchor domain encompassing the first 57 amino acids from the Nterminal, a highly structured central core domain encompassing amino acid residues 58-147, and a unstructures flexible C-terminal domain encompassing amino acid residues 148-180

Nef was first described as a negative regulator of the HIV-1 replication since in the absence of its expression levels of transcription from the viral LTR and viral replication were reported to be higher than in cells infected with the Nef positive virus counterpart (Ahmad and Venkatesan, 1988; Cheng-Mayer et al., 1989). Soon it was demonstrated that in fact the absence of Nef expression has a negative effect for virus spread in HIV-1-infected CD4 (+) cell cultures (Cheng-Mayer et al., 1989; Hammes et al., 1989; Kim et al., 1989; Lama et al., 1999; Ross et al., 1999). Moreover, evidences accumulated that the lack of Nef expression even in CD4 (-) cell cultures leads to reduction in the infectivity of the viral progeny (Chowers et al., 1995; Goldsmith et al., 1995). The effect of the Nef protein in virus infectivity is direct and specific since it can be rescued by providing Nef in *trans* to the virus producer cell (Miller et al., 1995; Pandori et al., 1996). The increase in virus infectivity by Nef is wellconserved amonsgst alleles from both HIVs and SIVs and can vary from 4-40-fold increase depending on the *nef* allele and the cell system being assayed. Nowdays, the increase in viral infectivity by Nef is the only consensus phenotype recognized for this primate lentiviral

The definitive proof of the crucial importance of Nef for HIV and SIV infections came from studies demonstrating that the disruption or the absence of the *nef* gene in both viruses was related to a slower or non-progression to AIDS in naturally infected humans and experimentally infected rhesus macaques, respectively (Daniel et al., 1992; Deacon et al., 1995; Kirchhoff et al., 1995). It was clearly demonstrated in these studies that in the absence of Nef expression infection with these viruses failed to mantain high levels of viremia and to progress to AIDS, defining Nef as a key factor for the pathogenesis of primate lentiviruses.

inhibiting viral release (Neil et al., 2006; Neil et al., 2007; Neil et al., 2008).

which is commonly named C-terminal flexible-loop (Geyer et al., 2001)

accessory protein.

As soon as it was demonstrated that Nef has an important role for HIV and SIV infections several studies were conducted to ellucidate the mechanism by which this protein influences viral replication. The several functions described for Nef in the last two decades are reviewed in the sections bellow.

Fig. 1. Representative genomic organization of the provirus of the primate lentiviruses. The main (*gag, pol and env*); regulatory (*tat and rev*); and accessory (*vif, vpr, vpu and nef*) ORFs are represented by horizontal lines. The black squares represent the LTRs present at the 5'and 3' extremities of the genome. The two exons of tat and rev are conected by angled lines. Note the extensive overlap of the structural, regulatory and accessory ORFs from the 3'half of the genome.

## **2. The classical functions of the HIV and SIV Nef proteins**

## **2.1 Downmodulation of CD4 and MHC-I**

The Nef protein from HIV and SIV have multiple functions and achieve its biochemical effects upon interactions with cellular components. Over thirty putative Nef targets have already been described. One of the first proteins that have been found associated with Nef is the human transmembrane CD4, which is downregulated from the cell surface by Nef (Garcia and Miller, 1991). Moreover, Nef also down-regulates other cell-surface proteins, as the major histocompatibility complex class I (MHC-I) molecules (Greenberg et al., 1998b; Swigut et al., 2000).

Observations from naturally HIV-1-infected individuals indicated that Nef functions on downmodulation of CD4 and MHC-I could be related to the pathogenesis of AIDS (Carl et al., 2001; Tobiume et al., 2002), however the real contribution of these functions still needs to be demonstrated (Crotti et al., 2006). The motifs in Nef that mediate CD4 downregulation were considered critical for SIVmac replication in rhesus macaques, however MHC-I downregulation by Nef is not sufficient for optimal virulence of SIVmac early in infection (Iafrate et al., 2000; Lang et al., 1997; Schindler et al., 2004). Moreover, the solely importance of the CD4 downmodulation for SIVmac pathogenesis in experimental models has been challenged (Jesus da Costa et al., 2009). The mechanisms by which Nef interferes with CD4 and MHC-I expression are described below.

CD4 is a type I integral membrane glycoprotein expressed primarily on T cells, thymocytes, and cells of the macrophage–monocyte lineage (Littman, 1987). CD4 is required for T-cell activation by the TCR signaling pathway and serves as the primary receptor for HIV and SIV. However, its continuous presence on the surface of HIV/SIV infected cell after viral entry is problematic for several reasons. First, because of their capacity to form complexes, co-expression of CD4 and the viral envelope protein gp120 disrupts the trafficking of both proteins (Lama et al., 1999; Ross et al., 1999). In addition, the presence of CD4 on the cell membrane reduces the ability of the newly formed particles to be properly released from the

Functions of the Lentiviral Accessory Protein

2010).

infection in a new host.

**2.2 Downmodulation of MHC-II** 

activate killing of the virus-infected cell (Kasper et al., 2005).

surveillance by HIV (DeGottardi et al., 2008; Lanier, 2005).

Nef During the Distinct Steps of HIV and SIV Replication Cycle 7

hypophosphorylated cytoplasmic tails, thus preventing completion of the secretory pathway that would finally provide an antigen-presenting receptor on the cell surface to

These models may not be mutually exclusive, a recent work shows that Nef simultaneously uses both antiretrograde and retrograde trafficking to down-regulate human leukocyte antigen class I (HLA-I) in the peripheral blood mononuclear cells and HeLa cells (Yi et al., 2010). Besides, another report used a small molecule to disrupt Nef-SFK binding and found that Nef orchestrates a highly regulated molecular program consisting of sequential use of signaling followed by stoichiometric modes to evade immune surveillance (Dikeakos et al.,

Human MHC-I main function is to regulate the development of an immune response. HLA-I genes include HLA-A, B, and C (Tripathi and Agrawal, 2007) and its removal from the cell surface increases the potential susceptibility of infected cells to elimination by natural killer cells (NK). Nef downregulates highly polymorphic HLA-A and -B molecules as a mechanism of HIV-1 immune evasion, but not HLA-C or -E molecules which frequently serve as ligands to inhibit the activation of NK cells (Lanier, 2005). This selection is broadly conserved between SIV (smm/mac) and HIV-2 Nef alleles (DeGottardi et al., 2008). Moreover, differences in HLA downmodulation by Nef are based on amino acid modifications in HLA cytoplasmic domains, which implies that diverse properties of Nef are required to achieve the simultaneous evasion of the CTL and natural killer cell

Nef is sufficiently adaptable to maintain downregulation of MHC-I and CD4 as a two independent functions and neither activity is optimized by the transmission event, despite the changing in immune pressure associated with sexual transmission (Noviello et al., 2007). In conclusion, the dramatic reduction of CD4 and MHCI expression in infected cells achieved by Nef seems to be in some degree important for the successful establishment of

In many ways Nef act in order to disrupt immune communication. Besides downmodulating MHC-I, CD80 and CD86, (Chaudhry et al., 2005; Greenberg et al., 1998a), Nef also interfere with MHC-II surface expression. However, for this surface marker, downmodulation is achieved by many mechanisms: i) Nef causes endocytosis of mature peptide-loaded MHC-II from the cell surface; ii) Nef can induce accumulation of immature MHC-II associated with the invariant chain (Ii) on the cell surface; iii) Nef reduces the rate of MHC-II delivery to the cell surface (Chaudhry et al., 2009; Stumptner-Cuvelette et al., 2001). MHC-II depends extensively on the endocytic machinery in order to be properly mounted on the cell surface. After its translocation to the endoplasmatic reticulum a mechanism is needed in order to prevent association with its own peptides. This is achieved by MHC-II association with the Ii, which blocks the MHC-II peptide cleft, forming the immature MHC-II complex. This immature MHC-II complex exits the ER and move through the Golgi apparatus. Finally, vesicles containing immature MHC-II fuse to MIIC vesicles, a specialized MHC-II peptide-loading compartment containing all the components needed to Ii degradation and foreign peptide loading onto the MHC-II cleft. The mature MHC-II is then directed to the cell membrane where interaction with the TCR of a CD4+ cell can happen. A fraction of immature MHC-II travels to the plasma membrane and is subsequently

infected cell therefore reducing viral infectivity (Cortes et al., 2002; Lama et al., 1999). Finally, decreasing the number of viral receptors on the surface of an infected cell seems to prevent reinfection by HIV/SIV particles (the so-called superinfection) (Le Guern and Levy, 1992; Michel et al., 2005).

Nef reduces the steady-state levels of CD4 by several proposed models based on a premise: Nef binds to the cytoplasmatic tail and promotes the endocytosis of CD4 from the cell surface, which in turn results in CD4 degradation in lysosomes. Thus myristoylation of Nef, which is necessary for its membrane localization, is crucial for the downmodulation of CD4 (Bentham et al., 2003). For binding, a cluster of residues in the N-terminal half of HIV-1 Nef recognizes a dileucine motif within the cytoplasmatic tail of CD4 (Leu-413-414) exposed only when the serine residues in CD4 are phosphorylated (Aiken et al., 1994; Bentham et al., 2003; Pitcher et al., 1999). Subsequently, Nef acts as connector between mature CD4 and components of clathrin-dependent trafficking pathways at the cell surface (and to a lesser degree in the Golgi apparatus). For this purpose, Nef bridges the CD4 cytoplasmic tail with the adaptor protein complex of endosomal clathrin-coated pits (CCPs), thereby triggering the formation of CD4-specific endocytic vesicles, and the catalytic unit of the vacuolar proton pump v-ATPase (Bresnahan et al., 1998; Foti et al., 1997; Lu et al., 1998; Mandic et al., 2001; Mangasarian et al., 1997; Piguet et al., 1998). Furthermore, to target CD4 for lysosomal degradation Nef connects the receptor with the β subunit of the COP-I coatomer in endosomes, diverting Nef-bound CD4 molecules from a recycling to a degradation pathway (Piguet et al., 1999).

Another conserved function of Nef across HIV/SIV is the disruption of the transport of MHC-I to the cell surface in infected T cells to avoid immune recognition by cytotoxic T lymphocytes (CTLs) (Greenberg et al., 1998b; Swigut et al., 2000). The pathway is initiated by Nef binding to the phosphorin acidic cluster sorting protein-2 (PACS-2), which controls the endosome-to-Golgi trafficking of cytosolic sorting proteins, then targeting Nef and its cargo to the perinuclear region to bind and activate Scr family kinase (SFK). This Nef-SFK complex then phosphorylates ZAP-70 (Syk in monocytes and heterologous cells) on tyrosine, enabling ZAP-70-SFK complex to bind the SH2 domain of Phosphatidyl Inositol 3 Kinase (PI3K) (Swann et al., 2001). MHC-I is constitutively internalized and recycled via a GTPase ADP ribosylation factor 6 (ARF6) pathway. This multi-kinase complex triggers internalization of cell-surface MHC-I through a clathrin-independent, ARF6-dependent pathway, which connects vesicle trafficking with actin cytoskeletal rearrangement (Atkins et al., 2008; Blagoveshchenskaya et al., 2002; Hung et al., 2007).

Other authors showed evidence that Nef acts early in the secretory pathway to redirect MHC-I from the trans-Golgi network (TGN) to the endolysosomal pathway known as anteretrograde trafficking. Nef directs the MHC-I into the trans-Golgi compartment through association with clathrin adaptor protein complex (AP-1), thereby sorting these proteins into specific clathrin-coated transport vesicles addressed to the endolysosomal pathway (Greenberg et al., 1998b), moreover MHC-I molecules in the trans-Golgi are probably targeted for degradation. Consistent with this model, RNA interference (RNAi) against AP-1 blocks Nef-mediated disruption of MHC-1. AP-1 was also demonstrated to co-precipitate with MHC-I and Nef in HIV-infected primary T cells (Roeth et al., 2004). Therefore, Nef binds to the MHC-I and stabilizes the interaction of a tyrosine in the cytoplasmic tail of MHC-I with the natural tyrosine-binding pocket in AP-1 (Wonderlich et al., 2008). Nef targets early types of MHC-I molecules in the ER by preferentially binding

infected cell therefore reducing viral infectivity (Cortes et al., 2002; Lama et al., 1999). Finally, decreasing the number of viral receptors on the surface of an infected cell seems to prevent reinfection by HIV/SIV particles (the so-called superinfection) (Le Guern and Levy,

Nef reduces the steady-state levels of CD4 by several proposed models based on a premise: Nef binds to the cytoplasmatic tail and promotes the endocytosis of CD4 from the cell surface, which in turn results in CD4 degradation in lysosomes. Thus myristoylation of Nef, which is necessary for its membrane localization, is crucial for the downmodulation of CD4 (Bentham et al., 2003). For binding, a cluster of residues in the N-terminal half of HIV-1 Nef recognizes a dileucine motif within the cytoplasmatic tail of CD4 (Leu-413-414) exposed only when the serine residues in CD4 are phosphorylated (Aiken et al., 1994; Bentham et al., 2003; Pitcher et al., 1999). Subsequently, Nef acts as connector between mature CD4 and components of clathrin-dependent trafficking pathways at the cell surface (and to a lesser degree in the Golgi apparatus). For this purpose, Nef bridges the CD4 cytoplasmic tail with the adaptor protein complex of endosomal clathrin-coated pits (CCPs), thereby triggering the formation of CD4-specific endocytic vesicles, and the catalytic unit of the vacuolar proton pump v-ATPase (Bresnahan et al., 1998; Foti et al., 1997; Lu et al., 1998; Mandic et al., 2001; Mangasarian et al., 1997; Piguet et al., 1998). Furthermore, to target CD4 for lysosomal degradation Nef connects the receptor with the β subunit of the COP-I coatomer in endosomes, diverting Nef-bound CD4 molecules from a recycling to a degradation pathway

Another conserved function of Nef across HIV/SIV is the disruption of the transport of MHC-I to the cell surface in infected T cells to avoid immune recognition by cytotoxic T lymphocytes (CTLs) (Greenberg et al., 1998b; Swigut et al., 2000). The pathway is initiated by Nef binding to the phosphorin acidic cluster sorting protein-2 (PACS-2), which controls the endosome-to-Golgi trafficking of cytosolic sorting proteins, then targeting Nef and its cargo to the perinuclear region to bind and activate Scr family kinase (SFK). This Nef-SFK complex then phosphorylates ZAP-70 (Syk in monocytes and heterologous cells) on tyrosine, enabling ZAP-70-SFK complex to bind the SH2 domain of Phosphatidyl Inositol 3 Kinase (PI3K) (Swann et al., 2001). MHC-I is constitutively internalized and recycled via a GTPase ADP ribosylation factor 6 (ARF6) pathway. This multi-kinase complex triggers internalization of cell-surface MHC-I through a clathrin-independent, ARF6-dependent pathway, which connects vesicle trafficking with actin cytoskeletal rearrangement (Atkins et

Other authors showed evidence that Nef acts early in the secretory pathway to redirect MHC-I from the trans-Golgi network (TGN) to the endolysosomal pathway known as anteretrograde trafficking. Nef directs the MHC-I into the trans-Golgi compartment through association with clathrin adaptor protein complex (AP-1), thereby sorting these proteins into specific clathrin-coated transport vesicles addressed to the endolysosomal pathway (Greenberg et al., 1998b), moreover MHC-I molecules in the trans-Golgi are probably targeted for degradation. Consistent with this model, RNA interference (RNAi) against AP-1 blocks Nef-mediated disruption of MHC-1. AP-1 was also demonstrated to co-precipitate with MHC-I and Nef in HIV-infected primary T cells (Roeth et al., 2004). Therefore, Nef binds to the MHC-I and stabilizes the interaction of a tyrosine in the cytoplasmic tail of MHC-I with the natural tyrosine-binding pocket in AP-1 (Wonderlich et al., 2008). Nef targets early types of MHC-I molecules in the ER by preferentially binding

al., 2008; Blagoveshchenskaya et al., 2002; Hung et al., 2007).

1992; Michel et al., 2005).

(Piguet et al., 1999).

hypophosphorylated cytoplasmic tails, thus preventing completion of the secretory pathway that would finally provide an antigen-presenting receptor on the cell surface to activate killing of the virus-infected cell (Kasper et al., 2005).

These models may not be mutually exclusive, a recent work shows that Nef simultaneously uses both antiretrograde and retrograde trafficking to down-regulate human leukocyte antigen class I (HLA-I) in the peripheral blood mononuclear cells and HeLa cells (Yi et al., 2010). Besides, another report used a small molecule to disrupt Nef-SFK binding and found that Nef orchestrates a highly regulated molecular program consisting of sequential use of signaling followed by stoichiometric modes to evade immune surveillance (Dikeakos et al., 2010).

Human MHC-I main function is to regulate the development of an immune response. HLA-I genes include HLA-A, B, and C (Tripathi and Agrawal, 2007) and its removal from the cell surface increases the potential susceptibility of infected cells to elimination by natural killer cells (NK). Nef downregulates highly polymorphic HLA-A and -B molecules as a mechanism of HIV-1 immune evasion, but not HLA-C or -E molecules which frequently serve as ligands to inhibit the activation of NK cells (Lanier, 2005). This selection is broadly conserved between SIV (smm/mac) and HIV-2 Nef alleles (DeGottardi et al., 2008). Moreover, differences in HLA downmodulation by Nef are based on amino acid modifications in HLA cytoplasmic domains, which implies that diverse properties of Nef are required to achieve the simultaneous evasion of the CTL and natural killer cell surveillance by HIV (DeGottardi et al., 2008; Lanier, 2005).

Nef is sufficiently adaptable to maintain downregulation of MHC-I and CD4 as a two independent functions and neither activity is optimized by the transmission event, despite the changing in immune pressure associated with sexual transmission (Noviello et al., 2007). In conclusion, the dramatic reduction of CD4 and MHCI expression in infected cells achieved by Nef seems to be in some degree important for the successful establishment of infection in a new host.

## **2.2 Downmodulation of MHC-II**

In many ways Nef act in order to disrupt immune communication. Besides downmodulating MHC-I, CD80 and CD86, (Chaudhry et al., 2005; Greenberg et al., 1998a), Nef also interfere with MHC-II surface expression. However, for this surface marker, downmodulation is achieved by many mechanisms: i) Nef causes endocytosis of mature peptide-loaded MHC-II from the cell surface; ii) Nef can induce accumulation of immature MHC-II associated with the invariant chain (Ii) on the cell surface; iii) Nef reduces the rate of MHC-II delivery to the cell surface (Chaudhry et al., 2009; Stumptner-Cuvelette et al., 2001). MHC-II depends extensively on the endocytic machinery in order to be properly mounted on the cell surface. After its translocation to the endoplasmatic reticulum a mechanism is needed in order to prevent association with its own peptides. This is achieved by MHC-II association with the Ii, which blocks the MHC-II peptide cleft, forming the immature MHC-II complex. This immature MHC-II complex exits the ER and move through the Golgi apparatus. Finally, vesicles containing immature MHC-II fuse to MIIC vesicles, a specialized MHC-II peptide-loading compartment containing all the components needed to Ii degradation and foreign peptide loading onto the MHC-II cleft. The mature MHC-II is then directed to the cell membrane where interaction with the TCR of a CD4+ cell can happen. A fraction of immature MHC-II travels to the plasma membrane and is subsequently

Functions of the Lentiviral Accessory Protein

**2.3 Cellular activation by Nef** 

HIV infection (Gould et al., 2003).

Nef During the Distinct Steps of HIV and SIV Replication Cycle 9

It is now clear that Nef is capable of mimicking transcriptional programs in order to manipulate the cell activation status in a way that favors HIV pathogenesis in a myriad of ways. The Nef protein can deliver antiapoptotic signals to the infected cells in order to sustain a prolonged infection, create a viral reservoir, or increase the number of permissive cells for viral infection (Mahlknecht et al., 2000). In a contrary fashion, it can stimulate apoptosis in bystander CD8+ T lymphocytes to evade immune response (Xu et al., 1999). In any case, the cell reprogramming induced by Nef is not only limited to manipulation of apoptosis. It also includes the activation or suppression of cell signaling pathways

(Mahlknecht et al., 2000; Mangino et al., 2007; Percario et al., 2003; Varin et al., 2003).

a mechanism of regulating Nef's effects (Breuer et al., 2006; Dennis et al., 2005).

Exogenous Nef have been shown to enter monocytes/macrophages, B cells and Dendritic Cells (DC) by adsorptive endocytosis. Nef also binds to the surface of T cells, but is not internalized by this cell type. No specific receptor to this protein has yet been identified (Alessandrini et al., 2000). The finding that Nef is found in sera of HIV+ positive subjects in a concentration of 10ng/ml show that this protein is somehow secreted and that this event should have a role in pathogenesis. It is assumed that this concentration could be even higher in lymphnodes or other sites where virus-producing and target cells are tightly packaged (Fujii et al., 1996). Until now no mechanism by which Nef is secreted from cells have been identified. However, since it is involved in cellular trafficking and the biogenesis of the Multi-Vesicular Bodies (MVBs), it is possible that Nef is packaged in some of these vesicles that are later exocyted from the cell. Uninfected cells could internalize Nef by endocytosis, pinocytosis or other unknown mechanisms. Exogenous Nef recapitulates in part the effects described for endogenous produced Nef (Quaranta et al., 2006; Varin et al., 2003). Still, some divergences appear, as some evidences showing that Nef can have distinct effects on an infected or uninfected cell (as the stimulation of apoptotic or non apoptotic status). Thus, Nef's delivering route is also a determining factor of the phenotype modulated by Nef. Therefore, Nef fits the Trojan horse hypothesis, which suggests that the virus take advantage of the deliberate secretion of highly immunogenic virions proteins, not packaged into viral particles, capable of modulating phenotypes on nearby cells, favoring

Nef activates many cells types to, ultimately, activate CD4+ T cells (and also activates the CD4+ T cell itself). The effect of Nef on cellular activation has been described in DCs, B cells, and macrophages, all cell types known to activate CD4+ T cells (Mahlknecht et al., 2000;

Although the effects of Nef sometimes seem contradictory it must be stressed out that its functions are the product of many protein-protein interactions that can take place during different steps of the viral replication cycle in the infected cell and are subjected to Nef cellular trafficking and conformational status. As a myristoylated protein, Nef is addressed to cellular membranes. However, it has been shown that less than 50% of the protein locates at this site, and that the majority of Nef is cytossolic (Fackler et al., 1997; Kaminchik et al., 1994; Niederman et al., 1993; Welker et al., 1998). After membrane targeting, Nef is rapidly internalized though interaction of its C-terminal flexible loop and cell proteins involved in the endocytic pathways. It has been shown that while Nef is in its membrane-bound form it appears more compactly folded, suggesting that membrane-bound and cytossolic Nef shown distinct accessible interaction domains (Breuer et al., 2006), which could explain the distinct phenotypes of Nef. Nef's ability to dimerize or even oligomerize can also function as

internalized and directed to MIIC vesicles. A similar endocytosis mechanism was described to recycle mature MHC-II from the cell surface (Reid and Watts, 1992).

Nef selectively distinguishes between mature and immature MHC-II. While induces a twofold reduction of peptide-loaded MHC-II from the cell surface, Nef induces a 15-fold more accumulation of immature MHC-II complexes composed of Ii, alpha and beta MHC-II chains. The motifs in Nef needed for one function or another are different. While for mature MHC-II endocytosis, the acidic motif EEEE65 is important, for upregulation of immature MHC-II WL57,58 motif is required. For both of them the poliproline (PxxP) and dileucine (LL164) motifs in Nef are required. The amount of Nef needed also vary depending on the phenomena. It was observed in transient expression experiments that even small quantities of Nef were enough for Ii upregulation, while for MHC-II downmodulation high expression of Nef was required. Of note, those high levels of Nef expression were considered physiologically relevant, as experiments using HIV infected cells showed quantities of Nef similar to that seen on super expression experiments (Stumptner-Cuvelette et al., 2001).

Nef also acts slowing the delivery rate of newly synthesized MHC-II molecules to the plasma membrane, however this molecular mechanism is less explored (Chaudhry et al., 2009).

Loss of cell surface MHC-II induced by Nef is observed during HIV-1 host cell infection. In the presence of Nef, mature MHC-II are found at high levels in intracellular lysossomal compartments, marked with Lamp-1, while delivery of immature MHC-II to the endolysossomal compartment is impaired. MHC-II Nef-mediated endocytosis was described as dependent on Rab5, Lyst, cholesterol and Phosphatidyl Inositol Kinases, and dispenses Dynamin 2 (Dyn2) (Chaudhry et al., 2009).

This downmodulation of mature and upregulation of immature MHC-II is a function conserved in many *nef* alleles from HIV-1/2 and SIVs. Contrary to what is seen in MHCI downmodulation, Nef activity on MHC-II and Ii do not change significantly during the stages of infection. Of note, the strong Ii upregulation observed in the presence of wild type Nef is lost in some *nef* alleles derived from Long-term infected HIV-1 patients which do not progress to AIDS, suggesting that this feature of Nef may contribute to immune evasion (Schindler et al., 2003).

Although Nef has been described to directly bind to the citoplasmatic tail of many receptors that it modulates (Chaudhry et al., 2005), there is still no evidence that it can bind MHC-II, suggesting that Nef might interact with proteins involved in the physiological turnover pathways of this surface marker, disrupting the natural balance of MHC-II surface expression. The model postulates that recycling MHC-II enters sorting endossomes for a decision on whether they will be recycled back to surface or not, which is where Nef intervene and reroute them to the Golgi apparatus. This model takes advantage of the observation that mature MHC-II is short-lived on the cell surface and thus, Nef subversive action on the endocytic program responsible for the removal of MHC-II is plausible (Chaudhry et al., 2009).

Another model has been proposed for Ii upregulation. In this simpler model, Nef would increase Ii surface expression by competing for the endocytic machinery. More specifically, the dileucine motif in Nef would tritate AP-2, the adaptor protein responsible for Ii endocytosis (Mitchell et al., 2008). Although elegant, this model encounters some conflicting data, showing that a small amount of Nef is able to induce strong accumulation of Ii. Also, other receptors that depend on AP-2 for its endocytosis, such as the Transferrin receptor, are not upregulated by Nef (Schindler et al., 2003). Nevertheless, this could be another mechanism used for Ii upregulation and still cannot be discarded.

#### **2.3 Cellular activation by Nef**

8 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

internalized and directed to MIIC vesicles. A similar endocytosis mechanism was described

Nef selectively distinguishes between mature and immature MHC-II. While induces a twofold reduction of peptide-loaded MHC-II from the cell surface, Nef induces a 15-fold more accumulation of immature MHC-II complexes composed of Ii, alpha and beta MHC-II chains. The motifs in Nef needed for one function or another are different. While for mature MHC-II endocytosis, the acidic motif EEEE65 is important, for upregulation of immature MHC-II WL57,58 motif is required. For both of them the poliproline (PxxP) and dileucine (LL164) motifs in Nef are required. The amount of Nef needed also vary depending on the phenomena. It was observed in transient expression experiments that even small quantities of Nef were enough for Ii upregulation, while for MHC-II downmodulation high expression of Nef was required. Of note, those high levels of Nef expression were considered physiologically relevant, as experiments using HIV infected cells showed quantities of Nef similar to that seen on super expression experiments (Stumptner-Cuvelette et al., 2001). Nef also acts slowing the delivery rate of newly synthesized MHC-II molecules to the plasma

membrane, however this molecular mechanism is less explored (Chaudhry et al., 2009).

dispenses Dynamin 2 (Dyn2) (Chaudhry et al., 2009).

(Schindler et al., 2003).

Loss of cell surface MHC-II induced by Nef is observed during HIV-1 host cell infection. In the presence of Nef, mature MHC-II are found at high levels in intracellular lysossomal compartments, marked with Lamp-1, while delivery of immature MHC-II to the endolysossomal compartment is impaired. MHC-II Nef-mediated endocytosis was described as dependent on Rab5, Lyst, cholesterol and Phosphatidyl Inositol Kinases, and

This downmodulation of mature and upregulation of immature MHC-II is a function conserved in many *nef* alleles from HIV-1/2 and SIVs. Contrary to what is seen in MHCI downmodulation, Nef activity on MHC-II and Ii do not change significantly during the stages of infection. Of note, the strong Ii upregulation observed in the presence of wild type Nef is lost in some *nef* alleles derived from Long-term infected HIV-1 patients which do not progress to AIDS, suggesting that this feature of Nef may contribute to immune evasion

Although Nef has been described to directly bind to the citoplasmatic tail of many receptors that it modulates (Chaudhry et al., 2005), there is still no evidence that it can bind MHC-II, suggesting that Nef might interact with proteins involved in the physiological turnover pathways of this surface marker, disrupting the natural balance of MHC-II surface expression. The model postulates that recycling MHC-II enters sorting endossomes for a decision on whether they will be recycled back to surface or not, which is where Nef intervene and reroute them to the Golgi apparatus. This model takes advantage of the observation that mature MHC-II is short-lived on the cell surface and thus, Nef subversive action on the endocytic

Another model has been proposed for Ii upregulation. In this simpler model, Nef would increase Ii surface expression by competing for the endocytic machinery. More specifically, the dileucine motif in Nef would tritate AP-2, the adaptor protein responsible for Ii endocytosis (Mitchell et al., 2008). Although elegant, this model encounters some conflicting data, showing that a small amount of Nef is able to induce strong accumulation of Ii. Also, other receptors that depend on AP-2 for its endocytosis, such as the Transferrin receptor, are not upregulated by Nef (Schindler et al., 2003). Nevertheless, this could be another

program responsible for the removal of MHC-II is plausible (Chaudhry et al., 2009).

mechanism used for Ii upregulation and still cannot be discarded.

to recycle mature MHC-II from the cell surface (Reid and Watts, 1992).

It is now clear that Nef is capable of mimicking transcriptional programs in order to manipulate the cell activation status in a way that favors HIV pathogenesis in a myriad of ways. The Nef protein can deliver antiapoptotic signals to the infected cells in order to sustain a prolonged infection, create a viral reservoir, or increase the number of permissive cells for viral infection (Mahlknecht et al., 2000). In a contrary fashion, it can stimulate apoptosis in bystander CD8+ T lymphocytes to evade immune response (Xu et al., 1999). In any case, the cell reprogramming induced by Nef is not only limited to manipulation of apoptosis. It also includes the activation or suppression of cell signaling pathways (Mahlknecht et al., 2000; Mangino et al., 2007; Percario et al., 2003; Varin et al., 2003).

Although the effects of Nef sometimes seem contradictory it must be stressed out that its functions are the product of many protein-protein interactions that can take place during different steps of the viral replication cycle in the infected cell and are subjected to Nef cellular trafficking and conformational status. As a myristoylated protein, Nef is addressed to cellular membranes. However, it has been shown that less than 50% of the protein locates at this site, and that the majority of Nef is cytossolic (Fackler et al., 1997; Kaminchik et al., 1994; Niederman et al., 1993; Welker et al., 1998). After membrane targeting, Nef is rapidly internalized though interaction of its C-terminal flexible loop and cell proteins involved in the endocytic pathways. It has been shown that while Nef is in its membrane-bound form it appears more compactly folded, suggesting that membrane-bound and cytossolic Nef shown distinct accessible interaction domains (Breuer et al., 2006), which could explain the distinct phenotypes of Nef. Nef's ability to dimerize or even oligomerize can also function as a mechanism of regulating Nef's effects (Breuer et al., 2006; Dennis et al., 2005).

Exogenous Nef have been shown to enter monocytes/macrophages, B cells and Dendritic Cells (DC) by adsorptive endocytosis. Nef also binds to the surface of T cells, but is not internalized by this cell type. No specific receptor to this protein has yet been identified (Alessandrini et al., 2000). The finding that Nef is found in sera of HIV+ positive subjects in a concentration of 10ng/ml show that this protein is somehow secreted and that this event should have a role in pathogenesis. It is assumed that this concentration could be even higher in lymphnodes or other sites where virus-producing and target cells are tightly packaged (Fujii et al., 1996). Until now no mechanism by which Nef is secreted from cells have been identified. However, since it is involved in cellular trafficking and the biogenesis of the Multi-Vesicular Bodies (MVBs), it is possible that Nef is packaged in some of these vesicles that are later exocyted from the cell. Uninfected cells could internalize Nef by endocytosis, pinocytosis or other unknown mechanisms. Exogenous Nef recapitulates in part the effects described for endogenous produced Nef (Quaranta et al., 2006; Varin et al., 2003). Still, some divergences appear, as some evidences showing that Nef can have distinct effects on an infected or uninfected cell (as the stimulation of apoptotic or non apoptotic status). Thus, Nef's delivering route is also a determining factor of the phenotype modulated by Nef. Therefore, Nef fits the Trojan horse hypothesis, which suggests that the virus take advantage of the deliberate secretion of highly immunogenic virions proteins, not packaged into viral particles, capable of modulating phenotypes on nearby cells, favoring HIV infection (Gould et al., 2003).

Nef activates many cells types to, ultimately, activate CD4+ T cells (and also activates the CD4+ T cell itself). The effect of Nef on cellular activation has been described in DCs, B cells, and macrophages, all cell types known to activate CD4+ T cells (Mahlknecht et al., 2000;

Functions of the Lentiviral Accessory Protein

delivered to B cells (Xu et al., 2009).

**2.4 Downmodulation of TCR/CD3** 

pathogenicity factor of HIV.

et al., 1999; Sousa et al., 2002).

al., 1998; Swigut et al., 2003).

Schmokel et al., 2011).

Nef During the Distinct Steps of HIV and SIV Replication Cycle 11

formation on infected nearby macrophages, and trafficking through them in vesicles that are

AIDS is by many authors considered as the outcome of many immunological disorders. The solely expression of Nef in SCID human/mouse model recapitulates many of the AIDS symptoms (Hanna et al., 1998; Lindemann et al., 1994; Skowronski et al., 1993). Nef completely transfigures signaling of DCs, T cells, monocytes/macrophages, B cells and probably many others cell types. These attributes make this protein probably the major

HIV-1 infection in humans and SIVmac infection in rhesus macaques induces overall levels of immune activation associated with accelerated T cell turnover rates and increased susceptibility to apoptotic cell death that culminates in progression to AIDS in the absence of an effective anti-HIV therapy. Moreover, the accessory protein Nef from both HIV and SIV has been implicated in immune activation and disease progression as discussed previously. HIV-1 Nef has also been reported to directly enhance the responsiveness of T cells to activation (Fenard et al., 2005; Fortin et al., 2004; Wang et al., 2000). Nonetheless, non-human primates naturally infected with their species-specific SIVs (e.g., sooty mangabeys (SMs) and African green monkeys (AGMs)) generally do not show signs of progression to SAIDS (Silvestri et al., 2007; VandeWoude and Apetrei, 2006). The mechanism underlying the remarkable difference in the outcome of infection between these primate hosts include T cell activation as a strong predictor of progression to AIDS (Giorgi

The signaling cascade which leads to T-cell activation and differentiation depends on the immunoreceptor tyrosine activation motifs (ITAMs) of the TCR/CD3 complex localized in cell surface for initiation of signaling cascades, thereby resulting in recruitment and activation of multiple protein tyrosine kinases, signaling intermediates, and adapter molecules (Guy and Vignali, 2009). TCR/CD3 complexes are composed of the clonotypic αβ heterodimer, the CD3 ε and ε heterodimers, the ζ homodimer and contain a total of 10 ITAMs (Alcover and Alarcon, 2000). TCR/CD3 complexes are relatively stable but are constantly internalized and recycled back to the cell surface by the endocytosis and intracellular trafficking pathway of membrane receptors. Besides the host TCR/CD3 complexes equilibrium, SIV and HIV-2 Nef bypasses the mechanisms that normally mediate the recruitment of TCR/CD3 complexes to the endocytic machinery, therefore, SIV and HIV-2 Nef target the CD3-ζ subunit and accelerate its endocytosis rate (Howe et al., 1998; Swigut et al., 2003). The proposed mechanisms for TCR-CD3 endocytosis induced by SIVmac and HIV-2 Nef proteins are based on the interaction of Nef with the CD3-ζ subunit via the AP-2 clathrin adaptor pathway (Bell et al., 1998; Howe et

Although HIV-1 Nef fails to downregulate CD3 (Foster and Garcia, 2008) essentially all SIV Nefs, except for a small subset, downmodulate TCR/CD3 complexes to suppress T cell activation and programmed cell death (Kirchhoff et al., 2008). The exceptions include the chimpanzee precursor of HIV-1, SIVcpz, as well as SIVgsn, SIVmus, SIVmon, SIVgor and SIVolc. In contrast, the majority of *nef* alleles, including those of SIVsmm, SIVmac, and HIV-2 but also those of SIVrcm, SIVdeb, SIVsyk, SIVblu, SIVsun, SIVtan, SIVsab, SIVden, SIVwrc, SIVgri, SIVlho, and SIVasc downmodulated TCR/CD3 efficiently (Schindler et al., 2006;

Quaranta et al., 2006; Xu et al., 2009). By activating CD4+ T cells, Nef favours infection spread, as well as increases the pool of permissive cells and transcription from the virus promoter on cells already infected.

Nef interacts with a number of serine and tyrosine protein kinases usually by its PxxP domain, known to bind proteins with SH3 domains (Saksela et al., 1995). Binding of Nef to these proteins lead to their activation, and activation of the pathway in which they are included. Some of these kinases include Hck, PAK1, Lyn, c-Raf, and p53, but many more have been described (Arold et al., 1998; Baur et al., 1997; Cheng et al., 1999; Greenway et al., 2002; Hodge et al., 1998; Lu et al., 1996; Saksela et al., 1995). Nef recruits several molecules involved in TCR signaling, as Lck and Vav (Baur et al., 1997; Djordjevic et al., 2004; Fackler et al., 1999), to glycolipid-enriched domains, leading the T-cell to a preactivation state (Simmons et al., 2001). Furthermore, Nef can activate T cells independently of TCR, stimulating calcium-dependent signaling by interaction with inositol triphosfate receptor on these cells (Manninen and Saksela, 2002).

Interestinglly, some effects of Nef on signaling can be seen even in the absence of the PxxP domain, as STAT3 phosphorilation. For STAT3 phosphorilation the domains necessary for interaction with the endocytic machinery, as the diacid domains EE155 and DD174 and the dileucine domain LL164 were needed, showing that interaction with SH3 domain containing proteins is not the only event related to manipulation of signaling pathways by Nef (Percario et al., 2003).

Activation of the signaling pathways by Nef leads, for instance, to the induction NFkB nuclear translocation by inducing phosphorilation of IKK alpha and beta and their subsequent degradation (Mangino et al., 2007; Varin et al., 2003). This activation leads to the secretion of pro-inflammatory mediators, as IL-1beta, IL-6 and TNFalpha, and chemokines as MIP1alpha and beta. Other pathways activated by Nef include AP-1, c-Jun, and MAPK ERK1/2, JNK and p38 (Mangino et al., 2007). This effect can take place in many cell types as promonocytic cells, Monocyte-Derived Macrophages (MDMs), and DCs (Olivetta et al., 2003; Quaranta et al., 2006; Varin et al., 2003). The soluble mediators synthesized as the result of the activation of these pathways, as IL-6 and MIP-1alpha, activate JAK, leading to dimmerization of STAT1 and 3 (Mangino et al., 2007; Percario et al., 2003). Also, as Nef activates IRF3, leading to the synthesis of IFNbeta, it also leads to STAT2 activation (Mangino et al., 2007). NFkB activation also contributes to synthesis of viral proteins, since NFkB is one of the main regulators of the viral promoter (Nabel and Baltimore, 1987).

Signaling through NFkB and STAT3 are responsible for regulation of antiapoptotic genes on infected cells, preventing their death, prolonging viral production and spread (Quaranta et al., 2006). Along with TNF, Nef stimulation of NFkB may act blocking caspase 8 activation (Wang et al., 1998).

It has been described that treatment of cells with exogenous Nef are similar to the treatment with TNFalpha, and lead to the same outcomes (Mahlknecht et al., 2000; Varin et al., 2003), as NFkB, AP-1 and JNK activation. It is thus believed that Nef might engage interactions with some actor of the TNFalpha signaling pathway, but such protein have not yet been identified (Varin et al., 2003).

Nef was found in the germinal centers of infected lymphoid follicules, and its presence has been observed in IgD+ B cells, although these cells showed no signs of infection, as they lack any other viral protein or RNA. On B cells, Nef inhibits CD40-dependent activation and IgG2 and IgA class-switch. Interestingly, Nef can reach these cells by inducing conduit formation on infected nearby macrophages, and trafficking through them in vesicles that are delivered to B cells (Xu et al., 2009).

AIDS is by many authors considered as the outcome of many immunological disorders. The solely expression of Nef in SCID human/mouse model recapitulates many of the AIDS symptoms (Hanna et al., 1998; Lindemann et al., 1994; Skowronski et al., 1993). Nef completely transfigures signaling of DCs, T cells, monocytes/macrophages, B cells and probably many others cell types. These attributes make this protein probably the major pathogenicity factor of HIV.

## **2.4 Downmodulation of TCR/CD3**

10 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Quaranta et al., 2006; Xu et al., 2009). By activating CD4+ T cells, Nef favours infection spread, as well as increases the pool of permissive cells and transcription from the virus

Nef interacts with a number of serine and tyrosine protein kinases usually by its PxxP domain, known to bind proteins with SH3 domains (Saksela et al., 1995). Binding of Nef to these proteins lead to their activation, and activation of the pathway in which they are included. Some of these kinases include Hck, PAK1, Lyn, c-Raf, and p53, but many more have been described (Arold et al., 1998; Baur et al., 1997; Cheng et al., 1999; Greenway et al., 2002; Hodge et al., 1998; Lu et al., 1996; Saksela et al., 1995). Nef recruits several molecules involved in TCR signaling, as Lck and Vav (Baur et al., 1997; Djordjevic et al., 2004; Fackler et al., 1999), to glycolipid-enriched domains, leading the T-cell to a preactivation state (Simmons et al., 2001). Furthermore, Nef can activate T cells independently of TCR, stimulating calcium-dependent signaling by interaction with inositol triphosfate receptor on

Interestinglly, some effects of Nef on signaling can be seen even in the absence of the PxxP domain, as STAT3 phosphorilation. For STAT3 phosphorilation the domains necessary for interaction with the endocytic machinery, as the diacid domains EE155 and DD174 and the dileucine domain LL164 were needed, showing that interaction with SH3 domain containing proteins is not the only event related to manipulation of signaling pathways by Nef

Activation of the signaling pathways by Nef leads, for instance, to the induction NFkB nuclear translocation by inducing phosphorilation of IKK alpha and beta and their subsequent degradation (Mangino et al., 2007; Varin et al., 2003). This activation leads to the secretion of pro-inflammatory mediators, as IL-1beta, IL-6 and TNFalpha, and chemokines as MIP1alpha and beta. Other pathways activated by Nef include AP-1, c-Jun, and MAPK ERK1/2, JNK and p38 (Mangino et al., 2007). This effect can take place in many cell types as promonocytic cells, Monocyte-Derived Macrophages (MDMs), and DCs (Olivetta et al., 2003; Quaranta et al., 2006; Varin et al., 2003). The soluble mediators synthesized as the result of the activation of these pathways, as IL-6 and MIP-1alpha, activate JAK, leading to dimmerization of STAT1 and 3 (Mangino et al., 2007; Percario et al., 2003). Also, as Nef activates IRF3, leading to the synthesis of IFNbeta, it also leads to STAT2 activation (Mangino et al., 2007). NFkB activation also contributes to synthesis of viral proteins, since NFkB is one of the main regulators of the viral promoter (Nabel and Baltimore, 1987). Signaling through NFkB and STAT3 are responsible for regulation of antiapoptotic genes on infected cells, preventing their death, prolonging viral production and spread (Quaranta et al., 2006). Along with TNF, Nef stimulation of NFkB may act blocking caspase 8 activation

It has been described that treatment of cells with exogenous Nef are similar to the treatment with TNFalpha, and lead to the same outcomes (Mahlknecht et al., 2000; Varin et al., 2003), as NFkB, AP-1 and JNK activation. It is thus believed that Nef might engage interactions with some actor of the TNFalpha signaling pathway, but such protein have not yet been

Nef was found in the germinal centers of infected lymphoid follicules, and its presence has been observed in IgD+ B cells, although these cells showed no signs of infection, as they lack any other viral protein or RNA. On B cells, Nef inhibits CD40-dependent activation and IgG2 and IgA class-switch. Interestingly, Nef can reach these cells by inducing conduit

promoter on cells already infected.

these cells (Manninen and Saksela, 2002).

(Percario et al., 2003).

(Wang et al., 1998).

identified (Varin et al., 2003).

HIV-1 infection in humans and SIVmac infection in rhesus macaques induces overall levels of immune activation associated with accelerated T cell turnover rates and increased susceptibility to apoptotic cell death that culminates in progression to AIDS in the absence of an effective anti-HIV therapy. Moreover, the accessory protein Nef from both HIV and SIV has been implicated in immune activation and disease progression as discussed previously. HIV-1 Nef has also been reported to directly enhance the responsiveness of T cells to activation (Fenard et al., 2005; Fortin et al., 2004; Wang et al., 2000). Nonetheless, non-human primates naturally infected with their species-specific SIVs (e.g., sooty mangabeys (SMs) and African green monkeys (AGMs)) generally do not show signs of progression to SAIDS (Silvestri et al., 2007; VandeWoude and Apetrei, 2006). The mechanism underlying the remarkable difference in the outcome of infection between these primate hosts include T cell activation as a strong predictor of progression to AIDS (Giorgi et al., 1999; Sousa et al., 2002).

The signaling cascade which leads to T-cell activation and differentiation depends on the immunoreceptor tyrosine activation motifs (ITAMs) of the TCR/CD3 complex localized in cell surface for initiation of signaling cascades, thereby resulting in recruitment and activation of multiple protein tyrosine kinases, signaling intermediates, and adapter molecules (Guy and Vignali, 2009). TCR/CD3 complexes are composed of the clonotypic αβ heterodimer, the CD3 ε and ε heterodimers, the ζ homodimer and contain a total of 10 ITAMs (Alcover and Alarcon, 2000). TCR/CD3 complexes are relatively stable but are constantly internalized and recycled back to the cell surface by the endocytosis and intracellular trafficking pathway of membrane receptors. Besides the host TCR/CD3 complexes equilibrium, SIV and HIV-2 Nef bypasses the mechanisms that normally mediate the recruitment of TCR/CD3 complexes to the endocytic machinery, therefore, SIV and HIV-2 Nef target the CD3-ζ subunit and accelerate its endocytosis rate (Howe et al., 1998; Swigut et al., 2003). The proposed mechanisms for TCR-CD3 endocytosis induced by SIVmac and HIV-2 Nef proteins are based on the interaction of Nef with the CD3-ζ subunit via the AP-2 clathrin adaptor pathway (Bell et al., 1998; Howe et al., 1998; Swigut et al., 2003).

Although HIV-1 Nef fails to downregulate CD3 (Foster and Garcia, 2008) essentially all SIV Nefs, except for a small subset, downmodulate TCR/CD3 complexes to suppress T cell activation and programmed cell death (Kirchhoff et al., 2008). The exceptions include the chimpanzee precursor of HIV-1, SIVcpz, as well as SIVgsn, SIVmus, SIVmon, SIVgor and SIVolc. In contrast, the majority of *nef* alleles, including those of SIVsmm, SIVmac, and HIV-2 but also those of SIVrcm, SIVdeb, SIVsyk, SIVblu, SIVsun, SIVtan, SIVsab, SIVden, SIVwrc, SIVgri, SIVlho, and SIVasc downmodulated TCR/CD3 efficiently (Schindler et al., 2006; Schmokel et al., 2011).

Functions of the Lentiviral Accessory Protein

intercellular route (Gerdes et al., 2007).

and subsequent cell motility (Stolp et al., 2010).

**3.1 Tetherin and Nef** 

improve immune evasion and viral spread in the infected host.

**3. SIV Nef to counteract a cellular restriction factor?** 

terminus (Kupzig et al., 2003; Rollason et al., 2007).

1999).

Nef During the Distinct Steps of HIV and SIV Replication Cycle 13

2009). Indeed, alteration of the endocytic and signaling pathways at the immunological synapse likely impacts the function and destiny of HIV-1-infected cells. T-cell chemotaxis constitutes an essential function of the immune response, since active secretion of chemokines controls homing and recruitment of leukocytes into tissues. A number of studies have reported that Nef affects T-cell chemotaxis through the modulation of Rho-GTPase-regulated signaling pathways (Janardhan et al., 2004; Lee et al., 2008; Swingler et al.,

HIV-1 infection of primary human macrophages induces the formation of tunneling nanotubes (TNT) by Nef (Lamers et al., 2010). TNT is a novel communication system observed in immune cells, including B, T and NK cells, neutrophils and monocytes, as well as in neurons and glial cells, which can be utilized by HIV to spread viral particles by an

The effect of Nef is dependent on its myristoylation and SH3 domains. While Nef myristoylation is required for its membrane association, the proline-rich SH3-binding domain is involved in Nef association with Vav, DOCK2-ELMO1, Rac and the cellular kinase Pak2 (Roeth and Collins, 2006). First, Vav is activated by the interaction between its C-terminal SH3 domain and PxxP motif in Nef leading to Vav's downstream effectors activation, resulting in morphological changes, cytoskeleton rearrangements and the activation of the c-Jun Nterminal kinase (JNK)/stress-activated protein kinase (SAPK) cascade (Fackler et al., 1999). Then, Nef activates Rac by binding the DOCK2-ELMO1 complex, a key activator of Rac in antigen- and chemokine-initiated signaling pathways, and this interaction is linked to the abilities of Nef to inhibit chemotaxis and promote T cell activation (Janardhan et al., 2004). Finally, Nef association with Pak2 prevents actin remodeling to impair host cell motility by disregulation of cofilin, which is an actin-depolymerizing factor that promotes actin turnover

The cytoskeleton reorganization induced by Nef is associated with an impairment of cell movements combined with induction of long filopodium-like structures in T lymphocytes (Stolp et al., 2010). In summary, Nef displays a variety of complex effects on the motility and cellular morphology of HIV-1-infectected T lymphocyte, thus resulting in a strategy to

The cellular protein Tetherin (also known as BST-2, CD317, or HM1.24) is a membrane protein with a number of distinct characteristics indicating that it plays a straight role in suppressing the release of virions from infected cells. Tetherin was discovered in 2008 when groups of Bieniasz and Guatelli were investigating the HIV-1 Vpu anti-Tetherin activity during the virus life cycle (Neil et al., 2008; Van Damme et al., 2008). The N-terminal of this protein is localized in the cytoplasm, followed by a transmembrane domain and a coiled-coil extracellular domain. The extracellular domain contains two N-linked glycosylation sites that are modified by a glycosyl phosphatidyl inositol (GPI) membrane anchor at the C-

Tetherin functions as a broadly acting antiviral factor because besides lentiviruses, Tetherin restricts the release of different retroviruses, including alpha-, beta-, delta-, spumaviruses, and other viruses as arena- (Lassa), filo- (Marbuburg, Ebola), and herpesviruses (KSHV) (Mansouri et al., 2009). Tetherin belongs to the three main classes of restriction factors in addition to

In a previous report, an association between Nef proteins of all *vpu*-containing viruses and the loss of ability to down-modulate the TCR/CD3 was demonstrated, hence only *vpu*containing viruses were predicted to be unable to down-modulate the TCR/CD3 complexes, e.g. HIV-1, SIVcpz, SIVgor, SIVgsn, SIVmus, SIVmon (Schindler et al., 2006). Exceptions to this association has been demonstrated since in SIVden strain, in which Nef retains the ability to downmodulate TCR/CD3, the *vpu* gene is present, whereas in SIVolc, which does not contain Vpu, Nef protein is unable to perform this function (Schmokel et al., 2011).

Besides the role of Nef in TCR/CD3 downmodulation a question remains to be understood: does CD3 downregulation contribute to the nonpathogenic phenotype of natural SIV infections? The reports suggest that a protective role of Nef-mediated TCR/CD3 downmodulation is needed to reduce the stimulation of virally infected CD4+ helper T cells by antigen-presenting cells and might contribute to the nonpathogenic phenotype of natural SIV infections. Inefficient CD4+ helper T cell activation would weaken the antiviral immune response and might allow the virus to persist at high levels. Indeed, reduced T cell activation, proliferation and apoptosis might also allow the host to maintain a functional immune system (Kirchhoff et al., 2008). However, two models of experimental SIV infection appear to be contradictory to this hypothesis. First, SIVmac, a virus inadvertently transmitted from naturally infected SMs to macaques in captivity (Daniel et al., 1985), causes immunodeficiency and SAIDS in macaques despite the fact that its Nef protein efficiently downmodulates TCR/CD3. Second, SIVmac viruses harboring the HIV-1 *nef* gene (the socalled Nef-SHIVs) do not induce greater pathogenicity than wild-type SIVmac in experimentally infected macaques (Alexander et al., 1999). Taken together, further analyses are required to clarify the biological significance of CD3 downregulation to the pathogenicity of SIV, HIV-1, and HIV-2 for their hosts.

Although compeling evidences that the functions of Nef described above can be related to primate lentiviral pathogenesis, each one of them individually can be excluded as the mechanism by which this protein increases viral infectivity by a number of other evidences. Nef is a multifunctional protein and besides interacting with a multitude of host cellular factors and to contribute to virus pathogenesis it seems to play a key role during the primate lentiviral replication cycle to make viral particles optimally infectious. The participation of Nef during the different steps of the primate lentiviral replication cycle will be reviwed in the next sections.

#### **2.5 Interference with cell cytoskeleton**

It has been demonstrated that HIV-1 takes advantage of the cytoskeleton dynamics in order to ensure viral entry and transport within and egress from target cells, as well as to interfere with other cellular processes (Fackler and Krausslich, 2006). The specific interference of Nef with cell cytoskeleton for virus entry will be discussed on section 4.1.

Nef's interference on the actin dynamics is also an important mechanism for Nef-induced alterations of T-cell receptor (TCR) signaling (Haller et al., 2006; Rudolph et al., 2009). TCR engagement triggers actin rearrangements that control receptor clustering for signal initiation and dynamic organization of signaling protein complexes to form an immunological synapse. Nef inhibits immunological synapse formation by a dynamic process involving rapid actin modifications (Thoulouze et al., 2006). TCR signaling events at the immunological synapse, including F-actin remodeling and re-localization of Lck, are evolutionary conserved activities of highly divergent lentiviral Nef proteins (Rudolph et al.,

In a previous report, an association between Nef proteins of all *vpu*-containing viruses and the loss of ability to down-modulate the TCR/CD3 was demonstrated, hence only *vpu*containing viruses were predicted to be unable to down-modulate the TCR/CD3 complexes, e.g. HIV-1, SIVcpz, SIVgor, SIVgsn, SIVmus, SIVmon (Schindler et al., 2006). Exceptions to this association has been demonstrated since in SIVden strain, in which Nef retains the ability to downmodulate TCR/CD3, the *vpu* gene is present, whereas in SIVolc, which does not contain Vpu, Nef protein is unable to perform this function (Schmokel et al., 2011). Besides the role of Nef in TCR/CD3 downmodulation a question remains to be understood: does CD3 downregulation contribute to the nonpathogenic phenotype of natural SIV infections? The reports suggest that a protective role of Nef-mediated TCR/CD3 downmodulation is needed to reduce the stimulation of virally infected CD4+ helper T cells by antigen-presenting cells and might contribute to the nonpathogenic phenotype of natural SIV infections. Inefficient CD4+ helper T cell activation would weaken the antiviral immune response and might allow the virus to persist at high levels. Indeed, reduced T cell activation, proliferation and apoptosis might also allow the host to maintain a functional immune system (Kirchhoff et al., 2008). However, two models of experimental SIV infection appear to be contradictory to this hypothesis. First, SIVmac, a virus inadvertently transmitted from naturally infected SMs to macaques in captivity (Daniel et al., 1985), causes immunodeficiency and SAIDS in macaques despite the fact that its Nef protein efficiently downmodulates TCR/CD3. Second, SIVmac viruses harboring the HIV-1 *nef* gene (the socalled Nef-SHIVs) do not induce greater pathogenicity than wild-type SIVmac in experimentally infected macaques (Alexander et al., 1999). Taken together, further analyses are required to clarify the biological significance of CD3 downregulation to the

Although compeling evidences that the functions of Nef described above can be related to primate lentiviral pathogenesis, each one of them individually can be excluded as the mechanism by which this protein increases viral infectivity by a number of other evidences. Nef is a multifunctional protein and besides interacting with a multitude of host cellular factors and to contribute to virus pathogenesis it seems to play a key role during the primate lentiviral replication cycle to make viral particles optimally infectious. The participation of Nef during the different steps of the primate lentiviral replication cycle will be reviwed in

It has been demonstrated that HIV-1 takes advantage of the cytoskeleton dynamics in order to ensure viral entry and transport within and egress from target cells, as well as to interfere with other cellular processes (Fackler and Krausslich, 2006). The specific interference of Nef

Nef's interference on the actin dynamics is also an important mechanism for Nef-induced alterations of T-cell receptor (TCR) signaling (Haller et al., 2006; Rudolph et al., 2009). TCR engagement triggers actin rearrangements that control receptor clustering for signal initiation and dynamic organization of signaling protein complexes to form an immunological synapse. Nef inhibits immunological synapse formation by a dynamic process involving rapid actin modifications (Thoulouze et al., 2006). TCR signaling events at the immunological synapse, including F-actin remodeling and re-localization of Lck, are evolutionary conserved activities of highly divergent lentiviral Nef proteins (Rudolph et al.,

with cell cytoskeleton for virus entry will be discussed on section 4.1.

pathogenicity of SIV, HIV-1, and HIV-2 for their hosts.

**2.5 Interference with cell cytoskeleton** 

the next sections.

2009). Indeed, alteration of the endocytic and signaling pathways at the immunological synapse likely impacts the function and destiny of HIV-1-infected cells. T-cell chemotaxis constitutes an essential function of the immune response, since active secretion of chemokines controls homing and recruitment of leukocytes into tissues. A number of studies have reported that Nef affects T-cell chemotaxis through the modulation of Rho-GTPase-regulated signaling pathways (Janardhan et al., 2004; Lee et al., 2008; Swingler et al., 1999).

HIV-1 infection of primary human macrophages induces the formation of tunneling nanotubes (TNT) by Nef (Lamers et al., 2010). TNT is a novel communication system observed in immune cells, including B, T and NK cells, neutrophils and monocytes, as well as in neurons and glial cells, which can be utilized by HIV to spread viral particles by an intercellular route (Gerdes et al., 2007).

The effect of Nef is dependent on its myristoylation and SH3 domains. While Nef myristoylation is required for its membrane association, the proline-rich SH3-binding domain is involved in Nef association with Vav, DOCK2-ELMO1, Rac and the cellular kinase Pak2 (Roeth and Collins, 2006). First, Vav is activated by the interaction between its C-terminal SH3 domain and PxxP motif in Nef leading to Vav's downstream effectors activation, resulting in morphological changes, cytoskeleton rearrangements and the activation of the c-Jun Nterminal kinase (JNK)/stress-activated protein kinase (SAPK) cascade (Fackler et al., 1999). Then, Nef activates Rac by binding the DOCK2-ELMO1 complex, a key activator of Rac in antigen- and chemokine-initiated signaling pathways, and this interaction is linked to the abilities of Nef to inhibit chemotaxis and promote T cell activation (Janardhan et al., 2004). Finally, Nef association with Pak2 prevents actin remodeling to impair host cell motility by disregulation of cofilin, which is an actin-depolymerizing factor that promotes actin turnover and subsequent cell motility (Stolp et al., 2010).

The cytoskeleton reorganization induced by Nef is associated with an impairment of cell movements combined with induction of long filopodium-like structures in T lymphocytes (Stolp et al., 2010). In summary, Nef displays a variety of complex effects on the motility and cellular morphology of HIV-1-infectected T lymphocyte, thus resulting in a strategy to improve immune evasion and viral spread in the infected host.

## **3. SIV Nef to counteract a cellular restriction factor?**

## **3.1 Tetherin and Nef**

The cellular protein Tetherin (also known as BST-2, CD317, or HM1.24) is a membrane protein with a number of distinct characteristics indicating that it plays a straight role in suppressing the release of virions from infected cells. Tetherin was discovered in 2008 when groups of Bieniasz and Guatelli were investigating the HIV-1 Vpu anti-Tetherin activity during the virus life cycle (Neil et al., 2008; Van Damme et al., 2008). The N-terminal of this protein is localized in the cytoplasm, followed by a transmembrane domain and a coiled-coil extracellular domain. The extracellular domain contains two N-linked glycosylation sites that are modified by a glycosyl phosphatidyl inositol (GPI) membrane anchor at the Cterminus (Kupzig et al., 2003; Rollason et al., 2007).

Tetherin functions as a broadly acting antiviral factor because besides lentiviruses, Tetherin restricts the release of different retroviruses, including alpha-, beta-, delta-, spumaviruses, and other viruses as arena- (Lassa), filo- (Marbuburg, Ebola), and herpesviruses (KSHV) (Mansouri et al., 2009). Tetherin belongs to the three main classes of restriction factors in addition to

Functions of the Lentiviral Accessory Protein

**4.1 Nef and virus entry and uncoating** 

membrane. Recent evidences favor the third model.

uncoating (Cavrois et al., 2004; Forshey and Aiken, 2003).

Nef During the Distinct Steps of HIV and SIV Replication Cycle 15

HIV and SIV enter target cells by fusion of the viral envelope with the cell membrane followed by the delivery of the virion core inside the cell cytoplasm. The host cell cytoskeleton imposes the first physical barrier to viral invasion upon entry and Retroviruses have evolved mechanisms to interfere with cytoskeleton arrangement. More specifically, it has been proposed that Nef could reorganize actin to ensure initial viral core movement. Association of Nef with viral cores (Kotov et al., 1999) and cellular proteins involved in actin cytoskeleton dynamics such as Vav and PAK (a member of the p21-activated kinase family) could account to the early movement of the viral cores through cortical actin and into microtubules (Roeth and Collins, 2006). Therefore, it has been proposed that this function of

Lentiviruses infect non-dividing cells implying that the recently formed viral DNA enters the nucleus through the nuclear pore. In fact, the so-called Pre-Integration Complex (PIC), formed by the double strand viral DNA associated with the viral Integrase (IN) and other viral and cellular proteins gains access to the nucleus by being actively transported through the nuclear pore. This phenomena imposes the necessity of an uncoating step because the diameter of the viral core (60 nm wide) exceeds that of the nuclear pore (30 nm) (Arhel, 2010). Uncoating is the process of core disassembly that takes place after virus entry into the host cell. Three distinct models of lentivirus uncoating have been proposed: i) in the first model it is predicted that disassembly occurs spontaneously and immediately after the viral core has entered the cell cytoplasm; ii) in the second model disassembly occurs in a time frame when the reverse transcription of the viral RNA has already started; iii) in the third model core disassembly occurs later on when the synthesis of the viral DNA is already completed and the Reverse Transcription Complex (RTC) is in close proximity of the nuclear

Disassembly of the viral cores has to occur in an optimal rate to ensure that reverse transcription is successfully completed. This was evident from studies demonstrating that mutations in Gag affecting core stability reduced reverse transcription in cells (Brun et al., 2008; Forshey et al., 2002). Since Nef-deleted viruses have a defect in reverse transcription unrelated to a direct role of Nef in the Reverse Transcriptase (RT) activity (which will be discussed in section 4.2), it was proposed that the absence of Nef in the incoming viruses would affect core stability, compromising uncoating and therefore reverse transcription. However, studies failed to show this effect of Nef ruling out a role for Nef during viral

Interestingly, the route of viral entry seems to dictate the Nef requirement for optimal infectivity. Whereas wild type HIV-1 or amphotropic murine leukemia glycoproteinpseudotyped HIV-1 virions that promote membrane fusion and cell entry through the plasma membrane are dependent on Nef to be fully infectious, HIV-1 virions pseudotyped with the glycoproteins for which fusion and entry take place after endocytosis and upon endosome acidification (e.g. Vesicular Stomatitis Virus glycoprotein - VSV-G) do not require Nef to increase infectivity (Aiken, 1997; Chazal et al., 2001; Luo et al., 1998). As pointed out previously, the treatment of target cells with drugs that disrupt the cortical actin cytoskeleton complements the infectivity defect of Nef-deleted virus (Campbell et al., 2004). Therefore, taken together, and discarding the role of Nef in facilitating viral uncoating (Cavrois et al., 2004; Forshey and Aiken, 2003), these findings have been interpreted as

**4. Nef and the basic steps of the HIV and SIV replication cycle** 

Nef could account to the increase in virus infectivity (Campbell et al., 2004).

APOBEC3G, which induces hypermutation in the retroviral genome, and the Tripartide Motif Protein 5α (TRIM 5α), which acts as new incoming retroviral capsid restriction factor (Neil et al., 2008). HIV-1 overcomes these restrictions factors by the action of accessory proteins as Vif and Vpu that act against cellular substrates APOBEC3G and Tetherin, respectively, to ensure viral persistence, replication, dissemination, and transmission (Malim and Emerman, 2008).

Tetherin is antagonized by the HIV-1 protein Vpu (Neil et al., 2008; Van Damme et al., 2008). Vpu interact directly with the transmembrane domain of Tetherin with a high specificity (Gupta et al., 2009; McNatt et al., 2009). The mechanism by which Vpu remove Tetherin from the cell surface was proposed as Vpu recruits β-TrCP, a substrate adaptor for an SCF E3 ubiquitin ligase complex, to remove Tetherin via post-endocytic membrane trafficking events (Douglas et al., 2009; Mitchell et al., 2009). As a consequence, Vpu leads to moderately reduced levels of Tetherin at the host cell surface and a modest decrease in total cellular Tetherin (Douglas et al., 2009; Mitchell et al., 2009; Van Damme et al., 2008).

Anti-Tetherin factors are also found in other viruses. Kaposi's sarcoma-associated herpesvirus (KSHV) uses K5/MIR2 protein to ubiquitinate and target Tetherin for degradation (Mansouri et al., 2009). Ebola virus glycoprotein antagonizes Tetherin-mediated restriction (Kaletsky et al., 2009; Lopez et al., 2010). Vpu functions as an anti-Tetherin factor in lentiviruses as SIV from Skye's monkeys (Lopez et al., 2010). Since lentiviruses of the SIVsmm/mac/HIV-2 lineage do not have a *vpu* gene to impair Tetherin aactivity, this role was taken by other viral proteins. Some primate lentiviruses (e.g., SIVsmm, SIVmac, and SIVagm) use the Nef protein to antagonize Tetherin (Jia et al., 2009). SIVcpz from chimpanzees and SIVgor from gorillas, which contain the *vpu* gene and are the ancestors of HIV-1, also use Nef to antagonize Tetherin (Yang et al., 2010). HIV-2 on the other hand, uses its envelope glycoprotein Env (and not Nef) to downmodulate Tetherin (Bour et al., 1996; Le Tortorec and Neil, 2009; Ritter et al., 1996). SIVtan from Tantalus monkeys uses both Env and Nef to antagonize Tetherin (Gupta et al., 2009; Zhang et al., 2009).

Analyses of the interactions between Tetherins from different primate species and the antagonist proteins used by viruses that infect those hosts have revealed a high degree of species-specificity. For example, the HIV-1 Vpu protein antagonizes human but not monkey Tetherin (Gupta et al., 2009; McNatt et al., 2009). These antiviral factors have sequence divergences that may constitute barriers to zoonotic viral transmission from animal reservoirs. For instance, the specificity of SIV Nef for rhesus Tetherin mapped to a four amino acid sequence in the cytoplasmatic domain that is missing from the human protein, whereas the specificity of HIV-1 Vpu for human Tetherin mapped to amino acid differences in the transmembrane domain (Jia et al., 2009).

Tetherin is usually only expressed efficiently in plasmacytoid dendritic cells, some cancer cells, terminally differentiated B cells, and bone marrow stromal cells (Blasius et al., 2006; Goto et al., 1994; Ishikawa et al., 1995) and its expression is strongly induced by type I Interferons (Neil et al., 2007). Tetherin is constitutively expressed on the surface of HeLa, Hep-2 and Jurkat cells lines but is not detected in other cell lines, such as 293T, HOS and Cos-7 cells (Van Damme et al., 2008). The absence of Tetherin expression in some cell lines suggest that the anti-Tetherin function of Vpu or Nef may not be obligatory for efficient viral replication *in vitro* and disease progression *in vivo*. It also remains to be determined whether Tetherin will promote or block cell-to-cell transmission (Gummuluru et al., 2000; Neil et al., 2006; Neil et al., 2008; Vendrame et al., 2009). Thus, further studies on the effect of Tetherin for primate lentiviral replication are necessary.

## **4. Nef and the basic steps of the HIV and SIV replication cycle**

## **4.1 Nef and virus entry and uncoating**

14 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

APOBEC3G, which induces hypermutation in the retroviral genome, and the Tripartide Motif Protein 5α (TRIM 5α), which acts as new incoming retroviral capsid restriction factor (Neil et al., 2008). HIV-1 overcomes these restrictions factors by the action of accessory proteins as Vif and Vpu that act against cellular substrates APOBEC3G and Tetherin, respectively, to ensure viral persistence, replication, dissemination, and transmission (Malim and Emerman, 2008). Tetherin is antagonized by the HIV-1 protein Vpu (Neil et al., 2008; Van Damme et al., 2008). Vpu interact directly with the transmembrane domain of Tetherin with a high specificity (Gupta et al., 2009; McNatt et al., 2009). The mechanism by which Vpu remove Tetherin from the cell surface was proposed as Vpu recruits β-TrCP, a substrate adaptor for an SCF E3 ubiquitin ligase complex, to remove Tetherin via post-endocytic membrane trafficking events (Douglas et al., 2009; Mitchell et al., 2009). As a consequence, Vpu leads to moderately reduced levels of Tetherin at the host cell surface and a modest decrease in total

cellular Tetherin (Douglas et al., 2009; Mitchell et al., 2009; Van Damme et al., 2008).

and Nef to antagonize Tetherin (Gupta et al., 2009; Zhang et al., 2009).

in the transmembrane domain (Jia et al., 2009).

Tetherin for primate lentiviral replication are necessary.

Anti-Tetherin factors are also found in other viruses. Kaposi's sarcoma-associated herpesvirus (KSHV) uses K5/MIR2 protein to ubiquitinate and target Tetherin for degradation (Mansouri et al., 2009). Ebola virus glycoprotein antagonizes Tetherin-mediated restriction (Kaletsky et al., 2009; Lopez et al., 2010). Vpu functions as an anti-Tetherin factor in lentiviruses as SIV from Skye's monkeys (Lopez et al., 2010). Since lentiviruses of the SIVsmm/mac/HIV-2 lineage do not have a *vpu* gene to impair Tetherin aactivity, this role was taken by other viral proteins. Some primate lentiviruses (e.g., SIVsmm, SIVmac, and SIVagm) use the Nef protein to antagonize Tetherin (Jia et al., 2009). SIVcpz from chimpanzees and SIVgor from gorillas, which contain the *vpu* gene and are the ancestors of HIV-1, also use Nef to antagonize Tetherin (Yang et al., 2010). HIV-2 on the other hand, uses its envelope glycoprotein Env (and not Nef) to downmodulate Tetherin (Bour et al., 1996; Le Tortorec and Neil, 2009; Ritter et al., 1996). SIVtan from Tantalus monkeys uses both Env

Analyses of the interactions between Tetherins from different primate species and the antagonist proteins used by viruses that infect those hosts have revealed a high degree of species-specificity. For example, the HIV-1 Vpu protein antagonizes human but not monkey Tetherin (Gupta et al., 2009; McNatt et al., 2009). These antiviral factors have sequence divergences that may constitute barriers to zoonotic viral transmission from animal reservoirs. For instance, the specificity of SIV Nef for rhesus Tetherin mapped to a four amino acid sequence in the cytoplasmatic domain that is missing from the human protein, whereas the specificity of HIV-1 Vpu for human Tetherin mapped to amino acid differences

Tetherin is usually only expressed efficiently in plasmacytoid dendritic cells, some cancer cells, terminally differentiated B cells, and bone marrow stromal cells (Blasius et al., 2006; Goto et al., 1994; Ishikawa et al., 1995) and its expression is strongly induced by type I Interferons (Neil et al., 2007). Tetherin is constitutively expressed on the surface of HeLa, Hep-2 and Jurkat cells lines but is not detected in other cell lines, such as 293T, HOS and Cos-7 cells (Van Damme et al., 2008). The absence of Tetherin expression in some cell lines suggest that the anti-Tetherin function of Vpu or Nef may not be obligatory for efficient viral replication *in vitro* and disease progression *in vivo*. It also remains to be determined whether Tetherin will promote or block cell-to-cell transmission (Gummuluru et al., 2000; Neil et al., 2006; Neil et al., 2008; Vendrame et al., 2009). Thus, further studies on the effect of HIV and SIV enter target cells by fusion of the viral envelope with the cell membrane followed by the delivery of the virion core inside the cell cytoplasm. The host cell cytoskeleton imposes the first physical barrier to viral invasion upon entry and Retroviruses have evolved mechanisms to interfere with cytoskeleton arrangement. More specifically, it has been proposed that Nef could reorganize actin to ensure initial viral core movement. Association of Nef with viral cores (Kotov et al., 1999) and cellular proteins involved in actin cytoskeleton dynamics such as Vav and PAK (a member of the p21-activated kinase family) could account to the early movement of the viral cores through cortical actin and into microtubules (Roeth and Collins, 2006). Therefore, it has been proposed that this function of Nef could account to the increase in virus infectivity (Campbell et al., 2004).

Lentiviruses infect non-dividing cells implying that the recently formed viral DNA enters the nucleus through the nuclear pore. In fact, the so-called Pre-Integration Complex (PIC), formed by the double strand viral DNA associated with the viral Integrase (IN) and other viral and cellular proteins gains access to the nucleus by being actively transported through the nuclear pore. This phenomena imposes the necessity of an uncoating step because the diameter of the viral core (60 nm wide) exceeds that of the nuclear pore (30 nm) (Arhel, 2010). Uncoating is the process of core disassembly that takes place after virus entry into the host cell. Three distinct models of lentivirus uncoating have been proposed: i) in the first model it is predicted that disassembly occurs spontaneously and immediately after the viral core has entered the cell cytoplasm; ii) in the second model disassembly occurs in a time frame when the reverse transcription of the viral RNA has already started; iii) in the third model core disassembly occurs later on when the synthesis of the viral DNA is already completed and the Reverse Transcription Complex (RTC) is in close proximity of the nuclear membrane. Recent evidences favor the third model.

Disassembly of the viral cores has to occur in an optimal rate to ensure that reverse transcription is successfully completed. This was evident from studies demonstrating that mutations in Gag affecting core stability reduced reverse transcription in cells (Brun et al., 2008; Forshey et al., 2002). Since Nef-deleted viruses have a defect in reverse transcription unrelated to a direct role of Nef in the Reverse Transcriptase (RT) activity (which will be discussed in section 4.2), it was proposed that the absence of Nef in the incoming viruses would affect core stability, compromising uncoating and therefore reverse transcription. However, studies failed to show this effect of Nef ruling out a role for Nef during viral uncoating (Cavrois et al., 2004; Forshey and Aiken, 2003).

Interestingly, the route of viral entry seems to dictate the Nef requirement for optimal infectivity. Whereas wild type HIV-1 or amphotropic murine leukemia glycoproteinpseudotyped HIV-1 virions that promote membrane fusion and cell entry through the plasma membrane are dependent on Nef to be fully infectious, HIV-1 virions pseudotyped with the glycoproteins for which fusion and entry take place after endocytosis and upon endosome acidification (e.g. Vesicular Stomatitis Virus glycoprotein - VSV-G) do not require Nef to increase infectivity (Aiken, 1997; Chazal et al., 2001; Luo et al., 1998). As pointed out previously, the treatment of target cells with drugs that disrupt the cortical actin cytoskeleton complements the infectivity defect of Nef-deleted virus (Campbell et al., 2004). Therefore, taken together, and discarding the role of Nef in facilitating viral uncoating (Cavrois et al., 2004; Forshey and Aiken, 2003), these findings have been interpreted as

Functions of the Lentiviral Accessory Protein

reverse transcription (Warrilow et al., 2009).

**4.3 Nef and virus assembly** 

assembly of HIV-1.

incorporated into DRMs.

the virus producer cell (Laguette et al., 2009; Pizzato et al., 2008).

Nef During the Distinct Steps of HIV and SIV Replication Cycle 17

physically interacting and increasing the processivity of the RT (Dobard et al., 2007; Wu et al., 1999). Therefore, not only the high stoichiometry of the RT must be maintained during reverse transcription but also that of the IN. Other viral proteins have been implicated in facilitating or participating in reverse transcription. For instance, NC and Vpr physically interact with RT as well as with IN and may play an important role during the initiation of

The presence of Nef in the RTC is well documented (Forshey and Aiken, 2003; Kotov et al., 1999), as well as its function in stimulating the synthesis of the viral dsDNA during reverse transcription in the target cell (Aiken and Trono, 1995; Schwartz et al., 1995). However, Nef does not influence RT activity *per se* since the *in vitro* activity of RT from Nef-deleted viruses is not altered and the treatment of these viruses with deoxyribonucleotides previous to infection of the target cells restores viral infectivity (Aiken and Trono, 1995; Khan et al., 2001). These observations prompt to the conclusion that Nef would function in an early step during the viral replication cycle before reverse transcription. Studies have failed to demonstrate any influence of Nef in virus entry, delivery of the viral cores to the cell cytoplasm or core disassembly (Cavrois et al., 2004; Forshey and Aiken, 2003; Miller et al., 1995). Other study had shown however that this early effect of Nef can be attributed to a post-entry event like facilitating the movement of the viral core through cortical actin located beneath the plasma membrane (Campbell et al., 2004), however this has recently being disputed since two different studies demonstrated that the effect of Nef in increasing viral infectivity derives not from its presence in the viral particles but from some effect on

The viral genomic RNA is transported to the cytoplasm were it leads to the synthesis of the Gag and GagPol polyprotein precursors. These precursors oligomerize and traffic to the plasma membrane by still not completely understood pathway(s). Concomitantly, the Env glycoproteins are translated in the ER and are transported to the plasma membrane via the secretory pathway. Membrane-targeted Gag and GagPol polyproteins recruit the viral genomic RNAs and assemble at the plasma membrane, leading to the induction of membrane curvature at the site of assembly, while the Env glycoproteins are incorporated into the budding particles during the assembly process. Experimental evidences suggest an important role for lipids and especialized cell membrane microdomains for the optimal

Also, HIV and SIV and many other viruses infect their target cells by interacting with the surface membrane microdomains enriched with cholesterol, sphingolipids and other saturated lipids, as well as specific types of proteins, referred as lipid rafts or detergentresistance membranes (DRMs) (Nayak and Hui, 2004). Lipid rafts form a liquid-ordered state through lipid-lipid interactions and are central for attachment of proteins when membranes are moved around inside the cell and during signaling transduction (Verkade and Simons, 1997). Glycosylphosphatidylinositol (GPI)-anchored proteins, transmembrane proteins and doubly acylated tyrosine kinases of the Src family all associate and are

Moreover, Nef has been proposed to have a role during the assembly step of the viral replication cycle. Several studies reported that HIV-1 Nef expression alters the lipid composition of virions by increasing cholesterol biosynthesis and its incorporation into

evidence for a role of Nef in facilitating the penetration of the viral cores through the cortical acting barrier, a function that would become dispensable if entry occurs through endocytosis. However, it was recently demonstrated that the presence of Nef itself in viral particles by means of its incorporation through Vpr (Nef.Vpr fusion protein) was not sufficient to increase viral infectivity (Laguette et al., 2009). Moreover, it has been shown that in some cases the requirement for Nef to achieve optimal viral infectivity is not circumvent by directing viral entry through endocytocis followed by exposure to low pH. In an elegant study it was shown that pseudotyping HIV-1 cores with the Rous Sarcoma Virus A (RSV-A) receptor, the Tva molecule, and using this pseudotypes to infect cells harboring the RSV-A glycoprotein Nef was still necessary for optimal infectivity (Pizzato et al., 2008). Therefore it remains to be fully established that the importance of Nef to the increase of viral infectivity is solely related to its effect on the actin rearrangement upon viral entry and whether other components of the viral core also contribute in the process.

#### **4.2 Nef and reverse transcription**

Upon entry to the target cell the reverse transcription step of the Retroviral replication cycle is initiated. Reverse transcription is defined as the synthesis of the viral double strand DNA (dsDNA) from the viral single strand RNA genome (ssRNA), which is catalyzed by the two sub unities of the viral Reverse Transcriptase (RT), p66 (polymerase and RNase H) and p51 (polymerase). A pre-requisite to reverse transcription is the formation of a pre-initiation RTC during viral maturation within the producer cell. Minimally, the pre-initiation RTC is composed by two copies of the viral ssRNA genome, the tRNA primer and the viral enzymes RT and IN. Other viral proteins including nucleocapsid (NC) and the accessory proteins Vpr and Nef are also part of the pre-initiation RTC (Warrilow et al., 2009). An important characteristic of the pre-initiation RTC for most of the retroviruses is that, in virions, it undergoes minimal reverse transcription and this event is trigged in the target cell by factors that are still not completely understood. One of the major events triggering reverse transcription seems to be the exposure of the RTC to the non-limiting concentration of deoxyribonucleotides within the target cells. Once the reverse transcription is initiated structural changes occur releasing most of the protein content of the pre-initiation RTC, turning it into a mature RTC. For instance, most of the RT and the Nef content is shed at this time. It is being now recognized that cellular proteins such as helicases and other cellular factors are also present at the RTC and must play important roles during reverse transcription (Warrilow et al., 2010). The RTC migrates towards the nuclear pore through association with the cell cytoskeleton and once reverse transcription is completed the RTC is fully maturated in the PIC to enter the nucleus for the next step of viral dsDNA integration (Warrilow et al., 2009). One of the models of uncoating discussed previously predicts that only at the stage of fully maturation of the RTC into PIC is that most of the CA protein content is shed. Therefore, uncoating occurs later after reverse transcription and implies that an optimal core microenvironment is maintained in order to avoid dilution of the crucial RTC contents and the attack of deleterious cellular factors (Arhel, 2010).

Reverse transcription occur in two distinct phases; the early phase encompasses the formation of the negative strand DNA (cDNA) from the genomic RNA, while in the late phase the positive strand DNA is synthesized from the cDNA generating the dsDNA. RT is the sole enzyme that catalyses the DNA synthesis and the degradation of the viral RNA template. However, IN has a crucial role during the initiation of the reverse transcription by

evidence for a role of Nef in facilitating the penetration of the viral cores through the cortical acting barrier, a function that would become dispensable if entry occurs through endocytosis. However, it was recently demonstrated that the presence of Nef itself in viral particles by means of its incorporation through Vpr (Nef.Vpr fusion protein) was not sufficient to increase viral infectivity (Laguette et al., 2009). Moreover, it has been shown that in some cases the requirement for Nef to achieve optimal viral infectivity is not circumvent by directing viral entry through endocytocis followed by exposure to low pH. In an elegant study it was shown that pseudotyping HIV-1 cores with the Rous Sarcoma Virus A (RSV-A) receptor, the Tva molecule, and using this pseudotypes to infect cells harboring the RSV-A glycoprotein Nef was still necessary for optimal infectivity (Pizzato et al., 2008). Therefore it remains to be fully established that the importance of Nef to the increase of viral infectivity is solely related to its effect on the actin rearrangement upon viral entry and

Upon entry to the target cell the reverse transcription step of the Retroviral replication cycle is initiated. Reverse transcription is defined as the synthesis of the viral double strand DNA (dsDNA) from the viral single strand RNA genome (ssRNA), which is catalyzed by the two sub unities of the viral Reverse Transcriptase (RT), p66 (polymerase and RNase H) and p51 (polymerase). A pre-requisite to reverse transcription is the formation of a pre-initiation RTC during viral maturation within the producer cell. Minimally, the pre-initiation RTC is composed by two copies of the viral ssRNA genome, the tRNA primer and the viral enzymes RT and IN. Other viral proteins including nucleocapsid (NC) and the accessory proteins Vpr and Nef are also part of the pre-initiation RTC (Warrilow et al., 2009). An important characteristic of the pre-initiation RTC for most of the retroviruses is that, in virions, it undergoes minimal reverse transcription and this event is trigged in the target cell by factors that are still not completely understood. One of the major events triggering reverse transcription seems to be the exposure of the RTC to the non-limiting concentration of deoxyribonucleotides within the target cells. Once the reverse transcription is initiated structural changes occur releasing most of the protein content of the pre-initiation RTC, turning it into a mature RTC. For instance, most of the RT and the Nef content is shed at this time. It is being now recognized that cellular proteins such as helicases and other cellular factors are also present at the RTC and must play important roles during reverse transcription (Warrilow et al., 2010). The RTC migrates towards the nuclear pore through association with the cell cytoskeleton and once reverse transcription is completed the RTC is fully maturated in the PIC to enter the nucleus for the next step of viral dsDNA integration (Warrilow et al., 2009). One of the models of uncoating discussed previously predicts that only at the stage of fully maturation of the RTC into PIC is that most of the CA protein content is shed. Therefore, uncoating occurs later after reverse transcription and implies that an optimal core microenvironment is maintained in order to avoid dilution of the crucial

whether other components of the viral core also contribute in the process.

RTC contents and the attack of deleterious cellular factors (Arhel, 2010).

Reverse transcription occur in two distinct phases; the early phase encompasses the formation of the negative strand DNA (cDNA) from the genomic RNA, while in the late phase the positive strand DNA is synthesized from the cDNA generating the dsDNA. RT is the sole enzyme that catalyses the DNA synthesis and the degradation of the viral RNA template. However, IN has a crucial role during the initiation of the reverse transcription by

**4.2 Nef and reverse transcription** 

physically interacting and increasing the processivity of the RT (Dobard et al., 2007; Wu et al., 1999). Therefore, not only the high stoichiometry of the RT must be maintained during reverse transcription but also that of the IN. Other viral proteins have been implicated in facilitating or participating in reverse transcription. For instance, NC and Vpr physically interact with RT as well as with IN and may play an important role during the initiation of reverse transcription (Warrilow et al., 2009).

The presence of Nef in the RTC is well documented (Forshey and Aiken, 2003; Kotov et al., 1999), as well as its function in stimulating the synthesis of the viral dsDNA during reverse transcription in the target cell (Aiken and Trono, 1995; Schwartz et al., 1995). However, Nef does not influence RT activity *per se* since the *in vitro* activity of RT from Nef-deleted viruses is not altered and the treatment of these viruses with deoxyribonucleotides previous to infection of the target cells restores viral infectivity (Aiken and Trono, 1995; Khan et al., 2001). These observations prompt to the conclusion that Nef would function in an early step during the viral replication cycle before reverse transcription. Studies have failed to demonstrate any influence of Nef in virus entry, delivery of the viral cores to the cell cytoplasm or core disassembly (Cavrois et al., 2004; Forshey and Aiken, 2003; Miller et al., 1995). Other study had shown however that this early effect of Nef can be attributed to a post-entry event like facilitating the movement of the viral core through cortical actin located beneath the plasma membrane (Campbell et al., 2004), however this has recently being disputed since two different studies demonstrated that the effect of Nef in increasing viral infectivity derives not from its presence in the viral particles but from some effect on the virus producer cell (Laguette et al., 2009; Pizzato et al., 2008).

## **4.3 Nef and virus assembly**

The viral genomic RNA is transported to the cytoplasm were it leads to the synthesis of the Gag and GagPol polyprotein precursors. These precursors oligomerize and traffic to the plasma membrane by still not completely understood pathway(s). Concomitantly, the Env glycoproteins are translated in the ER and are transported to the plasma membrane via the secretory pathway. Membrane-targeted Gag and GagPol polyproteins recruit the viral genomic RNAs and assemble at the plasma membrane, leading to the induction of membrane curvature at the site of assembly, while the Env glycoproteins are incorporated into the budding particles during the assembly process. Experimental evidences suggest an important role for lipids and especialized cell membrane microdomains for the optimal assembly of HIV-1.

Also, HIV and SIV and many other viruses infect their target cells by interacting with the surface membrane microdomains enriched with cholesterol, sphingolipids and other saturated lipids, as well as specific types of proteins, referred as lipid rafts or detergentresistance membranes (DRMs) (Nayak and Hui, 2004). Lipid rafts form a liquid-ordered state through lipid-lipid interactions and are central for attachment of proteins when membranes are moved around inside the cell and during signaling transduction (Verkade and Simons, 1997). Glycosylphosphatidylinositol (GPI)-anchored proteins, transmembrane proteins and doubly acylated tyrosine kinases of the Src family all associate and are incorporated into DRMs.

Moreover, Nef has been proposed to have a role during the assembly step of the viral replication cycle. Several studies reported that HIV-1 Nef expression alters the lipid composition of virions by increasing cholesterol biosynthesis and its incorporation into

Functions of the Lentiviral Accessory Protein

Miller et al., 1997; Pandori et al., 1998).

**4.4 Nef and virus budding** 

Schwedler et al., 2003).

Nef During the Distinct Steps of HIV and SIV Replication Cycle 19

(Pizzato et al., 2007). Interestingly, this was the same domain considered crucial for sorting of Nef on clathrin coated vesicles, showing that Nef may have multiple mechanisms in order

Furthermore, the increase in viral infectivity by Nef through binding to Dyn2 is dependent on clathrin and the proposed mechanism predicts that by binding to Dyn2 Nef would gain access and selectively modify specific membrane domains in the infected cell from which viral assembly occurs (Pizzato et al., 2007). On the other hand, clathrin by its turn may also have another direct role during HIV infection. At least for HIV-2 and SIVmac, the p6 peptide within Gag is thought to directly engage clathrin, by a motif that resembles the classic clathrin box, LLpL(-), where p is a polar residue and (-) a negatively charged one (Kirchhausen, 2000; Popov et al., 2011). It was also shown that although HIV-1 lacks such sequence in Gag or GagPol, it may also engage clathrin through GagPol, in a mechanism dependent of the IN region of this polyprotein (Popov et al., 2011). This might be explained by the enhanced GagPol dimerization rate that a GagPol precursor deleted in the IN domain possess, which could occlude a putative clathrin interaction surface (Bukovsky and Gottlinger, 1996). Since Nef interacts with GagPol and PR, it may also decrease GagPol dimerization, resulting in a better-exposed clathrin interacting surface (Costa et al., 2004;

The endo-lysosomal sorting machinery was first described in yeasts, but is well conserved in mammals. In yeasts it is called VPS pathway (for Vacuolar protein sorting), and the proteins of this pathway are denominated class E proteins, as their inactivation leads to the formation of an enlarged and abnormal membrane compartment. Class E proteins can present themselves as isolated proteins or as hetero-oligomeric complexes of high molecular weight known as ESCRT (Endosomal Sorting Complex Requires for Transport) (von

In mammals, this machinery is involved in the sorting of proteins to many cellular compartments, as endosomes, lysosomes, Golgi complex and endoplasmatic reticullum. The pathway initiates with the ESCRT-I recruitment to the already ubiquitinated (ESCRT tagging) cytoplasmatic portion of the protein to be sorted. This first recruitment is made by the cellular proteins STAM and HRS (referred by some authors as ESCRT-0). From this point, two pathways might be taken: ESCRT-I activates a second complex named ESCRT-II, or it recruits Alix/AIP-1, a central protein of the ESCRT pathway, but that is not a stable part of any of the three complexes. In both cases, the ESCRT-III is recruited, whose factors bind directly to intracellular membranes and promote its invagination, usually to an endosome. Due to its morphology, endosomal compartments that contain vesicles in its interior are called Multivesicular Bodies (MVBs). This structure might have two fates, lysosomes fusion, delivering its content to degradation, or fusion with the plasmatic

The topology of viral budding is analogous to the MVBs formation, and to the membrane fission event that happens on mitosis. Therefore it is reasonable that the same cytoplasmatic machinery used for the MVBs biogenesis is co-opted by viral specific motifs in viral proteins

Three types of late domains have been characterized in a variety of viral proteins: P(T/S)AP, responsible for the interaction with the ESCRT-I component, Tsg101; PPxY, that engage

membrane, liberating its vesicles as exosomes (Strack et al., 2003).

named late domains in order to perform viral budding (Fujii et al., 2007).

to gain access to clathrin-dependent endocytosis (Greenberg et al., 1998a).

DRMs, therefore Nef increases the concentration of Gag and colocalizes with viral structural components in the DRMs. These features were linked to the increase in viral infectivity and the facilitation of virus spread (Wang JK, 2000; Zheng et al., 2003). Also, it seems that infectivity enhancement by Nef requires its association with components of the assembling HIV/SIV particles. Gag from HIV/SIV associates with DRMs and disruption of Gag-raft interactions impairs virus particle production (Chukkapalli et al., 2010). It has been demonstrated that fusion of the host protein cyclophilin A (CypA) to Nef allowed controlled incorporation of Nef into HIV-1 particles via association with Gag during viral particle assembly for enhancement of HIV-1 infectivity (Qi and Aiken, 2008).

Besides the effect of Nef association with DRMs to viral infectivity, Nef also associates with these membrane microdomains together with several proteins involved in the initiation and propagation of T cell signaling and could therefore affect the later steps of the replication cycle. Nef was shown to interact with Pak-2 in lipid rafts (Krautkramer et al., 2004), which may result in increased frequency of cells expressing transcriptionally active forms of NF-κB and NFAT and increased T cell activation (Fenard et al., 2005). The interaction of Nef with PAK2 is conserved for many Nef proteins derived from HIV-1, HIV-2, and SIV strains (Sawai et al., 1995), and was demonstrated to be mediated by Cdc42 and Rac, which are PAK 2 activators, and being dependent on raft integrity (Krautkramer et al., 2004). While delivers activation stimulus to CD4 T cells Nef also mediates exclusion of molecules such as Lck, Vav, and TCRζ E2 known as ubiquitin-conjugating enzyme (UbcH7) from rafts in lipid rafts to avoid the negative regulation of T cell signaling (Simmons et al., 2005).

Rafts were also initially proposed to act as platforms for virus entry, facilitating interactions between CD4 receptors and the incoming virions (Chukkapalli et al., 2010). This role has been, however, questioned, because CD4 molecules unable to associate with rafts still allow virus entry (Percherancier et al., 2003; Popik and Alce, 2004). Moreover, Sol-Foulon and coworkers proposed that the effects of Nef on CD4 downregulation and the increase in viral infectivity were independent of lipid rafts (Sol-Foulon et al., 2004). Discrepancies in methodological approaches from these authors with the reports described before could account for the observed differences on this effect of Nef. Therefore, more detailed studies are needed in order to establish a connection between the localization and the effects of Nef in DRMs and its importance for virus replication and infectivity.

Endocytosis may be classified into two categories, clathrin dependent and independent. The role of Nef in inducing clathrin-dependent endocytosis of some surface receptors, as CD4 (Aiken et al., 1994; Chaudhuri et al., 2007; Garcia and Miller, 1991; Mariani and Skowronski, 1993), triggering the *de novo* formation of clathrin coated pits and acting as a connector between receptor cargo and the endocytic machinery (Foti et al., 1997) has been addressed here. Dyn2 was identified as a Nef binding partner through immunopreciptation assays, and is intrinsically related to clathrin-dependent endocytosis (Pizzato et al., 2007). It is a ubiquitously expressed member of large GTPases that is thought to aid the fission step that separates clathrin-coated vesicles from the plasma membrane (Hinshaw, 2000). Nef distinguishes between the three isoforms of Dyn, binding specifically to Dyn2, and this interaction is conserved among different *nef* alleles. The interaction is dependent of the Middle and GTPase Effector domains of Dyn2 and surface-exposed core domain residues in Nef, as L112, F121 and D123. Loss of Dyn2 interaction leads to loss of infectivity, however, Nef mutants that are able to bind Dyn2, as LL164,165AA, also show infectivity impairment, showing that Dyn2 interaction is not sufficient for Nef induced infectivity enhancement (Pizzato et al., 2007). Interestingly, this was the same domain considered crucial for sorting of Nef on clathrin coated vesicles, showing that Nef may have multiple mechanisms in order to gain access to clathrin-dependent endocytosis (Greenberg et al., 1998a).

Furthermore, the increase in viral infectivity by Nef through binding to Dyn2 is dependent on clathrin and the proposed mechanism predicts that by binding to Dyn2 Nef would gain access and selectively modify specific membrane domains in the infected cell from which viral assembly occurs (Pizzato et al., 2007). On the other hand, clathrin by its turn may also have another direct role during HIV infection. At least for HIV-2 and SIVmac, the p6 peptide within Gag is thought to directly engage clathrin, by a motif that resembles the classic clathrin box, LLpL(-), where p is a polar residue and (-) a negatively charged one (Kirchhausen, 2000; Popov et al., 2011). It was also shown that although HIV-1 lacks such sequence in Gag or GagPol, it may also engage clathrin through GagPol, in a mechanism dependent of the IN region of this polyprotein (Popov et al., 2011). This might be explained by the enhanced GagPol dimerization rate that a GagPol precursor deleted in the IN domain possess, which could occlude a putative clathrin interaction surface (Bukovsky and Gottlinger, 1996). Since Nef interacts with GagPol and PR, it may also decrease GagPol dimerization, resulting in a better-exposed clathrin interacting surface (Costa et al., 2004; Miller et al., 1997; Pandori et al., 1998).

## **4.4 Nef and virus budding**

18 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

DRMs, therefore Nef increases the concentration of Gag and colocalizes with viral structural components in the DRMs. These features were linked to the increase in viral infectivity and the facilitation of virus spread (Wang JK, 2000; Zheng et al., 2003). Also, it seems that infectivity enhancement by Nef requires its association with components of the assembling HIV/SIV particles. Gag from HIV/SIV associates with DRMs and disruption of Gag-raft interactions impairs virus particle production (Chukkapalli et al., 2010). It has been demonstrated that fusion of the host protein cyclophilin A (CypA) to Nef allowed controlled incorporation of Nef into HIV-1 particles via association with Gag during viral particle

Besides the effect of Nef association with DRMs to viral infectivity, Nef also associates with these membrane microdomains together with several proteins involved in the initiation and propagation of T cell signaling and could therefore affect the later steps of the replication cycle. Nef was shown to interact with Pak-2 in lipid rafts (Krautkramer et al., 2004), which may result in increased frequency of cells expressing transcriptionally active forms of NF-κB and NFAT and increased T cell activation (Fenard et al., 2005). The interaction of Nef with PAK2 is conserved for many Nef proteins derived from HIV-1, HIV-2, and SIV strains (Sawai et al., 1995), and was demonstrated to be mediated by Cdc42 and Rac, which are PAK 2 activators, and being dependent on raft integrity (Krautkramer et al., 2004). While delivers activation stimulus to CD4 T cells Nef also mediates exclusion of molecules such as Lck, Vav, and TCRζ E2 known as ubiquitin-conjugating enzyme (UbcH7) from rafts in lipid

Rafts were also initially proposed to act as platforms for virus entry, facilitating interactions between CD4 receptors and the incoming virions (Chukkapalli et al., 2010). This role has been, however, questioned, because CD4 molecules unable to associate with rafts still allow virus entry (Percherancier et al., 2003; Popik and Alce, 2004). Moreover, Sol-Foulon and coworkers proposed that the effects of Nef on CD4 downregulation and the increase in viral infectivity were independent of lipid rafts (Sol-Foulon et al., 2004). Discrepancies in methodological approaches from these authors with the reports described before could account for the observed differences on this effect of Nef. Therefore, more detailed studies are needed in order to establish a connection between the localization and the effects of Nef

Endocytosis may be classified into two categories, clathrin dependent and independent. The role of Nef in inducing clathrin-dependent endocytosis of some surface receptors, as CD4 (Aiken et al., 1994; Chaudhuri et al., 2007; Garcia and Miller, 1991; Mariani and Skowronski, 1993), triggering the *de novo* formation of clathrin coated pits and acting as a connector between receptor cargo and the endocytic machinery (Foti et al., 1997) has been addressed here. Dyn2 was identified as a Nef binding partner through immunopreciptation assays, and is intrinsically related to clathrin-dependent endocytosis (Pizzato et al., 2007). It is a ubiquitously expressed member of large GTPases that is thought to aid the fission step that separates clathrin-coated vesicles from the plasma membrane (Hinshaw, 2000). Nef distinguishes between the three isoforms of Dyn, binding specifically to Dyn2, and this interaction is conserved among different *nef* alleles. The interaction is dependent of the Middle and GTPase Effector domains of Dyn2 and surface-exposed core domain residues in Nef, as L112, F121 and D123. Loss of Dyn2 interaction leads to loss of infectivity, however, Nef mutants that are able to bind Dyn2, as LL164,165AA, also show infectivity impairment, showing that Dyn2 interaction is not sufficient for Nef induced infectivity enhancement

assembly for enhancement of HIV-1 infectivity (Qi and Aiken, 2008).

rafts to avoid the negative regulation of T cell signaling (Simmons et al., 2005).

in DRMs and its importance for virus replication and infectivity.

The endo-lysosomal sorting machinery was first described in yeasts, but is well conserved in mammals. In yeasts it is called VPS pathway (for Vacuolar protein sorting), and the proteins of this pathway are denominated class E proteins, as their inactivation leads to the formation of an enlarged and abnormal membrane compartment. Class E proteins can present themselves as isolated proteins or as hetero-oligomeric complexes of high molecular weight known as ESCRT (Endosomal Sorting Complex Requires for Transport) (von Schwedler et al., 2003).

In mammals, this machinery is involved in the sorting of proteins to many cellular compartments, as endosomes, lysosomes, Golgi complex and endoplasmatic reticullum. The pathway initiates with the ESCRT-I recruitment to the already ubiquitinated (ESCRT tagging) cytoplasmatic portion of the protein to be sorted. This first recruitment is made by the cellular proteins STAM and HRS (referred by some authors as ESCRT-0). From this point, two pathways might be taken: ESCRT-I activates a second complex named ESCRT-II, or it recruits Alix/AIP-1, a central protein of the ESCRT pathway, but that is not a stable part of any of the three complexes. In both cases, the ESCRT-III is recruited, whose factors bind directly to intracellular membranes and promote its invagination, usually to an endosome. Due to its morphology, endosomal compartments that contain vesicles in its interior are called Multivesicular Bodies (MVBs). This structure might have two fates, lysosomes fusion, delivering its content to degradation, or fusion with the plasmatic membrane, liberating its vesicles as exosomes (Strack et al., 2003).

The topology of viral budding is analogous to the MVBs formation, and to the membrane fission event that happens on mitosis. Therefore it is reasonable that the same cytoplasmatic machinery used for the MVBs biogenesis is co-opted by viral specific motifs in viral proteins named late domains in order to perform viral budding (Fujii et al., 2007).

Three types of late domains have been characterized in a variety of viral proteins: P(T/S)AP, responsible for the interaction with the ESCRT-I component, Tsg101; PPxY, that engage

Functions of the Lentiviral Accessory Protein

al., 2004; Jesus da Costa et al., 2009).

by the viral PR.

Nef During the Distinct Steps of HIV and SIV Replication Cycle 21

After dimerization, the first cleavages events occur in *cis*. The first cleavage site is located between sp1 and NC, and the second is inside the p6\* region of the GagPol precursor polyprotein, divinding p6\* in an octapeptide (sometimes referred as Transframe Peptide, TFP) and a 48 amino acids region that is immediately upstream the PR. These two cleavages generate the processing intermediates MA-CA-sp1 (42KDa), NC-TFP (7,4KDa) from Gag and p6\*-PR-RT-IN (113KDa) from Pol (Pettit et al., 2004). It is thought that at this point p6\* acts as a zymogen, lying on the catalityc center and blocking further cleavages (Partin et al., 1991). In order to continue processing an event capable of dislodging p6\* from the catalic site is needed. Many events have been alleged as capable of so. The pH decay that occurs during the budding is the fit theory. However, it is not excluded that some cellular or viral protein may participate in this process. For instance, it has been demonstrated that the viral accessory protein Nef binds specifically to the p6\* region of the GagPol precursor (Costa et

It is well established that the Nef protein from both HIV-1 (Chen et al., 1998; Freund et al., 1994; Miller et al., 1997; Pandori et al., 1998) and -2 (Schorr et al., 1996) is cleaved by the viral PR. Initially, the cleavage site within Nef was demonstrated to localize to the Nterminal region generating a 9kDa membrane anchor domain and a 20kDa Nef core domain (30kDa core domain in the case of HIV-2). These cleavage forms of Nef are recognized only inside the viral particles, implying that cleavage takes place during the viral maturation step that culminates with the formation of mature infectious viruses. Although the HIV-2 PR cleaves Nef from HIV-1 (Schorr et al., 1996), there is no amino acid sequence conservation of the cleavage sites between Nef from HIV-1 and HIV-2. While in the HIV-1 Nef the well established cleavage site localizes between the Tryptophan 57 and Leucine 58, in the amino acid sequence context 54DCAW\*LEAQ61, in HIV-2 Nef the cleavage site is localized between the Tyrosine 39 and Serine 40 in the amino acid sequence context 36GGEY\*SQFQ43. Another cleavage site within HIV-1 Nef must exist since two independent groups reported a C-terminal cleavage form of Nef with an apparent molecular weight of 13kDa (Laguette et al., 2009; Miller et al., 1997). Recent data from our group demonstrated that the SIVcpz PR also cleaves Nef from SIVcpz during the virus replication cycle probably at the same residues as in HIV-1 Nef. Moreover, we have also observed that the SIVcpz PR has the capacity of cleaving Nef from HIV-1 generating a Nef core domain with the same apparent molecular weight when cleaved by the HIV-1 PR (Sampaio, manuscript in preparation). Since there is a great degree of conservation between the *nef* genes from HIV-2 and SIVmac it is likely that the Nef protein from SIVmac is also cleaved

The fact that Nef is cleaved by the viral PR implicates that these proteins interact during the viral replication cycle, however the consequences of this cleavage for viral infectivity is still a matter of controversy. Mutations introduced at the main cleavage site on Nef, which prevent PR cleavage, does not necessarily correlates with a loss of infectivity. For instance, in one study the deletion of the cleavage site of Nef (DCAWL 54-58) prevented its cleavage and reduced viral infectivity by 80% (Miller et al., 1997), while in another study a similar deletion, which again prevented Nef cleavage, reduced viral infectivity by only 20% (Pandori et al., 1998) (Table 1). On the other hand, mutations at the same cleavage site that did not disrupt cleavage, like W57A, had a great impact on viral infectivity (Chen et al., 1998; Miller et al., 1997). A summary of mutations and deletions introduced at the cleavage site of Nef and its impact on viral infectivity are listed on Table

interactions with the Nedd4 ubiquitin ligase family; and YPxL, that interact with Alix/AIP-1 (Freed and Mouland, 2006). The fact that these late domains maintain its functionality even when translocated to other regions of viral proteins or swapped between distinct viruses suggest that they serve as anchoring sites to cellular factors, rather than structural elements (Garrus et al., 2001).

On HIV-1 and SIV, the budding process initiates concomitantly with the end of the viral particle assembly, which is orchestrated by the different Gag domains. The PTAP motif contained in p6 peptide within Gag is considered the main late domain of HIV. However, the HIV genome holds two more late domains, the YPxL also located in p6 and the YPLT domain in Nef, both capable of interacting with Alix/AIP-1. Nef physical and direct interaction with Alix/AIP-1 has been demonstrated both *in vivo* and *in vitro* (Costa et al., 2006). Nef can substitute for the L domain of a p6-deleted Gag polyprotein when fused to its C-terminal, restoring viral budding. Also, a Vpr.Nef chimera was able to restore viral budding when the PTAP domain in p6 was disrupted (Costa et al., 2006).

In CEM and SupT1 cell lines, Nef was shown to increase biogenesis of MVBs, a feature that can be useful for budding (Costa et al., 2006; Stumptner-Cuvelette et al., 2001). HIV budding toward MVB was seen in many cell lines, and can be especially decisive in macrophages, a lineage known to have well-developed endosomal machinery. Accumulation of late endosomes was dependent of the YPLT domain in Nef, and requires Nef interaction with Alix/AIP-1.

#### **4.5 Nef and virus maturation**

The retrovirus enzymatic and structural proteins are produced by the translation of a polycistronic RNA and originate the polyprotein precursors Gag and GagPol (Frankel and Young, 1998). The viral PR (Navia et al., 1989) catalyses the hydrolysis of the peptide bonds of the polyproteins' cleavage sites originating the mature proteins capable of generating an infectious particle (Louis et al., 2000).

The lentiviral PR is composed of 99 amino acids, and is synthesized as part of the GagPol precursor polyprotein. As it contains an aspartic acid in the center of the catalytic domain, the HIV and SIV PR are classified as members of the aspartyl PR family (Navia et al., 1989). Some features separate cellular aspartyl PRs from primate lentiviral PR. Unlike cellular aspartyl PRs, which are monomeric, viral aspartyl PRs are dimeric and must dimerize in order to gain catalytic activity. Therefore, the first step of PR activation is the dimerization of two GagPol molecules (Pettit et al., 2005; Weber, 1990). Other feature is that the cleavage sites recognized by HIV and SIV PR do not share amino acid identity on the cleavage sequence, or on its flanking regions (Hellen et al., 1989; Krausslich et al., 1988). Finally, the last characteristic that separate HIV and SIV PR from its cellular relatives is that, as for cellular PRs the whole catalytic machinery is pre-formed, and its activation lies mostly on the cleavage of a zymogen (Tang and Wong, 1987), for HIV and SIV PR the activation is extremely controlled by mechanisms involving protein folding, zymogen cleavage, PR context, interactions and pH (Gatlin et al., 1998; Partin et al., 1991; Pettit et al., 2004). These abundant regulatory mechanisms point out that the correct PR activation is essential for the formation of infectious particles, and that a premature activation must be avoided. Such activation could lead to complete viral processing before the completion of budding, resulting in the diffusion of viral constituents on the cytosol and altering the protein ratio in the budding particle (Pettit et al., 2004).

interactions with the Nedd4 ubiquitin ligase family; and YPxL, that interact with Alix/AIP-1 (Freed and Mouland, 2006). The fact that these late domains maintain its functionality even when translocated to other regions of viral proteins or swapped between distinct viruses suggest that they serve as anchoring sites to cellular factors, rather than structural elements

On HIV-1 and SIV, the budding process initiates concomitantly with the end of the viral particle assembly, which is orchestrated by the different Gag domains. The PTAP motif contained in p6 peptide within Gag is considered the main late domain of HIV. However, the HIV genome holds two more late domains, the YPxL also located in p6 and the YPLT domain in Nef, both capable of interacting with Alix/AIP-1. Nef physical and direct interaction with Alix/AIP-1 has been demonstrated both *in vivo* and *in vitro* (Costa et al., 2006). Nef can substitute for the L domain of a p6-deleted Gag polyprotein when fused to its C-terminal, restoring viral budding. Also, a Vpr.Nef chimera was able to restore viral

In CEM and SupT1 cell lines, Nef was shown to increase biogenesis of MVBs, a feature that can be useful for budding (Costa et al., 2006; Stumptner-Cuvelette et al., 2001). HIV budding toward MVB was seen in many cell lines, and can be especially decisive in macrophages, a lineage known to have well-developed endosomal machinery. Accumulation of late endosomes was dependent of the YPLT domain in Nef, and requires Nef interaction with

The retrovirus enzymatic and structural proteins are produced by the translation of a polycistronic RNA and originate the polyprotein precursors Gag and GagPol (Frankel and Young, 1998). The viral PR (Navia et al., 1989) catalyses the hydrolysis of the peptide bonds of the polyproteins' cleavage sites originating the mature proteins capable of generating an

The lentiviral PR is composed of 99 amino acids, and is synthesized as part of the GagPol precursor polyprotein. As it contains an aspartic acid in the center of the catalytic domain, the HIV and SIV PR are classified as members of the aspartyl PR family (Navia et al., 1989). Some features separate cellular aspartyl PRs from primate lentiviral PR. Unlike cellular aspartyl PRs, which are monomeric, viral aspartyl PRs are dimeric and must dimerize in order to gain catalytic activity. Therefore, the first step of PR activation is the dimerization of two GagPol molecules (Pettit et al., 2005; Weber, 1990). Other feature is that the cleavage sites recognized by HIV and SIV PR do not share amino acid identity on the cleavage sequence, or on its flanking regions (Hellen et al., 1989; Krausslich et al., 1988). Finally, the last characteristic that separate HIV and SIV PR from its cellular relatives is that, as for cellular PRs the whole catalytic machinery is pre-formed, and its activation lies mostly on the cleavage of a zymogen (Tang and Wong, 1987), for HIV and SIV PR the activation is extremely controlled by mechanisms involving protein folding, zymogen cleavage, PR context, interactions and pH (Gatlin et al., 1998; Partin et al., 1991; Pettit et al., 2004). These abundant regulatory mechanisms point out that the correct PR activation is essential for the formation of infectious particles, and that a premature activation must be avoided. Such activation could lead to complete viral processing before the completion of budding, resulting in the diffusion of viral constituents on the cytosol and altering the protein ratio in

budding when the PTAP domain in p6 was disrupted (Costa et al., 2006).

(Garrus et al., 2001).

Alix/AIP-1.

**4.5 Nef and virus maturation** 

infectious particle (Louis et al., 2000).

the budding particle (Pettit et al., 2004).

After dimerization, the first cleavages events occur in *cis*. The first cleavage site is located between sp1 and NC, and the second is inside the p6\* region of the GagPol precursor polyprotein, divinding p6\* in an octapeptide (sometimes referred as Transframe Peptide, TFP) and a 48 amino acids region that is immediately upstream the PR. These two cleavages generate the processing intermediates MA-CA-sp1 (42KDa), NC-TFP (7,4KDa) from Gag and p6\*-PR-RT-IN (113KDa) from Pol (Pettit et al., 2004). It is thought that at this point p6\* acts as a zymogen, lying on the catalityc center and blocking further cleavages (Partin et al., 1991). In order to continue processing an event capable of dislodging p6\* from the catalic site is needed. Many events have been alleged as capable of so. The pH decay that occurs during the budding is the fit theory. However, it is not excluded that some cellular or viral protein may participate in this process. For instance, it has been demonstrated that the viral accessory protein Nef binds specifically to the p6\* region of the GagPol precursor (Costa et al., 2004; Jesus da Costa et al., 2009).

It is well established that the Nef protein from both HIV-1 (Chen et al., 1998; Freund et al., 1994; Miller et al., 1997; Pandori et al., 1998) and -2 (Schorr et al., 1996) is cleaved by the viral PR. Initially, the cleavage site within Nef was demonstrated to localize to the Nterminal region generating a 9kDa membrane anchor domain and a 20kDa Nef core domain (30kDa core domain in the case of HIV-2). These cleavage forms of Nef are recognized only inside the viral particles, implying that cleavage takes place during the viral maturation step that culminates with the formation of mature infectious viruses. Although the HIV-2 PR cleaves Nef from HIV-1 (Schorr et al., 1996), there is no amino acid sequence conservation of the cleavage sites between Nef from HIV-1 and HIV-2. While in the HIV-1 Nef the well established cleavage site localizes between the Tryptophan 57 and Leucine 58, in the amino acid sequence context 54DCAW\*LEAQ61, in HIV-2 Nef the cleavage site is localized between the Tyrosine 39 and Serine 40 in the amino acid sequence context 36GGEY\*SQFQ43. Another cleavage site within HIV-1 Nef must exist since two independent groups reported a C-terminal cleavage form of Nef with an apparent molecular weight of 13kDa (Laguette et al., 2009; Miller et al., 1997). Recent data from our group demonstrated that the SIVcpz PR also cleaves Nef from SIVcpz during the virus replication cycle probably at the same residues as in HIV-1 Nef. Moreover, we have also observed that the SIVcpz PR has the capacity of cleaving Nef from HIV-1 generating a Nef core domain with the same apparent molecular weight when cleaved by the HIV-1 PR (Sampaio, manuscript in preparation). Since there is a great degree of conservation between the *nef* genes from HIV-2 and SIVmac it is likely that the Nef protein from SIVmac is also cleaved by the viral PR.

The fact that Nef is cleaved by the viral PR implicates that these proteins interact during the viral replication cycle, however the consequences of this cleavage for viral infectivity is still a matter of controversy. Mutations introduced at the main cleavage site on Nef, which prevent PR cleavage, does not necessarily correlates with a loss of infectivity. For instance, in one study the deletion of the cleavage site of Nef (DCAWL 54-58) prevented its cleavage and reduced viral infectivity by 80% (Miller et al., 1997), while in another study a similar deletion, which again prevented Nef cleavage, reduced viral infectivity by only 20% (Pandori et al., 1998) (Table 1). On the other hand, mutations at the same cleavage site that did not disrupt cleavage, like W57A, had a great impact on viral infectivity (Chen et al., 1998; Miller et al., 1997). A summary of mutations and deletions introduced at the cleavage site of Nef and its impact on viral infectivity are listed on Table

Functions of the Lentiviral Accessory Protein

preserved in both HIV-1 and SIVmac.

viral particles is almost complete.

Nef During the Distinct Steps of HIV and SIV Replication Cycle 23

rescued by a Nef-Tsg101 fusion protein, indicating that, besides interacting early during the expression of GagPol, Nef and the viral structural/enzymatic precursors are present concomitantly at the sites of virus budding. While investigating what influence the binding of Nef and GagPol would have for HIV-1 infectivity we have also established that Nef from HIV binds the cellular Alix protein and correlated these findings with optimal viral replication in cells (Costa et al., 2006). We demonstrated that this interaction correlated with the property of Nef to stimulate the synthesis of MVBs in cells and the optimal infection of MDMs. Taken together, the fact that Nef binds both the viral precursor GagPol and to the cellular Alix protein, which is involved in virus budding, suggest that Nef actively participates in a late step of viral replication. We further extended these observations to SIVmac, demonstrating that the binding of Nef to both GagPol and Alix is conserved in Nef from this virus (Jesus da Costa et al., 2009). The functional relevance of these bindings was clearly demonstrated in rhesus macaques. First, informative mutations were introduced into the *nef* gene of infectious SIVmac and this mutant virus used to infect four rhesus macaques. Whereas rhesus macaques infected with the WT virus developed high viral loads and progressed rapidly to SAIDS, only two of four monkeys inoculated with the mutant virus displayed a similar picture, albeit with much delayed kinetics. Importantly, in both cases, we observed reversions in SIVNef that restored its binding to SIVGagPol and Alix. In two other rhesus macaques, mutant Nef sequences persisted and they developed neither high viral loads nor SAIDS. Further studies in cells provided additional support for these findings. Taken together these studies suggest that interactions between Nef, GagPol and Alix are important for optimal viral replication and progression to disease in the monkey model of AIDS and possibly also pertinent to the human disease since these interactions are

Still, what would be the mechanism by which interacting with GagPol and Alix, Nef would stimulate viral infectivity? Recent work from our group demonstrated that Nef that from SIVcpz also binds to GagPol from both SIVcpz and HIV-1. Furthermore, our results showed that Nef exerts a direct influence upon the viral PR activity: i) first by characterizing a SIVcpz provirus that expresses a N-terminal truncated peptide of Nef that is a potent inhibitor of viral processing and consequently viral infectivity and exerts a dominant negative activity both against PR from SIVcpz and HIV-1 (Sampaio et al. – manuscript in preparation); second by demonstrating that in the absence of Nef expression during the replication cycle of HIV-1 the processing kinetics of the Gag and GagPol precursors are accelerated, resulting in slightly lower concentration of the structural (p24-Capsid protein) and 2-fold lower concentration of the enzymatic (IN) components within viral particles (Mendonça et al. – manuscript in preparation). Moreover, the accelerated activity of the viral PR in the absence of Nef expression in the producer cells was also demonstrated by the fact that higher concentrations of PR inhibitors (e.g. Lopinavir) are required to inhibit virus replication at the same levels as of the wild type viruses. Based on this preliminary results a working model was drawn (Figure 2), in which the role of Nef for virus infectivity is explained by the following: upon binding of the p6\* and PR regions, within the GagPol precursor, Nef would function as a fine regulator of PR activation, holding it to an optimal timing when budding of the

Therefore, the right content of the viral constituents is achieved. In the absence of Nef, PR activation would occur earlier on before the completion of budding, consequently some of

1. From this data we can observe that most of the mutations that prevented cleavage correlated with a reduction in viral infectivity, however some mutations that still allow cleavage to happen also had an impact on viral infectivity. Based on these observations authors excluded that processing of Nef by the viral PR could be related to the capacity of the former to increase viral infectivity.

Nevertheless, one can draw some conclusions from these data. First, some of the mutations considered not disrupting cleavage of Nef did in fact altered the normal rate of cleavage, as for mutations W57A and WL57AA there was much less cleavage observed (Chen et al., 1998; Miller et al., 1997), while for mutation CAW55LLL it could be observed a higher cleavage rate of the Nef protein (Miller et al., 1997). Therefore, alteration of the cleavage rate could interfere with the properties of Nef to increase viral infectivity without disrupting cleavage entirely. Second, a complicating issue is that the cleavage site of Nef is also involved with downmodulation of CD4 therefore, disruption of this domain could have consequences for viral infectivity that could be related to this well established function of Nef. But it must be pointed out that the viral infectivity data described here was obtained from CD4 negative cells ruling out the effect that these mutations would have on CD4 downmodulation. Lastly, as pointed out previously 55CAWL58 is the main viral PR cleavage site but other sites exist within Nef (Laguette et al., 2009; Miller et al., 1997). This could explain that however impairing the cleavage at the major site viral infectivity increase by Nef would not be greatly affected.

It is not the presence of Nef in the viral particles *per se* that increases viral infectivity. This was proven by a study by Laguette and co-workers (Laguette et al., 2009) where it was demonstrated that increasing the amounts of Nef being incorporated within HIV-1 particles by means of a Nef-Vpr fusion protein was not sufficient to make viral particles more infectious. In any case, this Nef-Vpr fusion was normally processed by the viral PR and retained classical Nef functions such as downmodulation of CD4 from the surface of virus producer cells. This data therefore indicates that the role of Nef related to the increase in viral infectivity occurs during the late stages of the replication cycle in the virus producer cells, probably by optimally regulating/modifying the structural and/or enzymatic viral components. Some aspects related to this possible function of Nef will be discussed further. Interestingly, besides connecting viral PR since being recognized as a specific substrate, Nef can also connect the structural/enzymatic polyprotein precursor GagPol through different domains either in Nef and in GagPol (Costa et al., 2004; Jesus da Costa et al., 2009). These studies demonstrated that the Nef protein from both HIV-1 (Costa et al., 2004) and SIVmac (Jesus da Costa et al., 2009) can interact with the GagPol precursor through the flexible-loop domain in Nef and the p6\* regulatory region of GagPol. Although the specific amino acid residues involved in this interaction in both Nef and p6\* were not yet demonstrated, its specificity and the biological relevance were. Interaction of GagPol and Nef occurs in cells during the HIV-1 replication cycle and was sufficient to explain the dominant negative phenotype of an laboratory adapted allele of HIV-1 *nef* (Nef F12) which localizes to the ER and inhibits the release of Gag and its processing by the viral PR (Fackler et al., 2001; Olivetta et al., 2000). The F12 phenotype was recapitulated by a Nef with an ER retention signal, meaning that by interacting with GagPol Nef retained this polyprotein precursor inside the producer cell avoiding both release and maturation of viral particles (Costa et al., 2004). Furthermore, the relevance of this interaction was confirmed by an experiment

demonstrating that release of a late-domain defective HIV-1 from producer cells was

1. From this data we can observe that most of the mutations that prevented cleavage correlated with a reduction in viral infectivity, however some mutations that still allow cleavage to happen also had an impact on viral infectivity. Based on these observations authors excluded that processing of Nef by the viral PR could be related to the capacity of

Nevertheless, one can draw some conclusions from these data. First, some of the mutations considered not disrupting cleavage of Nef did in fact altered the normal rate of cleavage, as for mutations W57A and WL57AA there was much less cleavage observed (Chen et al., 1998; Miller et al., 1997), while for mutation CAW55LLL it could be observed a higher cleavage rate of the Nef protein (Miller et al., 1997). Therefore, alteration of the cleavage rate could interfere with the properties of Nef to increase viral infectivity without disrupting cleavage entirely. Second, a complicating issue is that the cleavage site of Nef is also involved with downmodulation of CD4 therefore, disruption of this domain could have consequences for viral infectivity that could be related to this well established function of Nef. But it must be pointed out that the viral infectivity data described here was obtained from CD4 negative cells ruling out the effect that these mutations would have on CD4 downmodulation. Lastly, as pointed out previously 55CAWL58 is the main viral PR cleavage site but other sites exist within Nef (Laguette et al., 2009; Miller et al., 1997). This could explain that however impairing the cleavage at the major site viral infectivity increase by Nef would not be

It is not the presence of Nef in the viral particles *per se* that increases viral infectivity. This was proven by a study by Laguette and co-workers (Laguette et al., 2009) where it was demonstrated that increasing the amounts of Nef being incorporated within HIV-1 particles by means of a Nef-Vpr fusion protein was not sufficient to make viral particles more infectious. In any case, this Nef-Vpr fusion was normally processed by the viral PR and retained classical Nef functions such as downmodulation of CD4 from the surface of virus producer cells. This data therefore indicates that the role of Nef related to the increase in viral infectivity occurs during the late stages of the replication cycle in the virus producer cells, probably by optimally regulating/modifying the structural and/or enzymatic viral components. Some aspects related to this possible function of Nef will be discussed further. Interestingly, besides connecting viral PR since being recognized as a specific substrate, Nef can also connect the structural/enzymatic polyprotein precursor GagPol through different domains either in Nef and in GagPol (Costa et al., 2004; Jesus da Costa et al., 2009). These studies demonstrated that the Nef protein from both HIV-1 (Costa et al., 2004) and SIVmac (Jesus da Costa et al., 2009) can interact with the GagPol precursor through the flexible-loop domain in Nef and the p6\* regulatory region of GagPol. Although the specific amino acid residues involved in this interaction in both Nef and p6\* were not yet demonstrated, its specificity and the biological relevance were. Interaction of GagPol and Nef occurs in cells during the HIV-1 replication cycle and was sufficient to explain the dominant negative phenotype of an laboratory adapted allele of HIV-1 *nef* (Nef F12) which localizes to the ER and inhibits the release of Gag and its processing by the viral PR (Fackler et al., 2001; Olivetta et al., 2000). The F12 phenotype was recapitulated by a Nef with an ER retention signal, meaning that by interacting with GagPol Nef retained this polyprotein precursor inside the producer cell avoiding both release and maturation of viral particles (Costa et al., 2004). Furthermore, the relevance of this interaction was confirmed by an experiment demonstrating that release of a late-domain defective HIV-1 from producer cells was

the former to increase viral infectivity.

greatly affected.

rescued by a Nef-Tsg101 fusion protein, indicating that, besides interacting early during the expression of GagPol, Nef and the viral structural/enzymatic precursors are present concomitantly at the sites of virus budding. While investigating what influence the binding of Nef and GagPol would have for HIV-1 infectivity we have also established that Nef from HIV binds the cellular Alix protein and correlated these findings with optimal viral replication in cells (Costa et al., 2006). We demonstrated that this interaction correlated with the property of Nef to stimulate the synthesis of MVBs in cells and the optimal infection of MDMs. Taken together, the fact that Nef binds both the viral precursor GagPol and to the cellular Alix protein, which is involved in virus budding, suggest that Nef actively participates in a late step of viral replication. We further extended these observations to SIVmac, demonstrating that the binding of Nef to both GagPol and Alix is conserved in Nef from this virus (Jesus da Costa et al., 2009). The functional relevance of these bindings was clearly demonstrated in rhesus macaques. First, informative mutations were introduced into the *nef* gene of infectious SIVmac and this mutant virus used to infect four rhesus macaques. Whereas rhesus macaques infected with the WT virus developed high viral loads and progressed rapidly to SAIDS, only two of four monkeys inoculated with the mutant virus displayed a similar picture, albeit with much delayed kinetics. Importantly, in both cases, we observed reversions in SIVNef that restored its binding to SIVGagPol and Alix. In two other rhesus macaques, mutant Nef sequences persisted and they developed neither high viral loads nor SAIDS. Further studies in cells provided additional support for these findings. Taken together these studies suggest that interactions between Nef, GagPol and Alix are important for optimal viral replication and progression to disease in the monkey model of AIDS and possibly also pertinent to the human disease since these interactions are preserved in both HIV-1 and SIVmac.

Still, what would be the mechanism by which interacting with GagPol and Alix, Nef would stimulate viral infectivity? Recent work from our group demonstrated that Nef that from SIVcpz also binds to GagPol from both SIVcpz and HIV-1. Furthermore, our results showed that Nef exerts a direct influence upon the viral PR activity: i) first by characterizing a SIVcpz provirus that expresses a N-terminal truncated peptide of Nef that is a potent inhibitor of viral processing and consequently viral infectivity and exerts a dominant negative activity both against PR from SIVcpz and HIV-1 (Sampaio et al. – manuscript in preparation); second by demonstrating that in the absence of Nef expression during the replication cycle of HIV-1 the processing kinetics of the Gag and GagPol precursors are accelerated, resulting in slightly lower concentration of the structural (p24-Capsid protein) and 2-fold lower concentration of the enzymatic (IN) components within viral particles (Mendonça et al. – manuscript in preparation). Moreover, the accelerated activity of the viral PR in the absence of Nef expression in the producer cells was also demonstrated by the fact that higher concentrations of PR inhibitors (e.g. Lopinavir) are required to inhibit virus replication at the same levels as of the wild type viruses. Based on this preliminary results a working model was drawn (Figure 2), in which the role of Nef for virus infectivity is explained by the following: upon binding of the p6\* and PR regions, within the GagPol precursor, Nef would function as a fine regulator of PR activation, holding it to an optimal timing when budding of the viral particles is almost complete.

Therefore, the right content of the viral constituents is achieved. In the absence of Nef, PR activation would occur earlier on before the completion of budding, consequently some of

Functions of the Lentiviral Accessory Protein

infectivity of the viral particles in the presence of Nef.

resistance mutations that is still not appreciated.

Nef During the Distinct Steps of HIV and SIV Replication Cycle 25

prompt to the conclusion that Nef participates in an early step of the replication cycle postentry and before reverse transcription to increase infectivity. However it is becoming more evident that the requirement for Nef occurs in the producer cell, possibly by modifying or regulating the last steps of the viral replication cycle (assembly, budding and/or maturation) Nef would promote the formation of optimally infectious virus progeny. In our model, the specific interaction of Nef with the precursor polyprotein GagPol and its influence on the kinetics of processing of the viral precursor proteins, predicts that in the absence of Nef viral PR will be activated earlier on during budding resulting in the incorporation of lower amounts of the viral enzymes into viral cores, especially IN. Since IN is crucial to the initiation of reverse transcription and that Nef-deleted viruses have a defect in viral DNA synthesis, our model conciliates these findings to explain the optimal

If that is the case, very important implications for this function of Nef in the current antiretroviral therapy can be predicted. First, Nef could become a target to anti-retroviral drugs that would act in synergism with PR inhibitors currently in use to achieve a more efficient inhibition of virus replication. Second, we should predict a co-evolution of both Nef and PR during the therapy with PR inhibitors, which will have a direct impact on the selection of

Fig. 2. Schematic representation of the working model for the functions of Nef during the last steps of the primate Lentiviral replication cycle. Nef associates with cellular membranes and classically exerts several functions that do not necessarily correlate with its role in increasing viral infectivity and promote disease progression (arrow 1). Nef from both HIV-1 and SIVmac binds to the p6\*PR within the GagPol precursor (Costa et al., 2004; Jesus da Costa et al., 2009) (arrow 2). In the presence of Nef the viral PR is activated at an optimal time during the budding step, assuring the correct amount of the viral IN and RT inside the viral core (arrow 3). IN influences the activity of RT during the initiation of reverse transcription, therefore the normal amounst of IN in viral particles will promote optimal cDNA synthesis and viral infectivity of the incoming viruses (arrow 4). On the other hand, in the absence of Nef viral PR is prematurely activated, consequently the processing of the GagPol precursor would occur before the formation of the viral cores leading to a lesser incorporation of IN (arrow 5), what

would impact reverse transcription and viral infectivity (arrow 6).

the protein content is lost during the formation of the viral particles, especially the IN, since it is one of the first enzymes to be processed out from the GagPol precursor. How would this explain the decreased infectivity of the Nef-deficient viruses? Since IN is important not only for the integration step of the viral DNA to the host genome but also participates during the Reverse Transcription of the viral RNA, lower concentrations of the IN would affect reverse transcription of the incoming virus impacting on viral infectivity. Our results conciliates previous data demonstrating an impairment in the HIV-1 ability to reverse transcribe the viral RNA genome in Nef-deficient viruses, which could not be explained by a role of Nef in facilitating an early step of the replication cycle as enhancement of viral capsid delivery to the cytosol of the target cell (Cavrois et al., 2004; Miller et al., 1995; Tobiume et al., 2001), trafficking of the viral cores through the cortical actin network since (Campbell EM, 2004; Pizzato et al., 2008), or capsid uncoating (Cavrois et al., 2004; Kotov et al., 1999).


Table 1. Mutations within the cleavage site of Nef and its effect on viral infectivity\* Less cleavage of the Nef protein was observed (Miller et al., 1997). \*\* A higher cleavage rate of the Nef protein was observed (Miller et al., 1997). \*\*\* A cleavage site (ATIM) from the viral Nucleocapsid protein was introduced at amino acid position 55 in Nef, accelerated cleavage of Nef was observed (Pandori et al., 1998).

## **5. Conclusion**

Nef is a multifunctional accessory protein only present in the primate lentiviruses. Its influence on viral infectivity and pathogenesis is undisputable. However, the mechanism by which Nef contributes to the optimal infectivity of these viruses is still a matter of controversy. The classical functions described for Nef are the downmodulation of the CD4 and MHC-I molecules from the cell surface and the activation of signaling cascades in Nefexpressing or HIV/SIV-infected cells. While the downmodulation of immune system molecules from the surface of cells, as described here, could all contribute to the effect of Nef on pathogenesis and disease progression, these functions failed to be correlated with the contribution of Nef to the optimal infectivity of HIV/SIV. Some experimental evidences

the protein content is lost during the formation of the viral particles, especially the IN, since it is one of the first enzymes to be processed out from the GagPol precursor. How would this explain the decreased infectivity of the Nef-deficient viruses? Since IN is important not only for the integration step of the viral DNA to the host genome but also participates during the Reverse Transcription of the viral RNA, lower concentrations of the IN would affect reverse transcription of the incoming virus impacting on viral infectivity. Our results conciliates previous data demonstrating an impairment in the HIV-1 ability to reverse transcribe the viral RNA genome in Nef-deficient viruses, which could not be explained by a role of Nef in facilitating an early step of the replication cycle as enhancement of viral capsid delivery to the cytosol of the target cell (Cavrois et al., 2004; Miller et al., 1995; Tobiume et al., 2001), trafficking of the viral cores through the cortical actin network since (Campbell EM, 2004; Pizzato et al., 2008), or capsid uncoating (Cavrois et al., 2004; Kotov et al., 1999).

L58A Yes 50 (Chen et al., 1998) WL57GG No 75 (Miller et al., 1997)

CAW (55-57) LLL Yes \*\* 70 (Miller et al., 1997) CAW (55-57) No 75 (Miller et al., 1997)

DCAWL (54-57) No 20 (Pandori et al., 1998) LEAQ (58-61) Yes 50 (Pandori et al., 1998) AWLEA (56-60) No 80 (Pandori et al., 1998)

ATIM\*\*\* Yes\*\* 30 (Pandori et al., 1998)

Nef is a multifunctional accessory protein only present in the primate lentiviruses. Its influence on viral infectivity and pathogenesis is undisputable. However, the mechanism by which Nef contributes to the optimal infectivity of these viruses is still a matter of controversy. The classical functions described for Nef are the downmodulation of the CD4 and MHC-I molecules from the cell surface and the activation of signaling cascades in Nefexpressing or HIV/SIV-infected cells. While the downmodulation of immune system molecules from the surface of cells, as described here, could all contribute to the effect of Nef on pathogenesis and disease progression, these functions failed to be correlated with the contribution of Nef to the optimal infectivity of HIV/SIV. Some experimental evidences

Table 1. Mutations within the cleavage site of Nef and its effect on viral infectivity\* Less cleavage of the Nef protein was observed (Miller et al., 1997). \*\* A higher cleavage rate of the Nef protein was observed (Miller et al., 1997). \*\*\* A cleavage site (ATIM) from the viral Nucleocapsid protein was introduced at amino acid position 55 in Nef, accelerated cleavage

reduction (%) Literature

(Miller et al., 1997) (Chen et al., 1998)

(Chen et al., 1998) (Miller et al., 1997)

(Fackler et al., 2006)

50

50 75

Mutation Nef cleavage Infectivity

Yes \*

W57A Yes \* <sup>70</sup>

WL57AA No

of Nef was observed (Pandori et al., 1998).

DCAWL (54-61)-

**5. Conclusion** 

prompt to the conclusion that Nef participates in an early step of the replication cycle postentry and before reverse transcription to increase infectivity. However it is becoming more evident that the requirement for Nef occurs in the producer cell, possibly by modifying or regulating the last steps of the viral replication cycle (assembly, budding and/or maturation) Nef would promote the formation of optimally infectious virus progeny.

In our model, the specific interaction of Nef with the precursor polyprotein GagPol and its influence on the kinetics of processing of the viral precursor proteins, predicts that in the absence of Nef viral PR will be activated earlier on during budding resulting in the incorporation of lower amounts of the viral enzymes into viral cores, especially IN. Since IN is crucial to the initiation of reverse transcription and that Nef-deleted viruses have a defect in viral DNA synthesis, our model conciliates these findings to explain the optimal infectivity of the viral particles in the presence of Nef.

If that is the case, very important implications for this function of Nef in the current antiretroviral therapy can be predicted. First, Nef could become a target to anti-retroviral drugs that would act in synergism with PR inhibitors currently in use to achieve a more efficient inhibition of virus replication. Second, we should predict a co-evolution of both Nef and PR during the therapy with PR inhibitors, which will have a direct impact on the selection of resistance mutations that is still not appreciated.

Fig. 2. Schematic representation of the working model for the functions of Nef during the last steps of the primate Lentiviral replication cycle. Nef associates with cellular membranes and classically exerts several functions that do not necessarily correlate with its role in increasing viral infectivity and promote disease progression (arrow 1). Nef from both HIV-1 and SIVmac binds to the p6\*PR within the GagPol precursor (Costa et al., 2004; Jesus da Costa et al., 2009) (arrow 2). In the presence of Nef the viral PR is activated at an optimal time during the budding step, assuring the correct amount of the viral IN and RT inside the viral core (arrow 3). IN influences the activity of RT during the initiation of reverse transcription, therefore the normal amounst of IN in viral particles will promote optimal cDNA synthesis and viral infectivity of the incoming viruses (arrow 4). On the other hand, in the absence of Nef viral PR is prematurely activated, consequently the processing of the GagPol precursor would occur before the formation of the viral cores leading to a lesser incorporation of IN (arrow 5), what would impact reverse transcription and viral infectivity (arrow 6).

Functions of the Lentiviral Accessory Protein

J Immunol 177, 3260-3265.

Biochemistry 45, 2339-2349.

assembly or stability. Retrovirology 5, 57.

viral protease. J Virol 70, 6820-6825.

infectivity. J Virol 78, 5745-5755.

adaptor. J Virol 81, 3877-3890.

and CD86 in APCs. J Immunol 175, 4566-4574.

constitutive pathway. J Immunol 183, 2415-2424.

Virol 70, 820-829.

78, 5745-5755.

36-44.

endocytic pathway. Cell 111, 853-866.

Nef During the Distinct Steps of HIV and SIV Replication Cycle 27

Blagoveshchenskaya, A.D., Thomas, L., Feliciangeli, S.F., Hung, C.H., Thomas, G., 2002.

Blasius, A.L., Giurisato, E., Cella, M., Schreiber, R.D., Shaw, A.S., Colonna, M., 2006. Bone

Bour, S., Schubert, U., Peden, K., Strebel, K., 1996. The envelope glycoprotein of human

Bresnahan, P.A., Yonemoto, W., Ferrell, S., Williams-Herman, D., Geleziunas, R., Greene,

Brun, S., Solignat, M., Gay, B., Bernard, E., Chaloin, L., Fenard, D., Devaux, C., Chazal, N.,

Bukovsky, A., Gˆttlinger, H., 1996. Lack of integrase can markedly affect human

Campbell EM, N.R., Hope TJ, 2004. Disruption of the actin cytoskeleton can complement the

Campbell, E.M., Nunez, R., Hope, T.J., 2004. Disruption of the actin cytoskeleton can

Carl, S., Greenough, T.C., Krumbiegel, M., Greenberg, M., Skowronski, J., Sullivan, J.L.,

Cavrois, M., Neidleman, J., Yonemoto, W., Fenard, D., Greene, W.C., 2004. HIV-1 virion

Chaudhry, A., Das, S.R., Hussain, A., Mayor, S., George, A., Bal, V., Jameel, S., Rath, S., 2005.

Chaudhry, A., Verghese, D.A., Das, S.R., Jameel, S., George, A., Bal, V., Mayor, S., Rath, S.,

Chaudhuri, R., Lindwasser, O.W., Smith, W.J., Hurley, J.H., Bonifacino, J.S., 2007.

Nef functions during progression to AIDS. J Virol 75, 3657-3665.

HIV-1 Nef downregulates MHC-I by a PACS-1- and PI3K-regulated ARF6

marrow stromal cell antigen 2 is a specific marker of type I IFN-producing cells in the naive mouse, but a promiscuous cell surface antigen following IFN stimulation.

immunodeficiency virus type 2 enhances viral particle release: a Vpu-like factor? J

W.C., 1998. A dileucine motif in HIV-1 Nef acts as an internalization signal for CD4 downregulation and binds the AP-1 clathrin adaptor. Curr Biol 8, 1235-1238. Breuer, S., Gerlach, H., Kolaric, B., Urbanke, C., Opitz, N., Geyer, M., 2006. Biochemical

indication for myristoylation-dependent conformational changes in HIV-1 Nef.

Briant, L., 2008. VSV-G pseudotyping rescues HIV-1 CA mutations that impair core

immunodeficiency virus type 1 particle production in the presence of an active

ability of Nef to enhance human immunodeficiency virus type 1 infectivity. J Virol

complement the ability of Nef to enhance human immunodeficiency virus type 1

Kirchhoff, F., 2001. Modulation of different human immunodeficiency virus type 1

fusion assay: uncoating not required and no effect of Nef on fusion. Virology 328,

The Nef protein of HIV-1 induces loss of cell surface costimulatory molecules CD80

2009. HIV-1 Nef promotes endocytosis of cell surface MHC class II molecules via a

Downregulation of CD4 by human immunodeficiency virus type 1 Nef is dependent on clathrin and involves direct interaction of Nef with the AP2 clathrin

#### **6. References**


Adachi, A., Ono, N., Sakai, H., Ogawa, K., Shibata, R., Kiyomasu, T., Masuike, H., Ueda, S.,

Aguiar, R.S., Peterlin, B.M., 2008. APOBEC3 proteins and reverse transcription. Virus

Ahmad, N., Venkatesan, S., 1988. Nef protein of HIV-1 is a transcriptional repressor of HIV-

Aiken, C., 1997. Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the

Aiken, C., Konner, J., Landau, N.R., Lenburg, M.E., Trono, D., 1994. Nef induces CD4

Aiken, C., Trono, D., 1995. Nef stimulates human immunodeficiency virus type 1 proviral

Alcover, A., Alarcon, B., 2000. Internalization and intracellular fate of TCR-CD3 complexes.

Alessandrini, L., Santarcangelo, A.C., Olivetta, E., Ferrantelli, F., d'Aloja, P., Pugliese, K.,

mechanism of HIV T-tropic emergence in AIDS. J Gen Virol 81, 2905-2917. Alexander, L., Du, Z., Howe, A.Y., Czajak, S., Desrosiers, R.C., 1999. Induction of AIDS in

Arold, S., O'Brien, R., Franken, P., Strub, M.P., Hoh, F., Dumas, C., Ladbury, J.E., 1998. RT

Atkins, K.M., Thomas, L., Youker, R.T., Harriff, M.J., Pissani, F., You, H., Thomas, G., 2008.

Bell, I., Ashman, C., Maughan, J., Hooker, E., Cook, F., Reinhart, T.A., 1998. Association of

Bentham, M., Mazaleyrat, S., Harris, M., 2003. The di-leucine motif in the cytoplasmic tail of

interfering RNA and knock-out mice. J Biol Chem 283, 11772-11784. Baur, A.S., Sass, G., Laffert, B., Willbold, D., Cheng-Mayer, C., Peterlin, B.M., 1997. The N-

to TCR down-modulation. J Gen Virol 79 ( Pt 11), 2717-2727.

is critical for CD4 down-modulation. J Gen Virol 84, 2705-2713.

of human immunodeficiency virus type 1. J Virol 73, 5814-5825.

Arhel, N., 2010. Revisiting HIV-1 uncoating. Retrovirology 7, 96.

and a serine kinase. Immunity 6, 283-291.

1991. Generation and characterization of the human immunodeficiency virus type 1

glycoprotein of vesicular stomatitis virus targets HIV-1 entry to an endocytic pathway and suppresses both the requirement for Nef and the sensitivity to

endocytosis: requirement for a critical dileucine motif in the membrane-proximal

Pelosi, E., Chelucci, C., Mattia, G., Peschle, C., Verani, P., Federico, M., 2000. Ttropic human immunodeficiency virus (HIV) type 1 Nef protein enters human monocyte-macrophages and induces resistance to HIV replication: a possible

rhesus monkeys by a recombinant simian immunodeficiency virus expressing nef

loop flexibility enhances the specificity of Src family SH3 domains for HIV-1 Nef.

HIV-1 Nef binds PACS-2 to assemble a multikinase cascade that triggers major histocompatibility complex class I (MHC-I) down-regulation: analysis using short

terminus of Nef from HIV-1/SIV associates with a protein complex containing Lck

simian immunodeficiency virus Nef with the T-cell receptor (TCR) zeta chain leads

CD4 is not required for binding to human immunodeficiency virus type 1 Nef, but

**6. References** 

mutants. Arch Virol 117, 45-58.

1 LTR. Science 241, 1481-1485.

cyclosporin A. J Virol 71, 5871-5877.

DNA synthesis. J Virol 69, 5048-5056.

Crit Rev Immunol 20, 325-346.

Biochemistry 37, 14683-14691.

CD4 cytoplasmic domain. Cell 76, 853-864.

research 134, 74-85.


Functions of the Lentiviral Accessory Protein

solution. Proteins 60, 658-669.

transcription. J Virol 81, 10037-10046.

localization. Eur J Biochem 247, 843-851.

infection. Virology 351, 322-339.

replication. J Virol 76, 5667-5677.

cytoskeleton. Curr Opin Microbiol 9, 409-415.

7947.

Virol 75, 6601-6608.

Cell 3, 729-739.

Virol 77, 4409-4414.

Nef During the Distinct Steps of HIV and SIV Replication Cycle 29

Dennis, C.A., Baron, A., Grossmann, J.G., Mazaleyrat, S., Harris, M., Jaeger, J., 2005. Co-

Dikeakos, J.D., Atkins, K.M., Thomas, L., Emert-Sedlak, L., Byeon, I.J., Jung, J., Ahn, J.,

translational myristoylation alters the quaternary structure of HIV-1 Nef in

Wortman, M.D., Kukull, B., Saito, M., Koizumi, H., Williamson, D.M., Hiyoshi, M., Barklis, E., Takiguchi, M., Suzu, S., Gronenborn, A.M., Smithgall, T.E., Thomas, G., 2010. Small molecule inhibition of HIV-1-induced MHC-I down-regulation identifies a temporally regulated switch in Nef action. Mol Biol Cell 21, 3279-3292. Djordjevic, J.T., Schibeci, S.D., Stewart, G.J., Williamson, P., 2004. HIV type 1 Nef increases

the association of T cell receptor (TCR)-signaling molecules with T cell rafts and promotes activation-induced raft fusion. AIDS Res Hum Retroviruses 20, 547-555. Dobard, C.W., Briones, M.S., Chow, S.A., 2007. Molecular mechanisms by which human

immunodeficiency virus type 1 integrase stimulates the early steps of reverse

Vpu directs the degradation of the human immunodeficiency virus restriction factor BST-2/Tetherin via a {beta}TrCP-dependent mechanism. J Virol 83, 7931-

immunodeficiency virus type 1(F12) inhibits viral production and infectivity. J

Mueller-Lantzsch, N., 1997. Association of human immunodeficiency virus Nef protein with actin is myristoylation dependent and influences its subcellular

Nef induces cytoskeletal rearrangements and downstream effector functions. Mol

2006. Functional characterization of HIV-1 Nef mutants in the context of viral

Nef is physically recruited into the immunological synapse and potentiates T cell

in vitro reveals association of Nef with the subviral ribonucleoprotein complex. J

immunodeficiency virus type 1 core of optimal stability is crucial for viral

responsiveness to stimulation of human immunodeficiency virus-infected CD4+ T cells requires Nef and Tat virus gene products and results from higher NFAT, NF-

Douglas, J.L., Viswanathan, K., McCarroll, M.N., Gustin, J.K., Fruh, K., Moses, A.V., 2009.

Fackler, O.T., d'Aloja, P., Baur, A.S., Federico, M., Peterlin, B.M., 2001. Nef from human

Fackler, O.T., Kienzle, N., Kremmer, E., Boese, A., Schramm, B., Klimkait, T., K¸cherer, C.,

Fackler, O.T., Krausslich, H.G., 2006. Interactions of human retroviruses with the host cell

Fackler, O.T., Luo, W., Geyer, M., Alberts, A.S., Peterlin, B.M., 1999. Activation of Vav by

Fackler, O.T., Moris, A., Tibroni, N., Giese, S.I., Glass, B., Schwartz, O., Krausslich, H.G.,

Fenard, D., Yonemoto, W., de Noronha, C., Cavrois, M., Williams, S.A., Greene, W.C., 2005.

Forshey, B.M., Aiken, C., 2003. Disassembly of human immunodeficiency virus type 1 cores

Forshey, B.M., von Schwedler, U., Sundquist, W.I., Aiken, C., 2002. Formation of a human

Fortin, J.F., Barat, C., Beausejour, Y., Barbeau, B., Tremblay, M.J., 2004. Hyper-

activation early after TCR engagement. J Immunol 175, 6050-6057.

kappaB, and AP-1 induction. J Biol Chem 279, 39520-39531.


Chazal, N., Singer, G., Aiken, C., Hammarskjold, M.L., Rekosh, D., 2001. Human

Cheng, H., Hoxie, J.P., Parks, W.P., 1999. The conserved core of human immunodeficiency

Cheng-Mayer, C., Iannello, P., Shaw, K., Luciw, P.A., Levy, J.A., 1989. Differential effects of

Chowers, M.Y., Pandori, M.W., Spina, C.A., Richman, D.D., Guatelli, J.C., 1995. The growth

Cortes, M.J., Wong-Staal, F., Lama, J., 2002. Cell surface CD4 interferes with the infectivity of

Costa, L.J., Chen, N., Lopes, A., Aguiar, R.S., Tanuri, A., Plemenitas, A., Peterlin, B.M., 2006.

Costa, L.J., Zheng, Y.H., Sabotic, J., Mak, J., Fackler, O.T., Peterlin, B.M., 2004. Nef binds p6\*

Crotti, A., Neri, F., Corti, D., Ghezzi, S., Heltai, S., Baur, A., Poli, G., Santagostino, E.,

Daniel, M.D., Kirchhoff, F., Czajak, S.C., Sehgal, P.K., Desrosiers, R.C., 1992. Protective

Daniel, M.D., Letvin, N.L., King, N.W., Kannagi, M., Sehgal, P.K., Hunt, R.D., Kanki, P.J.,

Deacon, N.J., Tsykin, A., Solomon, A., Smith, K., Ludford-Menting, M., Hooker, D.J.,

HIV-1 from a blood transfusion donor and recipients. Science 270, 988-991. DeGottardi, M.Q., Specht, A., Metcalf, B., Kaur, A., Kirchhoff, F., Evans, D.T., 2008. Selective

human immunodeficiency virus type 2. J Virol 82, 3139-3146.

virion infectivity. J Virol 72, 3178-3184.

egress of HIV-1. Retrovirology 3, 33.

from macaques. Science 228, 1201-1204.

1629-1632.

5311-5323.

10663-10674.

1938-1941.

replication in T-lymphocytes. Virology 264, 5-15.

matrix domain. Proc Natl Acad Sci U S A 107, 1600-1605.

HIV-1 particles released from T cells. J Biol Chem 277, 1770-1779.

immunodeficiency virus type 1 particles pseudotyped with envelope proteins that fuse at low pH no longer require Nef for optimal infectivity. J Virol 75, 4014-4018. Chen, Y.L., Trono, D., Camaur, D., 1998. The proteolytic cleavage of human

immunodeficiency virus type 1 Nef does not correlate with its ability to stimulate

virus type 1 Nef is essential for association with Lck and for enhanced viral

nef on HIV replication: implications for viral pathogenesis in the host. Science 246,

advantage conferred by HIV-1 nef is determined at the level of viral DNA formation and is independent of CD4 downregulation. Virology 212, 451-457. Chukkapalli, V., Oh, S.J., Ono, A., 2010. Opposing mechanisms involving RNA and lipids

regulate HIV-1 Gag membrane binding through the highly basic region of the

Interactions between Nef and AIP1 proliferate multivesicular bodies and facilitate

in GagPol during replication of human immunodeficiency virus type 1. J Virol 78,

Vicenzi, E., 2006. Nef alleles from human immunodeficiency virus type 1-infected long-term-nonprogressor hemophiliacs with or without late disease progression are defective in enhancing virus replication and CD4 down-regulation. J Virol 80,

effects of a live attenuated SIV vaccine with a deletion in the nef gene. Science 258,

Essex, M., Desrosiers, R.C., 1985. Isolation of T-cell tropic HTLV-III-like retrovirus

McPhee, D.A., Greenway, A.L., Ellett, A., Chatfield, C., Lawson, V.A., Crowe, S., Maerz, A., Sonza, S., Learmont, J., Sullivan, J.S., Cunningham, A., Dwyer, D., Dowton, D., Mills, J., 1995. Genomic structure of an attenuated quasi species of

downregulation of rhesus macaque and sooty mangabey major histocompatibility complex class I molecules by Nef alleles of simian immunodeficiency virus and


Functions of the Lentiviral Accessory Protein

virus. J Virol 74, 10882-10891.

complex. Immunol Rev 232, 7-21.

transgenic mice. Cell 95, 163-175.

Amalgamated sequences. Nature 340, 682.

76, 2692-2702.

281, 19618-19630.

483-519.

15733.

Virol 72, 9827-9834.

Cell Host Microbe 1, 121-133.

replication in vivo. J Virol 74, 9836-9844.

cell growth. Genomics 26, 527-534.

Nef During the Distinct Steps of HIV and SIV Replication Cycle 31

Gummuluru, S., Kinsey, C.M., Emerman, M., 2000. An in vitro rapid-turnover assay for

Gupta, R.K., Mlcochova, P., Pelchen-Matthews, A., Petit, S.J., Mattiuzzo, G., Pillay, D.,

Guy, C.S., Vignali, D.A., 2009. Organization of proximal signal initiation at the TCR:CD3

Haller, C., Rauch, S., Michel, N., Hannemann, S., Lehmann, M.J., Keppler, O.T., Fackler,

Hammes, S.R., Dixon, E.P., Malim, M.H., Cullen, B.R., Greene, W.C., 1989. Nef protein of

Hanna, Z., Kay, D.G., Rebai, N., Guimond, A., Jothy, S., Jolicoeur, P., 1998. Nef harbors a

Hellen, C.U., de Crombrugghe, M., Wimmer, E., Seeger, M.A., Kaufman, T.C., 1989.

Hinshaw, J.E., 2000. Dynamin and its role in membrane fission. Annu Rev Cell Dev Biol 16,

Hodge, D.R., Dunn, K.J., Pei, G.K., Chakrabarty, M.K., Heidecker, G., Lautenberger, J.A.,

Howe, A.Y., Jung, J.U., Desrosiers, R.C., 1998. Zeta chain of the T-cell receptor interacts with

Hung, C.H., Thomas, L., Ruby, C.E., Atkins, K.M., Morris, N.P., Knight, Z.A., Scholz, I.,

Iafrate, A.J., Carl, S., Bronson, S., Stahl-Hennig, C., Swigut, T., Skowronski, J., Kirchhoff, F.,

Ishikawa, J., Kaisho, T., Tomizawa, H., Lee, B.O., Kobune, Y., Inazawa, J., Oritani, K., Itoh,

sequestration. Proc Natl Acad Sci U S A 106, 20889-20894.

inhibitor. Proc Natl Acad Sci U S A 86, 9549-9553.

to tumor suppressor p53 and protects cells against p53-mediated apoptosis. J Virol

human immunodeficiency virus type 1 replication selects for cell-to-cell spread of

Takeuchi, Y., Marsh, M., Towers, G.J., 2009. Simian immunodeficiency virus envelope glycoprotein counteracts tetherin/BST-2/CD317 by intracellular

O.T., 2006. The HIV-1 pathogenicity factor Nef interferes with maturation of stimulatory T-lymphocyte contacts by modulation of N-Wasp activity. J Biol Chem

human immunodeficiency virus type 1: evidence against its role as a transcriptional

major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in

Samuel, K.P., 1998. Binding of c-Raf1 kinase to a conserved acidic sequence within the carboxyl-terminal region of the HIV-1 Nef protein. J Biol Chem 273, 15727-

nef of simian immunodeficiency virus and human immunodeficiency virus type 2. J

Barklis, E., Weinberg, A.D., Shokat, K.M., Thomas, G., 2007. HIV-1 Nef assembles a Src family kinase-ZAP-70/Syk-PI3K cascade to downregulate cell-surface MHC-I.

2000. Disrupting surfaces of nef required for downregulation of CD4 and for enhancement of virion infectivity attenuates simian immunodeficiency virus

M., Ochi, T., Ishihara, K., et al., 1995. Molecular cloning and chromosomal mapping of a bone marrow stromal cell surface gene, BST2, that may be involved in pre-B-

Foster, J.L., Garcia, J.V., 2008. HIV-1 Nef: at the crossroads. Retrovirology 5, 84.


Foti, M., Mangasarian, A., Piguet, V., Lew, D.P., Krause, K.H., Trono, D., Carpentier, J.L., 1997. Nef-mediated clathrin-coated pit formation. J Cell Biol 139, 37-47. Frankel, A.D., Young, J.A., 1998. HIV-1: fifteen proteins and an RNA. Annu Rev Biochem 67,

Freed, E.O., Mouland, A.J., 2006. The cell biology of HIV-1 and other retroviruses.

Freund, J., Kellner, R., Houthaeve, T., Kalbitzer, H.R., 1994. Stability and proteolytic

Fujii, K., Hurley, J.H., Freed, E.O., 2007. Beyond Tsg101: the role of Alix in 'ESCRTing' HIV-

Fujii, Y., Otake, K., Tashiro, M., Adachi, A., 1996. Soluble Nef antigen of HIV-1 is cytotoxic

Garcia, J.V., Miller, A.D., 1991. Serine phosphorylation-independent downregulation of cell-

Garrus, J.E., von Schwedler, U.K., Pornillos, O.W., Morham, S.G., Zavitz, K.H., Wang, H.E.,

Gatlin, J., Arrigo, S.J., Schmidt, M.G., 1998. HIV-1 protease regulation: the role of the major

Gerdes, H.H., Bukoreshtliev, N.V., Barroso, J.F., 2007. Tunneling nanotubes: a new route for the exchange of components between animal cells. FEBS Lett 581, 2194-2201. Geyer, M., Fackler, O.T., Peterlin, B.M., 2001. Structure--function relationships in HIV-1 Nef.

Giorgi, J.V., Hausner, M.A., Hultin, L.E., 1999. Detailed immunophenotype of CD8+

Gould, S.J., Booth, A.M., Hildreth, J.E., 2003. The Trojan exosome hypothesis. Proc Natl

Greenberg, M., DeTulleo, L., Rapoport, I., Skowronski, J., Kirchhausen, T., 1998a. A

Greenberg, M.E., Iafrate, A.J., Skowronski, J., 1998b. The SH3 domain-binding surface and

Greenway, A.L., McPhee, D.A., Allen, K., Johnstone, R., Holloway, G., Mills, J., Azad, A.,

CD45RA/RO, CD62L and CD28 antigens. Immunol Lett 66, 105-110. Goldsmith, M.A., Warmerdam, M.T., Atchison, R.E., Miller, M.D., Greene, W.C., 1995.

Wettstein, D.A., Stray, K.M., CÙtÈ, M., Rich, R.L., Myszka, D.G., Sundquist, W.I., 2001. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1

homology region and adjacent C-terminal capsid sequences. J Biomed Sci 5, 305-

memory cytotoxic T-lymphocytes (CTL) against HIV-1 with respect to expression of

Dissociation of the CD4 downregulation and viral infectivity enhancement functions of human immunodeficiency virus type 1 Nef. J Virol 69, 4112-4121. Goto, T., Kennel, S.J., Abe, M., Takishita, M., Kosaka, M., Solomon, A., Saito, S., 1994. A

novel membrane antigen selectively expressed on terminally differentiated human

dileucine motif in HIV-1 Nef is essential for sorting into clathrin-coated pits and for

an acidic motif in HIV-1 Nef regulate trafficking of class I MHC complexes. Embo J

Sankovich, S., Lambert, P., 2002. Human immunodeficiency virus type 1 Nef binds

domains of Nef protein from human immunodeficiency virus (HIV) type 1. Eur J

Foster, J.L., Garcia, J.V., 2008. HIV-1 Nef: at the crossroads. Retrovirology 5, 84.

1-25.

308.

Retrovirology 3, 77.

Biochem 221, 811-819.

budding. Cell 107, 55-65.

EMBO Rep 2, 580-585.

B cells. Blood 84, 1922-1930.

17, 2777-2789.

Acad Sci U S A 100, 10592-10597.

downregulation of CD4. Curr Biol 8, 1239-1242.

1. Nat Rev Microbiol 5, 912-916.

for human CD4+ T cells. FEBS Lett 393, 93-96.

surface CD4 by nef. Nature 350, 508-511.

to tumor suppressor p53 and protects cells against p53-mediated apoptosis. J Virol 76, 2692-2702.


Functions of the Lentiviral Accessory Protein

Curr Biol 9, 622-631.

Virol 83, 11966-11978.

Virol 84, 7243-7255.

HIV-1. Curr Biol 6, 1677-1684.

Immunol 165, 6437-6446.

transfer systems. Virology 241, 224-233.

hostile environment. Cell Host Microbe 3, 388-398.

584.

pathogenesis? Curr HIV Res 8, 638-640.

range. Proc Natl Acad Sci U S A 89, 363-367.

long terminal repeat transgene. J Exp Med 179, 797-807.

Nef During the Distinct Steps of HIV and SIV Replication Cycle 33

Laguette, N., Benichou, S., Basmaciogullari, S., 2009. Human immunodeficiency virus type 1 Nef incorporation into virions does not increase infectivity. J Virol 83, 1093-1104. Lama, J., Mangasarian, A., Trono, D., 1999. Cell-surface expression of CD4 reduces HIV-1

Lamers, S.L., Fogel, G.B., Huysentruyt, L.C., McGrath, M.S., 2010. HIV-1 nef protein visits B-

Lang, S.M., Iafrate, A.J., Stahl-Hennig, C., Kuhn, E.M., Nisslein, T., Kaup, F.J., Haupt, M.,

Le Tortorec, A., Neil, S.J., 2009. Antagonism to and intracellular sequestration of human

Lee, C.M., Gala, S., Stewart, G.J., Williamson, P., 2008. The proline-rich region of HIV-1 Nef affects CXCR4-mediated chemotaxis in Jurkat T cells. Viral Immunol 21, 347-354. Lindemann, D., Wilhelm, R., Renard, P., Althage, A., Zinkernagel, R., Mous, J., 1994. Severe

Littman, D.R., 1987. The structure of the CD4 and CD8 genes. Annu Rev Immunol 5, 561-

Lopez, L.A., Yang, S.J., Hauser, H., Exline, C.M., Haworth, K.G., Oldenburg, J., Cannon,

Louis, J.M., Weber, I.T., TˆzsÈr, J., Clore, G.M., Gronenborn, A.M., 2000. HIV-1 protease: maturation, enzyme specificity, and drug resistance. Adv Pharmacol 49, 111-146. Lu, X., Wu, X., Plemenitas, A., Yu, H., Sawai, E.T., Abo, A., Peterlin, B.M., 1996. CDC42 and

Lu, X., Yu, H., Liu, S.H., Brodsky, F.M., Peterlin, B.M., 1998. Interactions between HIV1 Nef and vacuolar ATPase facilitate the internalization of CD4. Immunity 8, 647-656. Luo, T., Douglas, J.L., Livingston, R.L., Garcia, J.V., 1998. Infectivity enhancement by HIV-1

Mahlknecht, U., Deng, C., Lu, M.C., Greenough, T.C., Sullivan, J.L., O'Brien, W.A., Herbein,

Malim, M.H., Emerman, M., 2008. HIV-1 accessory proteins--ensuring viral survival in a

for the development of AIDS in rhesus macaques. Nat Med 3, 860-865. Lanier, L.L., 2005. Missing self, NK cells, and The White Album. J Immunol 174, 6565. Le Guern, M., Levy, J.A., 1992. Human immunodeficiency virus (HIV) type 1 can superinfect

infectivity by blocking Env incorporation in a Nef- and Vpu-inhibitable manner.

cells via macrophage nanotubes: a mechanism for AIDS-related lymphoma

Hunsmann, G., Skowronski, J., Kirchhoff, F., 1997. Association of simian immunodeficiency virus Nef with cellular serine/threonine kinases is dispensable

HIV-2-infected cells: pseudotype virions produced with expanded cellular host

tetherin by the human immunodeficiency virus type 2 envelope glycoprotein. J

immunodeficiency associated with a human immunodeficiency virus 1 NEF/3'-

P.M., 2010. Ebola virus glycoprotein counteracts BST-2/Tetherin restriction in a sequence-independent manner that does not require tetherin surface removal. J

Rac1 are implicated in the activation of the Nef-associated kinase and replication of

Nef is dependent on the pathway of virus entry: implications for HIV-based gene

G., 2000. Resistance to apoptosis in HIV-infected CD4+ T lymphocytes is mediated by macrophages: role for Nef and immune activation in viral persistence. J


Janardhan, A., Swigut, T., Hill, B., Myers, M.P., Skowronski, J., 2004. HIV-1 Nef binds the

Jesus da Costa, L., Lopes Dos Santos, A., Mandic, R., Shaw, K., Santana de Aguiar, R.,

Jia, B., Serra-Moreno, R., Neidermyer, W., Rahmberg, A., Mackey, J., Fofana, I.B., Johnson,

Kaminchik, J., Margalit, R., Yaish, S., Drummer, H., Amit, B., Sarver, N., Gorecki, M., Panet,

Kasper, M.R., Roeth, J.F., Williams, M., Filzen, T.M., Fleis, R.I., Collins, K.L., 2005. HIV-1 Nef

Khan, M., Garcia-Barrio, M., Powell, M.D., 2001. Restoration of wild-type infectivity to

Kim, S., Ikeuchi, K., Byrn, R., Groopman, J., Baltimore, D., 1989. Lack of a negative influence

Kirchhoff, F., 2010. Immune evasion and counteraction of restriction factors by HIV-1 and

Kirchhoff, F., Greenough, T.C., Brettler, D.B., Sullivan, J.L., Desrosiers, R.C., 1995. Brief

Kirchhoff, F., Schindler, M., Specht, A., Arhel, N., Munch, J., 2008. Role of Nef in primate

Kotov, A., Zhou, J., Flicker, P., Aiken, C., 1999. Association of Nef with the human

Krausslich, H.G., Schneider, H., Zybarth, G., Carter, C.A., Wimmer, E., 1988. Processing of in

type 1 by HIV proteinase generated in Escherichia coli. J Virol 62, 4393-4397. Krautkramer, E., Giese, S.I., Gasteier, J.E., Muranyi, W., Fackler, O.T., 2004. Human

Kupzig, S., Korolchuk, V., Rollason, R., Sugden, A., Wilde, A., Banting, G., 2003. Bst-

lentiviral immunopathogenesis. Cell Mol Life Sci 65, 2621-2636.

immunodeficiency virus type 1 core. J Virol 73, 8824-8830.

Biol 2, E6.

12840-12848.

rhesus macaques. Virology 394, 47-56.

matrix. AIDS Res Hum Retroviruses 10, 1003-1010.

Acad Sci U S A 106, 2886-2891.

transcription. J Virol 75, 12081-12087.

Natl Acad Sci U S A 86, 9544-9548.

Kirchhausen, T., 2000. Clathrin. Annu Rev Biochem 69, 699-727.

HIV-1 infection. N Engl J Med 332, 228-232.

into lipid rafts. J Virol 78, 4085-4097.

Traffic 4, 694-709.

other primate lentiviruses. Cell Host Microbe 8, 55-67.

DOCK2-ELMO1 complex to activate rac and inhibit lymphocyte chemotaxis. PLoS

Tanuri, A., Luciw, P.A., Peterlin, B.M., 2009. Interactions between SIVNef, SIVGagPol and Alix correlate with viral replication and progression to AIDS in

W.E., Westmoreland, S., Evans, D.T., 2009. Species-specific activity of SIV Nef and HIV-1 Vpu in overcoming restriction by tetherin/BST2. PLoS Pathog 5, e1000429. Kaletsky, R.L., Francica, J.R., Agrawal-Gamse, C., Bates, P., 2009. Tetherin-mediated

restriction of filovirus budding is antagonized by the Ebola glycoprotein. Proc Natl

A., 1994. Cellular distribution of HIV type 1 Nef protein: identification of domains in Nef required for association with membrane and detergent-insoluble cellular

disrupts antigen presentation early in the secretory pathway. J Biol Chem 280,

human immunodeficiency virus type 1 strains lacking nef by intravirion reverse

on viral growth by the nef gene of human immunodeficiency virus type 1. Proc

report: absence of intact nef sequences in a long-term survivor with nonprogressive

vitro-synthesized gag precursor proteins of human immunodeficiency virus (HIV)

immunodeficiency virus type 1 Nef activates p21-activated kinase via recruitment

2/HM1.24 is a raft-associated apical membrane protein with an unusual topology.


Functions of the Lentiviral Accessory Protein

Biochem 37, 443-491.

PLoS Pathog 2, e39.

Nef During the Distinct Steps of HIV and SIV Replication Cycle 35

Nayak, D.P., Hui, E.K., 2004. The role of lipid microdomains in virus biology. Subcell

Neil, S.J., Eastman, S.W., Jouvenet, N., Bieniasz, P.D., 2006. HIV-1 Vpu promotes release and

Neil, S.J., Sandrin, V., Sundquist, W.I., Bieniasz, P.D., 2007. An interferon-alpha-induced

Niederman, T.M., Hastings, W.R., Ratner, L., 1993. Myristoylation-enhanced binding of the

Noviello, C.M., Pond, S.L., Lewis, M.J., Richman, D.D., Pillai, S.K., Yang, O.O., Little, S.J.,

Olivetta, E., Percario, Z., Fiorucci, G., Mattia, G., Schiavoni, I., Dennis, C., J‰ger, J., Harris,

endocytotic signals and NF-kappa B activation. J Immunol 170, 1716-1727. Olivetta, E., Pugliese, K., Bona, R., D'Aloja, P., Ferrantelli, F., Santarcangelo, A.C., Mattia, G.,

Pandori, M., Craig, H., Moutouh, L., Corbeil, J., Guatelli, J., 1998. Virological importance of

Pandori, M.W., Fitch, N.J., Craig, H.M., Richman, D.D., Spina, C.A., Guatelli, J.C., 1996.

Partin, K., Zybarth, G., Ehrlich, L., DeCrombrugghe, M., Wimmer, E., Carter, C., 1991.

Percario, Z., Olivetta, E., Fiorucci, G., Mangino, G., Peretti, S., Romeo, G., Affabris, E.,

Percherancier, Y., Lagane, B., Planchenault, T., Staropoli, I., Altmeyer, R., Virelizier, J.L.,

detergent-resistant, raft membrane domains. J Biol Chem 278, 3153-3161.

counteracted by the HIV-1 Vpu protein. Cell Host Microbe 2, 193-203. Neil, S.J., Zang, T., Bieniasz, P.D., 2008. Tetherin inhibits retrovirus release and is

HIV-1 Nef protein to T cell skeletal matrix. Virology 197, 420-425.

antagonized by HIV-1 Vpu. Nature 451, 425-430.

immunodeficiency virus type 1. J Virol 81, 4776-4786.

intracytoplasmic tails. J Virol 74, 483-492.

virion protein. J Virol 70, 4283-4290.

machinery. J Leukoc Biol 74, 821-832.

251, 302-316.

4776-4780.

prevents endocytosis of nascent retrovirus particles from the plasma membrane.

tethering mechanism inhibits HIV-1 and Ebola virus particle release but is

Smith, D.M., Guatelli, J.C., 2007. Maintenance of Nef-mediated modulation of major histocompatibility complex class I and CD4 after sexual transmission of human

M., Romeo, G., Affabris, E., Federico, M., 2003. HIV-1 Nef induces the release of inflammatory factors from human monocyte/macrophages: involvement of Nef

Verani, P., Federico, M., 2000. cis expression of the F12 human immunodeficiency virus (HIV) Nef allele transforms the highly productive NL4-3 HIV type 1 to a replication-defective strain: involvement of both Env gp41 and CD4

the protease-cleavage site in human immunodeficiency virus type 1 Nef is independent of both intravirion processing and CD4 down-regulation. Virology

Producer-cell modification of human immunodeficiency virus type 1: Nef is a

Deletion of sequences upstream of the proteinase improves the proteolytic processing of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A 88,

Federico, M., 2003. Human immunodeficiency virus type 1 (HIV-1) Nef activates STAT3 in primary human monocyte/macrophages through the release of soluble factors: involvement of Nef domains interacting with the cell endocytotic

Arenzana-Seisdedos, F., Hoessli, D.C., Bachelerie, F., 2003. HIV-1 entry into T-cells is not dependent on CD4 and CCR5 localization to sphingolipid-enriched,


Mandic, R., Fackler, O.T., Geyer, M., Linnemann, T., Zheng, Y.H., Peterlin, B.M., 2001.

internalize CD4 and to increase viral infectivity. Mol Biol Cell 12, 463-473. Mangasarian, A., Foti, M., Aiken, C., Chin, D., Carpentier, J.L., Trono, D., 1997. The HIV-1

Mangino, G., Percario, Z.A., Fiorucci, G., Vaccari, G., Manrique, S., Romeo, G., Federico, M.,

Manninen, A., Saksela, K., 2002. HIV-1 Nef interacts with inositol trisphosphate receptor to

Mansouri, M., Viswanathan, K., Douglas, J.L., Hines, J., Gustin, J., Moses, A.V., Fruh, K.,

Mariani, R., Skowronski, J., 1993. CD4 down-regulation by nef alleles isolated from human

McNatt, M.W., Zang, T., Hatziioannou, T., Bartlett, M., Fofana, I.B., Johnson, W.E., Neil, S.J.,

strategy to downregulate cell-surface CCR5 and CD4. Curr Biol 15, 714-723. Miller, M.D., Warmerdam, M.T., Ferrell, S.S., Benitez, R., Greene, W.C., 1997. Intravirion

Miller, M.D., Warmerdam, M.T., Page, K.A., Feinberg, M.B., Greene, W.C., 1995. Expression

Mitchell, R.S., Chaudhuri, R., Lindwasser, O.W., Tanaka, K.A., Lau, D., Murillo, R.,

Mitchell, R.S., Katsura, C., Skasko, M.A., Fitzpatrick, K., Lau, D., Ruiz, A., Stephens, E.B.,

Nabel, G., Baltimore, D., 1987. An inducible transcription factor activates expression of

Navia, M.A., Fitzgerald, P.M., McKeever, B.M., Leu, C.T., Heimbach, J.C., Herber, W.K.,

protease from human immunodeficiency virus HIV-1. Nature 337, 615-620.

tetherin transmembrane domain variants. PLoS Pathog 5, e1000300. Michel, N., Allespach, I., Venzke, S., Fackler, O.T., Keppler, O.T., 2005. The Nef protein of

insufficient to enhance viral infectivity. Virology 234, 215-225.

immunodeficiency virus type 1 Nef. J Virol 82, 7758-7767.

human immunodeficiency virus in T cells. Nature 326, 711-713.

activate calcium signaling in T cells. J Exp Med 195, 1023-1032.

Kaposi's sarcoma-associated herpesvirus. J Virol 83, 9672-9681.

plasma membrane. Immunity 6, 67-77.

2791.

5549-5553.

entry. J Virol 69, 579-584.

PLoS Pathog 5, e1000450.

Negative factor from SIV binds to the catalytic subunit of the V-ATPase to

Nef protein acts as a connector with sorting pathways in the Golgi and at the

Geyer, M., Affabris, E., 2007. In vitro treatment of human monocytes/macrophages with myristoylated recombinant Nef of human immunodeficiency virus type 1 leads to the activation of mitogen-activated protein kinases, IkappaB kinases, and interferon regulatory factor 3 and to the release of beta interferon. J Virol 81, 2777-

2009. Molecular mechanism of BST2/tetherin downregulation by K5/MIR2 of

immunodeficiency virus type 1-infected individuals. Proc Natl Acad Sci U S A 90,

Bieniasz, P.D., 2009. Species-specific activity of HIV-1 Vpu and positive selection of

human immunodeficiency virus establishes superinfection immunity by a dual

generation of the C-terminal core domain of HIV-1 Nef by the HIV-1 protease is

of the human immunodeficiency virus type 1 (HIV-1) nef gene during HIV-1 production increases progeny particle infectivity independently of gp160 or viral

Bonifacino, J.S., Guatelli, J.C., 2008. Competition model for upregulation of the major histocompatibility complex class II-associated invariant chain by human

Margottin-Goguet, F., Benarous, R., Guatelli, J.C., 2009. Vpu antagonizes BST-2 mediated restriction of HIV-1 release via beta-TrCP and endo-lysosomal trafficking.

Sigal, I.S., Darke, P.L., Springer, J.P., 1989. Three-dimensional structure of aspartyl


Functions of the Lentiviral Accessory Protein

Nef During the Distinct Steps of HIV and SIV Replication Cycle 37

Ross, T.M., Oran, A.E., Cullen, B.R., 1999. Inhibition of HIV-1 progeny virion release by cell-

conserved activities of lentiviral Nef proteins. J Virol 83, 11528-11539. Saksela, K., Cheng, G., Baltimore, D., 1995. Proline-rich (PxxP) motifs in HIV-1 Nef bind to

of Nef+ viruses but not for down-regulation of CD4. Embo J 14, 484-491. Sawai, E.T., Baur, A.S., Peterlin, B.M., Levy, J.A., Cheng-Mayer, C., 1995. A conserved

Schindler, M., Munch, J., Kutsch, O., Li, H., Santiago, M.L., Bibollet-Ruche, F., Muller-

Schindler, M., W¸rfl, S., Benaroch, P., Greenough, T.C., Daniels, R., Easterbrook, P., Brenner,

Schmokel, J., Sauter, D., Schindler, M., Leendertz, F.H., Bailes, E., Dazza, M.C., Saragosti, S.,

Schorr, J., Kellner, R., Fackler, O., Freund, J., Konvalinka, J., Kienzle, N., Krausslich, H.G.,

Schubert, U., Anton, L.C., Bacik, I., Cox, J.H., Bour, S., Bennink, J.R., Orlowski, M., Strebel,

Schwartz, O., Marechal, V., Danos, O., Heard, J.M., 1995. Human immunodeficiency virus

Silvestri, G., Paiardini, M., Pandrea, I., Lederman, M.M., Sodora, D.L., 2007. Understanding the benign nature of SIV infection in natural hosts. J Clin Invest 117, 3148-3154. Simmons, A., Aluvihare, V., McMichael, A., 2001. Nef triggers a transcriptional program in

Simmons, A., Gangadharan, B., Hodges, A., Sharrocks, K., Prabhakar, S., Garcia, A., Dwek,

not always linked in primate lentiviruses. J Virol 85, 742-752.

and the ubiquitin-conjugating pathway. J Virol 72, 2280-2288.

virus-infected macaques. J Virol 78, 10588-10597.

rise to HIV-1. Cell 125, 1055-1067.

70, 9051-9054.

Virol 69, 4053-4059.

mediators. Immunity 14, 763-777.

virus nef alleles. J Virol 77, 10548-10556.

surface CD4 is relieved by expression of the viral Nef protein. Curr Biol 9, 613-621. Rudolph, J.M., Eickel, N., Haller, C., Schindler, M., Fackler, O.T., 2009. Inhibition of T-cell

receptor-induced actin remodeling and relocalization of Lck are evolutionarily

SH3 domains of a subset of Src kinases and are required for the enhanced growth

domain and membrane targeting of Nef from HIV and SIV are required for association with a cellular serine kinase activity. J Biol Chem 270, 15307-15314. Schindler, M., Munch, J., Brenner, M., Stahl-Hennig, C., Skowronski, J., Kirchhoff, F., 2004.

Comprehensive analysis of nef functions selected in simian immunodeficiency

Trutwin, M.C., Novembre, F.J., Peeters, M., Courgnaud, V., Bailes, E., Roques, P., Sodora, D.L., Silvestri, G., Sharp, P.M., Hahn, B.H., Kirchhoff, F., 2006. Nefmediated suppression of T cell activation was lost in a lentiviral lineage that gave

M., M¸nch, J., Kirchhoff, F., 2003. Down-modulation of mature major histocompatibility complex class II and up-regulation of invariant chain cell surface expression are well-conserved functions of human and simian immunodeficiency

Bibollet-Ruche, F., Peeters, M., Hahn, B.H., Kirchhoff, F., 2011. The presence of a vpu gene and the lack of Nef-mediated downmodulation of T cell receptor-CD3 are

Mueller-Lantzsch, N., Kalbitzer, H.R., 1996. Specific cleavage sites of Nef proteins from human immunodeficiency virus types 1 and 2 for the viral proteases. J Virol

K., Yewdell, J.W., 1998. CD4 glycoprotein degradation induced by human immunodeficiency virus type 1 Vpu protein requires the function of proteasomes

type 1 Nef increases the efficiency of reverse transcription in the infected cell. J

T cells imitating single-signal T cell activation and inducing HIV virulence

R., Zitzmann, N., McMichael, A., 2005. Nef-mediated lipid raft exclusion of UbcH7


Pettit, S.C., Clemente, J.C., Jeung, J.A., Dunn, B.M., Kaplan, A.H., 2005. Ordered processing

Pettit, S.C., Everitt, L.E., Choudhury, S., Dunn, B.M., Kaplan, A.H., 2004. Initial cleavage of

Piguet, V., Gu, F., Foti, M., Demaurex, N., Gruenberg, J., Carpentier, J.L., Trono, D., 1999.

Pitcher, C., Honing, S., Fingerhut, A., Bowers, K., Marsh, M., 1999. Cluster of differentiation

Pizzato, M., Popova, E., Gottlinger, H.G., 2008. Nef can enhance the infectivity of receptor-

Popik, W., Alce, T.M., 2004. CD4 receptor localized to non-raft membrane microdomains

Popov, S., Strack, B., Sanchez-Merino, V., Popova, E., Rosin, H., Gˆttlinger, H.G., 2011.

Qi, M., Aiken, C., 2008. Nef enhances HIV-1 infectivity via association with the virus

Quaranta, M.G., Mattioli, B., Giordani, L., Viora, M., 2006. The immunoregulatory effects of HIV-1 Nef on dendritic cells and the pathogenesis of AIDS. FASEB J 20, 2198-2208. Reid, P.A., Watts, C., 1992. Constitutive endocytosis and recycling of major

Ritter, G.D., Jr., Yamshchikov, G., Cohen, S.J., Mulligan, M.J., 1996. Human

Roeth, J.F., Collins, K.L., 2006. Human immunodeficiency virus type 1 Nef: adapting to intracellular trafficking pathways. Microbiol Mol Biol Rev 70, 548-563. Roeth, J.F., Williams, M., Kasper, M.R., Filzen, T.M., Collins, K.L., 2004. HIV-1 Nef disrupts

Rollason, R., Korolchuk, V., Hamilton, C., Schu, P., Banting, G., 2007. Clathrin-mediated

Clathrin through Gag-Pol or Gag. J Virol 85, 3792-3801.

assembly complex. Virology 373, 287-297.

of the cytoplasmic domain. J Virol 70, 2669-2673.

protease occurs by an intramolecular mechanism. J Virol 78, 8477-8485. Piguet, V., Chen, Y.L., Mangasarian, A., Foti, M., Carpentier, J.L., Trono, D., 1998.

context of the embedded viral protease. J Virol 79, 10601-10607.

of adaptor complexes. Embo J 17, 2472-2481.

Natl Acad Sci U S A 104, 6812-6817.

Biol Chem 279, 704-712.

Immunology 77, 539-542.

motif. J Cell Sci 120, 3850-3858.

63-73.

10819.

903-913.

of the human immunodeficiency virus type 1 GagPol precursor is influenced by the

the human immunodeficiency virus type 1 GagPol precursor by its activated

Mechanism of Nef-induced CD4 endocytosis: Nef connects CD4 with the mu chain

Nef-induced CD4 degradation: a diacidic-based motif in Nef functions as a lysosomal targeting signal through the binding of beta-COP in endosomes. Cell 97,

antigen 4 (CD4) endocytosis and adaptor complex binding require activation of the CD4 endocytosis signal by serine phosphorylation. Mol Biol Cell 10, 677-691. Pizzato, M., Helander, A., Popova, E., Calistri, A., Zamborlini, A., Pal, G., Gˆttlinger, H.G.,

2007. Dynamin 2 is required for the enhancement of HIV-1 infectivity by Nef. Proc

pseudotyped human immunodeficiency virus type 1 particles. J Virol 82, 10811-

supports HIV-1 entry. Identification of a novel raft localization marker in CD4. J

Human Immunodeficiency Virus Type 1 and Related Primate Lentiviruses Engage

histocompatibility complex class II glycoproteins in human B-lymphoblastoid cells.

immunodeficiency virus type 2 glycoprotein enhancement of particle budding: role

MHC-I trafficking by recruiting AP-1 to the MHC-I cytoplasmic tail. J Cell Biol 167,

endocytosis of a lipid-raft-associated protein is mediated through a dual tyrosine


Functions of the Lentiviral Accessory Protein

Microbe 3, 245-252.

114, 701-713.

10492-10496.

3022.

Nef During the Distinct Steps of HIV and SIV Replication Cycle 39

Van Damme, N., Goff, D., Katsura, C., Jorgenson, R.L., Mitchell, R., Johnson, M.C., Stephens,

VandeWoude, S., Apetrei, C., 2006. Going wild: lessons from naturally occurring T-

Varin, A., Manna, S.K., Quivy, V., Decrion, A.Z., Van Lint, C., Herbein, G., Aggarwal, B.B.,

Vendrame, D., Sourisseau, M., Perrin, V., Schwartz, O., Mammano, F., 2009. Partial

Verkade, P., Simons, K., 1997. Robert Feulgen Lecture 1997. Lipid microdomains and membrane trafficking in mammalian cells. Histochem Cell Biol 108, 211-220. von Schwedler, U.K., Stuchell, M., M¸ller, B., Ward, D.M., Chung, H.Y., Morita, E., Wang,

Wang, C.Y., Mayo, M.W., Korneluk, R.G., Goeddel, D.V., Baldwin, A.S., 1998. NF-kappaB

Wang JK, K.E., Verdin E, Trono D., 2000. The Nef protein of HIV-1 associates with rafts and

Wang, J.K., Kiyokawa, E., Verdin, E., Trono, D., 2000. The Nef protein of HIV-1 associates with rafts and primes T cells for activation. Proc Natl Acad Sci U S A 97, 394-399. Warrilow, D., Tachedjian, G., Harrich, D., 2009. Maturation of the HIV reverse transcription

Warrilow, D., Warren, K., Harrich, D., 2010. Strand transfer and elongation of HIV-1 reverse transcription is facilitated by cell factors in vitro. PLoS One 5, e13229. Weber, I.T., 1990. Comparison of the crystal structures and intersubunit interactions of

Welker, R., Harris, M., Cardel, B., Kr‰usslich, H.G., 1998. Virion incorporation of human

Wu, X., Liu, H., Xiao, H., Conway, J.A., Hehl, E., Kalpana, G.V., Prasad, V., Kappes, J.C.,

human immunodeficiency and Rous sarcoma virus proteases. J Biol Chem 265,

immunodeficiency virus type 1 Nef is mediated by a bipartite membrane-targeting signal: analysis of its role in enhancement of viral infectivity. J Virol 72, 8833-8840. Willey, R.L., Maldarelli, F., Martin, M.A., Strebel, K., 1992. Human immunodeficiency virus type 1 Vpu protein induces rapid degradation of CD4. J Virol 66, 7193-7200. Wonderlich, E.R., Williams, M., Collins, K.L., 2008. The tyrosine binding pocket in the

adaptor protein 1 (AP-1) mu1 subunit is necessary for Nef to recruit AP-1 to the major histocompatibility complex class I cytoplasmic tail. J Biol Chem 283, 3011-

1999. Human immunodeficiency virus type 1 integrase protein promotes reverse transcription through specific interactions with the nucleoprotein reverse

primes T cells for activation. Proc Natl Acad Sci U S A 97, 394-399.

complex: putting the jigsaw together. Rev Med Virol 19, 324-337.

lymphotropic lentiviruses. Clin Microbiol Rev 19, 728-762.

impact of cell-to-cell viral transfer. J Virol 83, 10527-10537.

pathogenesis. J Biol Chem 278, 2219-2227.

caspase-8 activation. Science 281, 1680-1683.

transcription complex. J Virol 73, 2126-2135.

E.B., Guatelli, J., 2008. The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated from the cell surface by the viral Vpu protein. Cell Host

2003. Exogenous Nef protein activates NF-kappa B, AP-1, and c-Jun N-terminal kinase and stimulates HIV transcription in promonocytic cells. Role in AIDS

inhibition of human immunodeficiency virus replication by type I interferons:

H.E., Davis, T., He, G.P., Cimbora, D.M., Scott, A., Kr‰usslich, H.G., Kaplan, J., Morham, S.G., Sundquist, W.I., 2003. The protein network of HIV budding. Cell

antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress

inhibits Cbl activity in T cells to positively regulate signaling. Immunity 23, 621- 634.


Skowronski, J., Parks, D., Mariani, R., 1993. Altered T cell activation and development in

Sol-Foulon, N., Esnault, C., Percherancier, Y., Porrot, F., Metais-Cunha, P., Bachelerie, F.,

Sousa, A.E., Carneiro, J., Meier-Schellersheim, M., Grossman, Z., Victorino, R.M., 2002. CD4

1 and HIV-2 but only indirectly to the viral load. J Immunol 169, 3400-3406. Stolp, B., Abraham, L., Rudolph, J.M., Fackler, O.T., 2010. Lentiviral Nef proteins utilize

Strack, B., Calistri, A., Craig, S., Popova, E., Gˆttlinger, H.G., 2003. AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell 114, 689-699. Stumptner-Cuvelette, P., Morchoisne, S., Dugast, M., Le Gall, S., Raposo, G., Schwartz, O.,

Swann, S.A., Williams, M., Story, C.M., Bobbitt, K.R., Fleis, R., Collins, K.L., 2001. HIV-1 Nef

Swigut, T., Greenberg, M., Skowronski, J., 2003. Cooperative interactions of simian

Swigut, T., Iafrate, A.J., Muench, J., Kirchhoff, F., Skowronski, J., 2000. Simian and human

major histocompatibility complex antigen expression. J Virol 74, 5691-5701. Swingler, S., Mann, A., Jacque, J., Brichacek, B., Sasseville, V.G., Williams, K., Lackner, A.A.,

Tang, J., Wong, R.N., 1987. Evolution in the structure and function of aspartic proteases. J

Thoulouze, M.I., Sol-Foulon, N., Blanchet, F., Dautry-Varsat, A., Schwartz, O., Alcover, A.,

Tobiume, M., Takahoko, M., Yamada, T., Tatsumi, M., Iwamoto, A., Matsuda, M., 2002.

Tobiume, M., Tokunaga, K., Kiyokawa, E., Takahoko, M., Mochizuki, N., Tatsumi, M.,

Tripathi, P., Agrawal, S., 2007. The role of human leukocyte antigen E and G in HIV

surface expression. Proc Natl Acad Sci U S A 98, 12144-12149.

of T-cell receptor-CD3 endocytosis. J Virol 77, 8116-8126.

transgenic mice expressing the HIV-1 nef gene. Embo J 12, 703-713.

634.

103.

Cell Biochem 33, 53-63.

Virol 76, 5959-5965.

Arch Virol 146, 1739-1751.

infection. Aids 21, 1395-1404.

remodeling. J Virol 84, 3935-3948.

dependent pathway. Virology 282, 267-277.

immunological synapse. Immunity 24, 547-561.

inhibits Cbl activity in T cells to positively regulate signaling. Immunity 23, 621-

Schwartz, O., 2004. The effects of HIV-1 Nef on CD4 surface expression and viral infectivity in lymphoid cells are independent of rafts. J Biol Chem 279, 31398-31408.

T cell depletion is linked directly to immune activation in the pathogenesis of HIV-

PAK2-mediated deregulation of cofilin as a general strategy to interfere with actin

Benaroch, P., 2001. HIV-1 Nef impairs MHC class II antigen presentation and

blocks transport of MHC class I molecules to the cell surface via a PI 3-kinase-

immunodeficiency virus Nef, AP-2, and CD3-zeta mediate the selective induction

immunodeficiency virus Nef proteins use different surfaces to downregulate class I

Janoff, E.N., Wang, R., Fisher, D., Stevenson, M., 1999. HIV-1 Nef mediates lymphocyte chemotaxis and activation by infected macrophages. Nat Med 5, 997-

2006. Human immunodeficiency virus type-1 infection impairs the formation of the

Inefficient enhancement of viral infectivity and CD4 downregulation by human immunodeficiency virus type 1 Nef from Japanese long-term nonprogressors. J

Matsuda, M., 2001. Requirement of nef for HIV-1 infectivity is biased by the expression levels of Env in the virus-producing cells and CD4 in the target cells.


**2** 

*Brazil* 

**The Role of Human Immunodeficiency Virus** 

**Therapy in HIV-1-Induced Oxidative Stress** 

*Clinical Immunology and Molecular Diagnosis Laboratories, University Hospital of* 

Over 33 million people worldwide are infected with human immunodeficiency virus type 1 (HIV-1). In addition, over 2.7 million new cases are diagnosed each year with half of these infections occurring in individuals younger than 25 years (UNAIDS, 2008). Fortunately, since the emergence of highly active antiretroviral therapy (HAART) in 1996, morbidity and mortality associated with HIV-1 infection have been markedly decreased. HIV-1 infected patients have demonstrated dramatic decreases in viral burden and opportunistic infections, and an overall increase in life expectancy. Despite the positive HAART-associations outcomes, including the improvement of the clinical course, prognosis, and survival of patients infected with HIV-1, it has become increasingly clear that HIV-1 infected patients have an enhanced risk for developing noninfectious consequences of HIV-1 infection over time. In the last few years, lipodystrophy, characterized by redistribution of body fat, and insulin resistance, have been reported in many HIV-1 infected patients, and their relationship with antiretroviral drugs and HIV-1 infection *per se* have become a subject of debate and researches worldwide. Evidence suggests that HIV-1 infected patients are under chronic oxidative stress that may be involved in the development and progression of the disease. Oxidative stress is enhanced by the chronic inflammation that is associated with activation of lymphocytes and phagocytes, and is accompanied by the direct or indirect effects of several opportunistic pathogens. In addition, HIV-1 proteins and various components of current HAART regimes contribute to oxidative stress-induced disturbances such as cardiovascular disease (including metabolic syndrome and endothelial dysfunction), neurological disorders (HIV-1 dementia), and ocular complications (retinopathy). Cardiovascular complications are been recognized with increasing frequency and are associated with the greatest risk of death in HIV-1 patients. Studies demonstrated that not only do various components of HAART contribute to endothelial cell damage and vascular dysfunction in patients, but also the viral proteins themselves increase cardiovascular risk. HIV-1-associated cardiovascular disease progression is thus most likely a multifactorial process, resulting from a combination of distinct HIV-1 proteins as well as various

components of current multidrug antiretroviral therapy (Kline et al., 2008).

**1. Introduction** 

Edna Maria Vissoci Reiche and Andréa Name Colado Simão

*Londrina, State University of Londrina,* 

**Type 1 (HIV-1) Proteins and Antiretroviral Drug** 


## **The Role of Human Immunodeficiency Virus Type 1 (HIV-1) Proteins and Antiretroviral Drug Therapy in HIV-1-Induced Oxidative Stress**

Edna Maria Vissoci Reiche and Andréa Name Colado Simão *Clinical Immunology and Molecular Diagnosis Laboratories, University Hospital of Londrina, State University of Londrina, Brazil* 

## **1. Introduction**

40 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Xu, W., Santini, P.A., Sullivan, J.S., He, B., Shan, M., Ball, S.C., Dyer, W.B., Ketas, T.J.,

Xu, X.N., Laffert, B., Screaton, G.R., Kraft, M., Wolf, D., Kolanus, W., Mongkolsapay, J.,

Yang, S.J., Lopez, L.A., Hauser, H., Exline, C.M., Haworth, K.G., Cannon, P.M., 2010. Antitetherin activities in Vpu-expressing primate lentiviruses. Retrovirology 7, 13. Yi, L., Rosales, T., Rose, J.J., Chowdhury, B., Knutson, J.R., Venkatesan, S., 2010. HIV-1 Nef

Zhang, F., Wilson, S.J., Landford, W.C., Virgen, B., Gregory, D., Johnson, M.C., Munch, J.,

10, 1008-1017.

1489-1496.

Chem 285, 30884-30905.

Acad Sci U S A 100, 8460-8465.

Chadburn, A., Cohen-Gould, L., Knowles, D.M., Chiu, A., Sanders, R.W., Chen, K., Cerutti, A., 2009. HIV-1 evades virus-specific IgG2 and IgA responses by targeting systemic and intestinal B cells via long-range intercellular conduits. Nat Immunol

McMichael, A.J., Baur, A.S., 1999. Induction of Fas ligand expression by HIV involves the interaction of Nef with the T cell receptor zeta chain. J Exp Med 189,

binds a subpopulation of MHC-I throughout its trafficking itinerary and downregulates MHC-I by perturbing both anterograde and retrograde trafficking. J Biol

Kirchhoff, F., Bieniasz, P.D., Hatziioannou, T., 2009. Nef proteins from simian immunodeficiency viruses are tetherin antagonists. Cell Host Microbe 6, 54-67. Zheng, Y.H., Plemenitas, A., Fielding, C.J., Peterlin, B.M., 2003. Nef increases the synthesis

of and transports cholesterol to lipid rafts and HIV-1 progeny virions. Proc Natl

Over 33 million people worldwide are infected with human immunodeficiency virus type 1 (HIV-1). In addition, over 2.7 million new cases are diagnosed each year with half of these infections occurring in individuals younger than 25 years (UNAIDS, 2008). Fortunately, since the emergence of highly active antiretroviral therapy (HAART) in 1996, morbidity and mortality associated with HIV-1 infection have been markedly decreased. HIV-1 infected patients have demonstrated dramatic decreases in viral burden and opportunistic infections, and an overall increase in life expectancy. Despite the positive HAART-associations outcomes, including the improvement of the clinical course, prognosis, and survival of patients infected with HIV-1, it has become increasingly clear that HIV-1 infected patients have an enhanced risk for developing noninfectious consequences of HIV-1 infection over time. In the last few years, lipodystrophy, characterized by redistribution of body fat, and insulin resistance, have been reported in many HIV-1 infected patients, and their relationship with antiretroviral drugs and HIV-1 infection *per se* have become a subject of debate and researches worldwide. Evidence suggests that HIV-1 infected patients are under chronic oxidative stress that may be involved in the development and progression of the disease. Oxidative stress is enhanced by the chronic inflammation that is associated with activation of lymphocytes and phagocytes, and is accompanied by the direct or indirect effects of several opportunistic pathogens. In addition, HIV-1 proteins and various components of current HAART regimes contribute to oxidative stress-induced disturbances such as cardiovascular disease (including metabolic syndrome and endothelial dysfunction), neurological disorders (HIV-1 dementia), and ocular complications (retinopathy). Cardiovascular complications are been recognized with increasing frequency and are associated with the greatest risk of death in HIV-1 patients. Studies demonstrated that not only do various components of HAART contribute to endothelial cell damage and vascular dysfunction in patients, but also the viral proteins themselves increase cardiovascular risk. HIV-1-associated cardiovascular disease progression is thus most likely a multifactorial process, resulting from a combination of distinct HIV-1 proteins as well as various components of current multidrug antiretroviral therapy (Kline et al., 2008).

The Role of Human Immunodeficiency Virus Type 1 (HIV-1)

well as intra- and intermolecular protein cross-links.

and urine (Halliwell & Gutteridge, 1999).

2005).

et al., 1998).

**2.1 NO and HIV-1 infection** 

Proteins and Antiretroviral Drug Therapy in HIV-1-Induced Oxidative Stress 43

metabolism. Under physiological conditions, proteins are more readily attacked by MDA than are free amino acids, resulting in modification of several residues, especially lysine, as

One particular class of toxic products of LPO is the isoprostanes, a series of prostaglandinlike compounds formed during peroxidation of arachidonic acid. Because they are structurally similar to prostaglandin F2α, isoprostanes are collectively referred as F2 isoprostane. F2-isoprostane is useful marker of LPO and can be measured in human plasma

Collectively, ROS can lead to oxidation of proteins, and DNA, peroxidation of lipids, and ultimately cell death (Butterfield et al., 2001). These protein carbonyl moieties result from a direct oxidation of many amino acids such as lysine, arginine, histidine, proline and threonine, -scission of the peptide backbone, or from binding of the LPO product 4 hydroxy-2-nonenal (HNE) to proteins. Alterations in proteins can lead to aggregation, changes in secondary and tertiary structure, susceptibility to proteolysis, fragmentation, and loss-of function. LPO produces large amounts of aldehydes, such as HNE, MDA, and acrolein, and leads to isoprostanes formation (Butterfield et. al, 2002). HNE and acrolein contribute to membrane damage and cell death induced by various oxidative insults, and through alterations of protein structure, these molecules are capable of inhibiting DNA, RNA, and protein synthesis, glycolysis, and degradation of enzymes (Pocernick et al.,

ROS produce a multiplicity of change in proteins, including oxidation of –SH groups, hydroxylation of thyrosine and phenylalanine, conversion of methionine to its sulphoxide and generation of protein peroxides. Several assays for damage to specific amino acid residues in proteins have been developed and can be used to assess steady-state levels of oxidative protein damage *in vivo*. The carbonyl assay is a general approach for evaluating oxidation protein damage. It is based on the fact that several ROS attack amino acid residues in proteins that results products with carbonyl groups, which can be measured after reaction with 2,4-dinitrophenylhydrazine (Halliwell & Gutteridge, 1999). Oxidative stress also increases the levels of protein oxidation measured by the Advanced Oxidation Protein Products (AOPPs). AOPPs are novel biomarkers of oxidative damage and are considered as reliable markers to estimate de degree of oxidant-mediated protein damage. AOPPs resulted from the interaction between oxidants and plasma proteins with the oxidation of amino acid residues such as tyrosine, leading to the formation of dityrosine-containing protein crosslinking products detected by spectrophotometry (Witko-Sarsat et al., 1998). Neutrophils that constitute the most important source of chlorined oxidants due to their high content in myeloperoxidase might be involved in plasma AOPPs formation. *In vivo* plasma levels of AOPPs closely correlate with level of dityrosine, a hallmark of oxidized proteins, and with pentosidine, a marker of protein glycation closely related with oxidative stress (Witko-Sarsat

NO is a free-radical gas, a diffusible messenger that displays a variety of physiological functions, including vasorelaxation, bronchodilatation, inhibition of platelet aggregation, and neurotransmission (Radi, 2004). Additionally, it appears to be involved in the macrophage-dependent killing of intracellular parasites and functions as a tumoricidal and antimicrobial molecule i*n vitro* and *in vivo* (Torre et al., 2002). NO represents an important

It is estimated that one-third of adults infected with HIV-1 develop dementia (Janssen et al., 1992). It was reported that oxidative stress has been demonstrated in the brain and cerebrospinal fluid (CSF) from HIV-1 infected individuals, showing important implications for therapeutic approaches for HIV-1-induced dementia (HIVD).

The aim of this chapter is to review the roles of both HIV-1 proteins and antiretroviral drugs in the development of oxidative stress-induced disturbances such as cardiovascular disease and neurological disorders. For this purpose, studies, *in vitro* and *in vivo*, were identified by a systematic search through PubMed for English-language literature, included original and review articles published up to 2011.

## **2. Oxidative stress and biomarkers**

Oxidative stress is defined as an imbalance between the antioxidant and pro-oxidant systems with the shift towards the pro-oxidant system. Oxidative stress is also defined as the modification and accumulation of biological molecules altered by various kinds of reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS and RNS affect gene transcription and cell growth/proliferation, and they have been considered intercellular signal molecules.

ROS and RNS are highly reactive, toxic oxygen or nitrogen moieties, respectively, such hydroxyl radical, peroxyl radical, superoxide anion, hydrogen peroxide, nitric oxid (NO), and peroxynitrite. The half-life of ROS species varies from nanoseconds for the hydroxyl radical to seconds for NO and peroxyl radicals. Because of the differences in half-lives, the ROS reactivity differs from the aqueous environment in which they were formed to reacting deep within the membrane (Pocernich et al., 2005).

In biological systems, the cellular membrane constitutes in a main target of the ROS and RNS. In addition to the cellular membrane, other intracellular membranes are important target of the oxidative stress such as mitochondrial, nuclear and endoplasmic reticulum membranes that can suffer the lesive action of the ROS and RNS by changing their form and function. Not only enzymes but also receptors and transport proteins can be important early targets of oxidative damage. While most ROS do not diffuse more than a few femtometres (fm), the lipid peroxides that are resulted from the ROS-induced peroxidation of membrane phospholipids, such as malondialdehyde (MDA), can transverse the circulation and cell membranes, with resultant dysfunction of vital cellular processes including membrane transport and mitochondrial respiration (Haliwell, 1987).

ROS can attack double bonds in polyunsaturated fatty acids (PUFAs), inducing lipid peroxidation (LPO), which may result in more oxidative cellular damage. LPO has been defined as the oxidative deterioration of polyunsaturated lipids and its measurement is a laboratorial approach for determining oxidative stress. Peroxides and aldehydes generated are not only passive biomarkers of oxidative stress, but also cytotoxic products (Zwart et al., 1999).

MDA is a three carbon, low molecular weight aldehyde that can be produced from free radicals that attack on PUFAs of biological membranes. The determination of MDA is used for monitoring LPO in biological samples. LPO has been the focus of attention in recent researches because it was commonly thought that the thiobarbituric acid (TBA) test, the commonest assay of LPO *in vitro*, measures free MDA. It arises largely from peroxidation of PUFAs with more than two double bonds, such as linolenic, arachidonic and docosahexaenoic acids. MDA can also be formed enzimatically during eicosanoid

It is estimated that one-third of adults infected with HIV-1 develop dementia (Janssen et al., 1992). It was reported that oxidative stress has been demonstrated in the brain and cerebrospinal fluid (CSF) from HIV-1 infected individuals, showing important implications

The aim of this chapter is to review the roles of both HIV-1 proteins and antiretroviral drugs in the development of oxidative stress-induced disturbances such as cardiovascular disease and neurological disorders. For this purpose, studies, *in vitro* and *in vivo*, were identified by a systematic search through PubMed for English-language literature, included original and

Oxidative stress is defined as an imbalance between the antioxidant and pro-oxidant systems with the shift towards the pro-oxidant system. Oxidative stress is also defined as the modification and accumulation of biological molecules altered by various kinds of reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS and RNS affect gene transcription and cell growth/proliferation, and they have been considered

ROS and RNS are highly reactive, toxic oxygen or nitrogen moieties, respectively, such hydroxyl radical, peroxyl radical, superoxide anion, hydrogen peroxide, nitric oxid (NO), and peroxynitrite. The half-life of ROS species varies from nanoseconds for the hydroxyl radical to seconds for NO and peroxyl radicals. Because of the differences in half-lives, the ROS reactivity differs from the aqueous environment in which they were formed to reacting

In biological systems, the cellular membrane constitutes in a main target of the ROS and RNS. In addition to the cellular membrane, other intracellular membranes are important target of the oxidative stress such as mitochondrial, nuclear and endoplasmic reticulum membranes that can suffer the lesive action of the ROS and RNS by changing their form and function. Not only enzymes but also receptors and transport proteins can be important early targets of oxidative damage. While most ROS do not diffuse more than a few femtometres (fm), the lipid peroxides that are resulted from the ROS-induced peroxidation of membrane phospholipids, such as malondialdehyde (MDA), can transverse the circulation and cell membranes, with resultant dysfunction of vital cellular processes including membrane

ROS can attack double bonds in polyunsaturated fatty acids (PUFAs), inducing lipid peroxidation (LPO), which may result in more oxidative cellular damage. LPO has been defined as the oxidative deterioration of polyunsaturated lipids and its measurement is a laboratorial approach for determining oxidative stress. Peroxides and aldehydes generated are not only passive biomarkers of oxidative stress, but also cytotoxic products (Zwart et al.,

MDA is a three carbon, low molecular weight aldehyde that can be produced from free radicals that attack on PUFAs of biological membranes. The determination of MDA is used for monitoring LPO in biological samples. LPO has been the focus of attention in recent researches because it was commonly thought that the thiobarbituric acid (TBA) test, the commonest assay of LPO *in vitro*, measures free MDA. It arises largely from peroxidation of PUFAs with more than two double bonds, such as linolenic, arachidonic and docosahexaenoic acids. MDA can also be formed enzimatically during eicosanoid

for therapeutic approaches for HIV-1-induced dementia (HIVD).

review articles published up to 2011.

intercellular signal molecules.

1999).

**2. Oxidative stress and biomarkers** 

deep within the membrane (Pocernich et al., 2005).

transport and mitochondrial respiration (Haliwell, 1987).

metabolism. Under physiological conditions, proteins are more readily attacked by MDA than are free amino acids, resulting in modification of several residues, especially lysine, as well as intra- and intermolecular protein cross-links.

One particular class of toxic products of LPO is the isoprostanes, a series of prostaglandinlike compounds formed during peroxidation of arachidonic acid. Because they are structurally similar to prostaglandin F2α, isoprostanes are collectively referred as F2 isoprostane. F2-isoprostane is useful marker of LPO and can be measured in human plasma and urine (Halliwell & Gutteridge, 1999).

Collectively, ROS can lead to oxidation of proteins, and DNA, peroxidation of lipids, and ultimately cell death (Butterfield et al., 2001). These protein carbonyl moieties result from a direct oxidation of many amino acids such as lysine, arginine, histidine, proline and threonine, -scission of the peptide backbone, or from binding of the LPO product 4 hydroxy-2-nonenal (HNE) to proteins. Alterations in proteins can lead to aggregation, changes in secondary and tertiary structure, susceptibility to proteolysis, fragmentation, and loss-of function. LPO produces large amounts of aldehydes, such as HNE, MDA, and acrolein, and leads to isoprostanes formation (Butterfield et. al, 2002). HNE and acrolein contribute to membrane damage and cell death induced by various oxidative insults, and through alterations of protein structure, these molecules are capable of inhibiting DNA, RNA, and protein synthesis, glycolysis, and degradation of enzymes (Pocernick et al., 2005).

ROS produce a multiplicity of change in proteins, including oxidation of –SH groups, hydroxylation of thyrosine and phenylalanine, conversion of methionine to its sulphoxide and generation of protein peroxides. Several assays for damage to specific amino acid residues in proteins have been developed and can be used to assess steady-state levels of oxidative protein damage *in vivo*. The carbonyl assay is a general approach for evaluating oxidation protein damage. It is based on the fact that several ROS attack amino acid residues in proteins that results products with carbonyl groups, which can be measured after reaction with 2,4-dinitrophenylhydrazine (Halliwell & Gutteridge, 1999). Oxidative stress also increases the levels of protein oxidation measured by the Advanced Oxidation Protein Products (AOPPs). AOPPs are novel biomarkers of oxidative damage and are considered as reliable markers to estimate de degree of oxidant-mediated protein damage. AOPPs resulted from the interaction between oxidants and plasma proteins with the oxidation of amino acid residues such as tyrosine, leading to the formation of dityrosine-containing protein crosslinking products detected by spectrophotometry (Witko-Sarsat et al., 1998). Neutrophils that constitute the most important source of chlorined oxidants due to their high content in myeloperoxidase might be involved in plasma AOPPs formation. *In vivo* plasma levels of AOPPs closely correlate with level of dityrosine, a hallmark of oxidized proteins, and with pentosidine, a marker of protein glycation closely related with oxidative stress (Witko-Sarsat et al., 1998).

## **2.1 NO and HIV-1 infection**

NO is a free-radical gas, a diffusible messenger that displays a variety of physiological functions, including vasorelaxation, bronchodilatation, inhibition of platelet aggregation, and neurotransmission (Radi, 2004). Additionally, it appears to be involved in the macrophage-dependent killing of intracellular parasites and functions as a tumoricidal and antimicrobial molecule i*n vitro* and *in vivo* (Torre et al., 2002). NO represents an important

The Role of Human Immunodeficiency Virus Type 1 (HIV-1)

T cell proliferation (Holt et al., 1991).

al., 1994).

**2.2 Antioxidants** 

acid (Butterfield, et al., 1997).

of experimental models (Gunnett et al., 2003).

necessary to confirm these previous results.

Proteins and Antiretroviral Drug Therapy in HIV-1-Induced Oxidative Stress 45

binding activity, and enhance activity of tyrosine kinase, p56, which is implicated in lymphocyte signaling events (Lander et al., 1993). Paradoxically, high concentrations of NO, which occur following macrophage activation, suppress antigen presenting cell activity and

In addition, vascular dysfunction and damage have been shown to be associated with impaired endothelial NO metabolism and function. Therefore, iNOS-derived NO mediates the inflammatory response and has been shown to cause vascular dysfunction in a number

The data of NO levels obtained in HIV-1 infected individual samples are controversial. Groeneveld et al. (1996) have shown that serum nitrate concentrations are higher in asymptomatic HIV-1 infected patients than in healthy individuals. In addition, increased production of NO was correlated with RNA-HIV-1 viral load and activation of mononuclear phagocytes in HIV-1 infected patients. Torre et al. (1996a, 1996b) have shown that NO production is increased in AIDS patients with opportunistic infection, whereas nitrite concentrations were normal in asymptomatic patients. These authors have also confirmed increased production of NO and IL-1β, TNF-α, and IFN-γ in the sera of children with HIV-1 infection and they postulated that the increase in the concentration of these cytokines may represent a substantial stimulation of NO production. Zangerle et al. (1995) noted high nitrite and nitrate concentrations in 39 patients with AIDS without opportunistic infections, especially in those with lower CD4+ T cell counts, whereas in asymptomatic patients no such increase was seen. However, a previous study showed no altered endogenous nitrate formation in eight patients with AIDS, most of whom had opportunistic infections (Evans et

However, some aspects must be taken in to account when these apparent controversial results are discussed including the fact that the oxidative stress was evaluated in HIV-1 infected individuals that differed in the clinical course of the disease and in the presence or absence of opportunistic infections. Increases in the NO production may not be observed due the consume resources by the oxidative stress. Anyway, further studies may be

To neutralize the damaging oxidative stress, natural antioxidant systems have evolved, including enzymes like glutathione (GSH) peroxidase, glutathione reductase, glutathione transferase, superoxide dismutase (SOD), S-methyl transferase, and catalase. Protection against free radicals can also come from small non-protein, cellular antioxidants, nonenzymatic, such as vitamin C, vitamin E, carotenoids, flavonoids, thioredoxin, and uric

GSH is a tripeptide ( glutamate-cysteine-glycine) present in high concentrations in all mammalian cells that has many critical protective and metabolic functions. GSH detoxifies electrophilic metabolites of xenobiotics and protects cells from the toxic effects of free radicals and ROS (Bleuter, 1989). It is also important in the immune response against infections and plays an important role in lymphocyte proliferation, antibody-dependent and cell-mediated cytotoxicity, and protection of lymphocytes against superoxides that are produced to destroy invading pathogens (Droge et al., 1991; Smyth, 1991). N-acetyl-Lcysteine (NAC) acts as an indirect precursor of GSH by raising levels of cysteine, a precursor of GSH. Whey proteins have been shown to increase GSH levels in human, most likely by

component of the host immune response against DNA and RNA viral infections, including HIV-1 infection (Mannick et al., 1995).

NO is synthesized by the family of enzymes called nitric oxide synthase (NOS). Various isoenzymes of NOS, such as endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) are localized in endothelium, macrophages, and the brain, respectively. In normal endothelial cells, the amino acid L-arginine is constitutively converted to L-citrulline and NO by eNOS.

The iNOS expression is increased by oxidative stress or pro-inflammatory cytokines (Nathan, 1997). However, interleukin 1 beta (IL-1β), interleukin 6 (IL-6), interferon alfa-2b (IFN-2b), interferon gamma (IFN-), and interleukin 17 (IL-17) induce iNOS, whereas transforming growth factor beta (TGF-), interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 11 (IL-11), and interleukin 13 (IL-13) suppress the induction of NO released from macrophages (Torre et al., 2002). In addition, HIV-1 also stimulates NO production by human macrophages, inasmuch as concentration of recombinant gp120 HIV-1 envelope glycoprotein *in vitro* increases production of NO by human monocyte-derived macrophages (Pietraforte et al., 1994).

The excessive production of NO by iNOS may contribute to tissue damage in several inflammatory and infectious diseases and this damage may be the price to pay for equipping so many host cells with the ability to deploy this compound against infections. Although NO production can be increased by the iNOS, the biodisponibility of NO can be impaired because NO is consumed in a reaction with superoxide anion yielding a strong oxidant species, the peroxynitrite (ONOO-), which in turn accelerates the LPO reaction (Li et al., 2007; Tao et al., 2007). Peroxynitrite production is also supported by the elevated levels of nitrotyrosine, a marker of endogenous peroxynitrite generation found in both human and animal models (Yamaguchi et al., 2006).

Since NO is a very labile free radical with a half-life of only a few seconds and is rapidly oxidized by tissue oxygen to the stable end products, nitrite (NO2- ) and nitrate (NO3 -), it is difficult to measure NO levels in the tissue directly with real time. NO can be evaluated by several methods, including the assessment of NO metabolite (NOx) levels. Commonly, serum NO levels are assessed on the basis of nitrite and nitrate concentration according to the Griess reaction supplemented by the enzymatic reduction of nitrate to nitrite with cadmium (Guevara et al., 1998; Navarro-Gonzales et al., 1998). Following up the changes in nitrite/nitrate levels in the human tissues and plasma samples can be an important tool in understanding NO involvement.

Although NO is an important mediator of the immune response against microorganisms, NO that is produced during the infectious diseases may be also deleterious, particularly in HIV-1 infection where may contribute to AIDS pathogenesis by enhancing viral replication in lymphocytes (Jimenez et al., 2001) and monocytes (Blond et al., 2000), increasing lymphocyte apoptosis (Mossalayi et al., 1999), and participating in the pathogenesis of AIDS-related dementia complex (Adamson et al., 1996). A study demonstrated impaired iNOS mRNA expression and NO levels in peripheral blood mononuclear cells from HIV-1 infected patients, either *in vivo* or *in vitro* HIV-1 infection of normal cells (Cairoli et al., 2008). Low levels of NO have been implicated in lymphocyte activation and proliferation (Barbul et al., 1990). NO donors such as sodium nitroprusside and to a lesser degree gaseous NO, increase lymphocyte uptake of glucose (an early event during lymphocyte activation), stimulate TNF- production and the transcriptional nuclear factor kappa beta (NF-kB) binding activity, and enhance activity of tyrosine kinase, p56, which is implicated in lymphocyte signaling events (Lander et al., 1993). Paradoxically, high concentrations of NO, which occur following macrophage activation, suppress antigen presenting cell activity and T cell proliferation (Holt et al., 1991).

In addition, vascular dysfunction and damage have been shown to be associated with impaired endothelial NO metabolism and function. Therefore, iNOS-derived NO mediates the inflammatory response and has been shown to cause vascular dysfunction in a number of experimental models (Gunnett et al., 2003).

The data of NO levels obtained in HIV-1 infected individual samples are controversial. Groeneveld et al. (1996) have shown that serum nitrate concentrations are higher in asymptomatic HIV-1 infected patients than in healthy individuals. In addition, increased production of NO was correlated with RNA-HIV-1 viral load and activation of mononuclear phagocytes in HIV-1 infected patients. Torre et al. (1996a, 1996b) have shown that NO production is increased in AIDS patients with opportunistic infection, whereas nitrite concentrations were normal in asymptomatic patients. These authors have also confirmed increased production of NO and IL-1β, TNF-α, and IFN-γ in the sera of children with HIV-1 infection and they postulated that the increase in the concentration of these cytokines may represent a substantial stimulation of NO production. Zangerle et al. (1995) noted high nitrite and nitrate concentrations in 39 patients with AIDS without opportunistic infections, especially in those with lower CD4+ T cell counts, whereas in asymptomatic patients no such increase was seen. However, a previous study showed no altered endogenous nitrate formation in eight patients with AIDS, most of whom had opportunistic infections (Evans et al., 1994).

However, some aspects must be taken in to account when these apparent controversial results are discussed including the fact that the oxidative stress was evaluated in HIV-1 infected individuals that differed in the clinical course of the disease and in the presence or absence of opportunistic infections. Increases in the NO production may not be observed due the consume resources by the oxidative stress. Anyway, further studies may be necessary to confirm these previous results.

## **2.2 Antioxidants**

44 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

component of the host immune response against DNA and RNA viral infections, including

NO is synthesized by the family of enzymes called nitric oxide synthase (NOS). Various isoenzymes of NOS, such as endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) are localized in endothelium, macrophages, and the brain, respectively. In normal endothelial cells, the amino acid L-arginine is constitutively

The iNOS expression is increased by oxidative stress or pro-inflammatory cytokines (Nathan, 1997). However, interleukin 1 beta (IL-1β), interleukin 6 (IL-6), interferon alfa-2b (IFN-2b), interferon gamma (IFN-), and interleukin 17 (IL-17) induce iNOS, whereas transforming growth factor beta (TGF-), interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 11 (IL-11), and interleukin 13 (IL-13) suppress the induction of NO released from macrophages (Torre et al., 2002). In addition, HIV-1 also stimulates NO production by human macrophages, inasmuch as concentration of recombinant gp120 HIV-1 envelope glycoprotein *in vitro* increases production of NO by human monocyte-derived macrophages

The excessive production of NO by iNOS may contribute to tissue damage in several inflammatory and infectious diseases and this damage may be the price to pay for equipping so many host cells with the ability to deploy this compound against infections. Although NO production can be increased by the iNOS, the biodisponibility of NO can be impaired because NO is consumed in a reaction with superoxide anion yielding a strong oxidant species, the peroxynitrite (ONOO-), which in turn accelerates the LPO reaction (Li et al., 2007; Tao et al., 2007). Peroxynitrite production is also supported by the elevated levels of nitrotyrosine, a marker of endogenous peroxynitrite generation found in both human and

Since NO is a very labile free radical with a half-life of only a few seconds and is rapidly oxidized by tissue oxygen to the stable end products, nitrite (NO2-) and nitrate (NO3-), it is difficult to measure NO levels in the tissue directly with real time. NO can be evaluated by several methods, including the assessment of NO metabolite (NOx) levels. Commonly, serum NO levels are assessed on the basis of nitrite and nitrate concentration according to the Griess reaction supplemented by the enzymatic reduction of nitrate to nitrite with cadmium (Guevara et al., 1998; Navarro-Gonzales et al., 1998). Following up the changes in nitrite/nitrate levels in the human tissues and plasma samples can be an important tool in

Although NO is an important mediator of the immune response against microorganisms, NO that is produced during the infectious diseases may be also deleterious, particularly in HIV-1 infection where may contribute to AIDS pathogenesis by enhancing viral replication in lymphocytes (Jimenez et al., 2001) and monocytes (Blond et al., 2000), increasing lymphocyte apoptosis (Mossalayi et al., 1999), and participating in the pathogenesis of AIDS-related dementia complex (Adamson et al., 1996). A study demonstrated impaired iNOS mRNA expression and NO levels in peripheral blood mononuclear cells from HIV-1 infected patients, either *in vivo* or *in vitro* HIV-1 infection of normal cells (Cairoli et al., 2008). Low levels of NO have been implicated in lymphocyte activation and proliferation (Barbul et al., 1990). NO donors such as sodium nitroprusside and to a lesser degree gaseous NO, increase lymphocyte uptake of glucose (an early event during lymphocyte activation), stimulate TNF- production and the transcriptional nuclear factor kappa beta (NF-kB)

HIV-1 infection (Mannick et al., 1995).

converted to L-citrulline and NO by eNOS.

animal models (Yamaguchi et al., 2006).

understanding NO involvement.

(Pietraforte et al., 1994).

To neutralize the damaging oxidative stress, natural antioxidant systems have evolved, including enzymes like glutathione (GSH) peroxidase, glutathione reductase, glutathione transferase, superoxide dismutase (SOD), S-methyl transferase, and catalase. Protection against free radicals can also come from small non-protein, cellular antioxidants, nonenzymatic, such as vitamin C, vitamin E, carotenoids, flavonoids, thioredoxin, and uric acid (Butterfield, et al., 1997).

GSH is a tripeptide ( glutamate-cysteine-glycine) present in high concentrations in all mammalian cells that has many critical protective and metabolic functions. GSH detoxifies electrophilic metabolites of xenobiotics and protects cells from the toxic effects of free radicals and ROS (Bleuter, 1989). It is also important in the immune response against infections and plays an important role in lymphocyte proliferation, antibody-dependent and cell-mediated cytotoxicity, and protection of lymphocytes against superoxides that are produced to destroy invading pathogens (Droge et al., 1991; Smyth, 1991). N-acetyl-Lcysteine (NAC) acts as an indirect precursor of GSH by raising levels of cysteine, a precursor of GSH. Whey proteins have been shown to increase GSH levels in human, most likely by

The Role of Human Immunodeficiency Virus Type 1 (HIV-1)

Aruoma, 1994; Israel & Gougerot-Pocidalo, 1997).

Proteins and Antiretroviral Drug Therapy in HIV-1-Induced Oxidative Stress 47

 and ROS, can cause the release of NF-kB from factor IkB, and NF-kB translocates to the nucleus and binds to DNA. In this way, the NF-kB is available to bind in the nuclear DNA and to induce HIV-1 gene transcription (Schereck et al., 1991). Thus, oxidative stress may potentially be involved in the pathogenesis of HIV-1 infection through direct effects of cells and through interactions with NF-kB and activation of HIV-1 replication (Greenspan &

The activation of phagocytes induced by HIV-1 is associated with oxidative stress, not only because ROS are released but also the fact that activated phagocytes may release prooxidant cytokines, such as TNF- and IL-1, which promote iron uptake by the monocyte macrophage system. TNF- is synthesized in infected host cells, produces pro-oxidant effects in mitochondria, and inhibits mitochondrial respiration at Site II, the site of superoxide production (Schulze-Osthoff et al., 1992). Other cytokine that is involved in the oxidative stress is the IL-1. Activated monocytes produce IL-1 that stimulates neutrophils to release lysosomal proteins, including lactoferrin. This protein rapidly binds iron and this complex accumulates in the monocyte macrophage system. If the accumulated iron exceeds cellular iron-binding capacity, unbound pro-oxidant iron could interact with the superoxide

Oxidative stress biomarkers (pro-oxidants and antioxidants) have been investigated in HIV-1 patients serum samples; however, previous studies show inconsistent findings regarding MDA levels in these patients. One study showed significantly elevated serum MDA concentration in HIV-1 infected patients, where HIV-1 symptomatic presented higher levels than asymptomatic patients, suggesting that the infection results in oxidative stress of the host lipids (Sönnerborg et al., 1988; Jordão Júnior et al. 1998; Suresh et al., 2009). The oxidative stress was evaluated by the LPO and GSH plasma levels in 150 HIV-1 infected individuals and in 30 healthy controls, and the results showed that the mean LPO plasma levels were significantly higher in HIV-1 infected individuals as compared to healthy controls, and the mean GSH level in HIV-1 infected individuals was significantly lower compared to healthy controls. In addition, there was a significant positive correlation between absolute CD4+ T cells and GSH levels. However, there was no significant difference in the levels of LPO and GSH among HIV-1 infected individuals receiving antiretroviral

Jordão Júnior et al. (1998) evaluated 28 serologically positive HIV-1 patients, 16 patients with AIDS (with < 200/mm3 CD4+T lymphocytes) and 12 HIV-1 infected and asymptomatic patients (with 200-500/mm3 CD4+ T lymphocytes). The control group consisted of 11 healthy individuals. All individuals showed normal plasma vitamin A levels. However, urinary excretion of vitamin A and MDA was higher in AIDS patients than in HIV-1 asymptomatic patients and considerably higher than in the control subjects. Therefore, the severe oxidative stress that occurs in the HIV-1 seropositive patients in comparison with seronegative individuals can exert a role in the progression of disease (Suresh et al., 2009)

**4. Oxidative stress and cardiovascular diseases associated with HIV-1** 

Endothelium dysfunction is an initial step in the development of cardiovascular diseases, especially atherosclerosis, and is associated with an increase in oxidative stress. HIV-1 infection is associated with increased ROS production and chronic oxidative stress,

via Fenton's reaction and produces hydroxyl radicals (Halliwell, 1987).

therapy (ART) and those without ART (Wanchu et al., 2009).

**infection** 

supplying the amino acid cysteine necessary for the synthesis of GSH (Pocernich et al., 2005).

Vitamin C represents the major water-soluble antioxidant in the human body. Ascorbate protects cell components from free radical damage by quenching water soluble radicals, scavenging lipid-peroxidation-derived radicals, or reducing tocopherol radical to tocopherol (Stehbens, 2004).

SOD is an endogenous antioxidant that catalyses de dismutation of the superoxide anion radical (Stambullian et al., 2007).

Vitamin E, a potent chain breaking lipid soluble antioxidant, reacts with lipid peroxyl radical eventually by terminating the peroxidation chain reaction and thereby by reducing oxidative damage. Vitamin E acts as an antioxidant on biomembranes and it is the principal lipid soluble chain-breaking antioxidant in mitochondria, microsomes, and lipoproteins.

Selenium is an essential nonmetal trace element that is necessary for normal immune function. Selenium also increases the GSH peroxidase activity and its deficiency diminishes cell-mediated immunity and depresses B-cell function (Stehbens, 2004).

#### **3. Evidences of oxidative stress in individuals infected with HIV-1**

The hallmark of HIV-1 infection is the cellular CD4+ T cell immunodeficiency; however, the real cause of the loss of these cells is unknown. The most widely accepted hypothesis is that HIV-1 primes the cell to apoptotic death. Different agents appear to trigger apoptosis in CD4+ T cells, including viral protein, inappropriate secretion of inflammatory cytokines by activated macrophages and toxins produced by opportunistic microorganisms. Since oxidative stress can also induce apoptosis, it can be hypothesized that it could participate in CD4+ T cell apoptosis observed in AIDS (Reppeto et al., 1996).

Evidence suggested that HIV-1 infected patients are under chronic oxidative stress. This effect is subsequent to depletion of endogenous antioxidant moieties and to an increased production of ROS. Observation of the multiple pathogenic interactions between ROS and the HIV-1 has drawn attention to the possibility that these types of the interaction may play a role in the pathogenesis of many other viruses as well. ROS has been suggested to be involved in many aspects of HIV-1 disease pathogenesis, including increase viral replication, inflammatory response, decrease of immune cell proliferation, loss of immune function, chronic weight loss, and increase sensitivity to drug toxicity. In addition, antiretroviral combination therapy increases protein oxidation as well as the level of oxidative stress already present in HIV-1 infection (Ngondi et al., 2006).

One aspect of the role of ROS in HIV-1 pathogenesis is the positive modulatory effect on the immune activation, important both in eradication of viral infection but also in immuneinduced cellular injury (Schwarz, 1996). HIV-1 infections causes a chronic inflammation as shown by high plasma levels of pro and inflammatory cytokines, chemokines and ROS in seropositive individuals (Israel & Gougerot-Pocidalo, 1997). Increased production of ROS such as superoxide anion, hydroxyl radical, and hydrogen peroxide may be related to an increased activation of polymorphonuclear leukocytes during HIV-1 infection or influenced by the pro-oxidant effect of pro-inflammatory cytokines produced by activated macrophages during the course of HIV-1 infection (Das et al., 1990)

In HIV-1 infected patients, the increased oxidative stress has been implicated in increased HIV-1 transcription through the activation of NF-kB. NF-kB is bound to kinase inhibitor nuclear factor-kB (IkB) in the cytoplasm in its active form, but various factors, such as TNF-

supplying the amino acid cysteine necessary for the synthesis of GSH (Pocernich et al.,

Vitamin C represents the major water-soluble antioxidant in the human body. Ascorbate protects cell components from free radical damage by quenching water soluble radicals, scavenging lipid-peroxidation-derived radicals, or reducing tocopherol radical to tocopherol

SOD is an endogenous antioxidant that catalyses de dismutation of the superoxide anion

Vitamin E, a potent chain breaking lipid soluble antioxidant, reacts with lipid peroxyl radical eventually by terminating the peroxidation chain reaction and thereby by reducing oxidative damage. Vitamin E acts as an antioxidant on biomembranes and it is the principal lipid soluble chain-breaking antioxidant in mitochondria, microsomes, and lipoproteins. Selenium is an essential nonmetal trace element that is necessary for normal immune function. Selenium also increases the GSH peroxidase activity and its deficiency diminishes

The hallmark of HIV-1 infection is the cellular CD4+ T cell immunodeficiency; however, the real cause of the loss of these cells is unknown. The most widely accepted hypothesis is that HIV-1 primes the cell to apoptotic death. Different agents appear to trigger apoptosis in CD4+ T cells, including viral protein, inappropriate secretion of inflammatory cytokines by activated macrophages and toxins produced by opportunistic microorganisms. Since oxidative stress can also induce apoptosis, it can be hypothesized that it could participate in

Evidence suggested that HIV-1 infected patients are under chronic oxidative stress. This effect is subsequent to depletion of endogenous antioxidant moieties and to an increased production of ROS. Observation of the multiple pathogenic interactions between ROS and the HIV-1 has drawn attention to the possibility that these types of the interaction may play a role in the pathogenesis of many other viruses as well. ROS has been suggested to be involved in many aspects of HIV-1 disease pathogenesis, including increase viral replication, inflammatory response, decrease of immune cell proliferation, loss of immune function, chronic weight loss, and increase sensitivity to drug toxicity. In addition, antiretroviral combination therapy increases protein oxidation as well as the level of

One aspect of the role of ROS in HIV-1 pathogenesis is the positive modulatory effect on the immune activation, important both in eradication of viral infection but also in immuneinduced cellular injury (Schwarz, 1996). HIV-1 infections causes a chronic inflammation as shown by high plasma levels of pro and inflammatory cytokines, chemokines and ROS in seropositive individuals (Israel & Gougerot-Pocidalo, 1997). Increased production of ROS such as superoxide anion, hydroxyl radical, and hydrogen peroxide may be related to an increased activation of polymorphonuclear leukocytes during HIV-1 infection or influenced by the pro-oxidant effect of pro-inflammatory cytokines produced by activated

In HIV-1 infected patients, the increased oxidative stress has been implicated in increased HIV-1 transcription through the activation of NF-kB. NF-kB is bound to kinase inhibitor nuclear factor-kB (IkB) in the cytoplasm in its active form, but various factors, such as TNF-

cell-mediated immunity and depresses B-cell function (Stehbens, 2004).

CD4+ T cell apoptosis observed in AIDS (Reppeto et al., 1996).

oxidative stress already present in HIV-1 infection (Ngondi et al., 2006).

macrophages during the course of HIV-1 infection (Das et al., 1990)

**3. Evidences of oxidative stress in individuals infected with HIV-1** 

2005).

(Stehbens, 2004).

radical (Stambullian et al., 2007).

 and ROS, can cause the release of NF-kB from factor IkB, and NF-kB translocates to the nucleus and binds to DNA. In this way, the NF-kB is available to bind in the nuclear DNA and to induce HIV-1 gene transcription (Schereck et al., 1991). Thus, oxidative stress may potentially be involved in the pathogenesis of HIV-1 infection through direct effects of cells and through interactions with NF-kB and activation of HIV-1 replication (Greenspan & Aruoma, 1994; Israel & Gougerot-Pocidalo, 1997).

The activation of phagocytes induced by HIV-1 is associated with oxidative stress, not only because ROS are released but also the fact that activated phagocytes may release prooxidant cytokines, such as TNF- and IL-1, which promote iron uptake by the monocyte macrophage system. TNF- is synthesized in infected host cells, produces pro-oxidant effects in mitochondria, and inhibits mitochondrial respiration at Site II, the site of superoxide production (Schulze-Osthoff et al., 1992). Other cytokine that is involved in the oxidative stress is the IL-1. Activated monocytes produce IL-1 that stimulates neutrophils to release lysosomal proteins, including lactoferrin. This protein rapidly binds iron and this complex accumulates in the monocyte macrophage system. If the accumulated iron exceeds cellular iron-binding capacity, unbound pro-oxidant iron could interact with the superoxide via Fenton's reaction and produces hydroxyl radicals (Halliwell, 1987).

Oxidative stress biomarkers (pro-oxidants and antioxidants) have been investigated in HIV-1 patients serum samples; however, previous studies show inconsistent findings regarding MDA levels in these patients. One study showed significantly elevated serum MDA concentration in HIV-1 infected patients, where HIV-1 symptomatic presented higher levels than asymptomatic patients, suggesting that the infection results in oxidative stress of the host lipids (Sönnerborg et al., 1988; Jordão Júnior et al. 1998; Suresh et al., 2009). The oxidative stress was evaluated by the LPO and GSH plasma levels in 150 HIV-1 infected individuals and in 30 healthy controls, and the results showed that the mean LPO plasma levels were significantly higher in HIV-1 infected individuals as compared to healthy controls, and the mean GSH level in HIV-1 infected individuals was significantly lower compared to healthy controls. In addition, there was a significant positive correlation between absolute CD4+ T cells and GSH levels. However, there was no significant difference in the levels of LPO and GSH among HIV-1 infected individuals receiving antiretroviral therapy (ART) and those without ART (Wanchu et al., 2009).

Jordão Júnior et al. (1998) evaluated 28 serologically positive HIV-1 patients, 16 patients with AIDS (with < 200/mm3 CD4+T lymphocytes) and 12 HIV-1 infected and asymptomatic patients (with 200-500/mm3 CD4+ T lymphocytes). The control group consisted of 11 healthy individuals. All individuals showed normal plasma vitamin A levels. However, urinary excretion of vitamin A and MDA was higher in AIDS patients than in HIV-1 asymptomatic patients and considerably higher than in the control subjects. Therefore, the severe oxidative stress that occurs in the HIV-1 seropositive patients in comparison with seronegative individuals can exert a role in the progression of disease (Suresh et al., 2009)

## **4. Oxidative stress and cardiovascular diseases associated with HIV-1 infection**

Endothelium dysfunction is an initial step in the development of cardiovascular diseases, especially atherosclerosis, and is associated with an increase in oxidative stress. HIV-1 infection is associated with increased ROS production and chronic oxidative stress,

The Role of Human Immunodeficiency Virus Type 1 (HIV-1)

vessel wall.

endothelium dysfunction.

immune system.

Proteins and Antiretroviral Drug Therapy in HIV-1-Induced Oxidative Stress 49

The vascular endothelium is exposed continually to a number of viral stimuli in the bloodstream. These stimuli include: a) HIV-1 infected CD4+ T cells, monocytes, and macrophages; b) freely circulating HIV-1 viruses; c) HIV-1 proteins released upon host cell lysis; d) actively secreted proteins (Tat and gp120); and e) viral-induced pro-inflammatory cytokines. HIV-1-induced cytokines may also activate the endothelium, leading to enhanced production of ROS, and the release of chemoattractant at localized areas of vascular inflammation. HIV-1-infected individuals have higher plasma levels of hydroperoxides and MDA compared with uninfected individuals, indicating enhanced ROS-mediated LPO. HIV-1-induced ROS likely contribute to endothelium dysfunction through direct effects on the endothelium and/or indirectly through monocytes and macrophages contacting the

Elevated ROS in HIV-1 infection could play a role in various signaling pathways, among which are the mitogen-activated protein kinases (MAPKs). MAPKs are serine and threonine protein kinases, which have three major classes, including extracellular signal-regulated kinase 1 and 2 (ERK1/2) and BMK1, c-Jun N-terminal protein kinases (JNKs) and p38. ROS may mediate activation of MAPKs in a variety of cells, leading to changes in gene expression (Blenis, 1993), downregulation of eNOS, and alteration of other gene expression involved in the endothelium dysfunction. Taken together, these data indicate that oxidative stress activating MAPKs, may be one of the major mechanisms in HIV-1-induced

HIV-1 infected patients have low circulating levels of the antioxidant vitamin C, cysteine, and GSH, a situation that can lead to increased oxidative stress. Serum GSH levels and GSH peroxidase activity are decreased in HIV-1 patients, while the LPO product MDA, DNA fragmentation in lymphocytes, and total hydroperoxides are increased. These observations have important implications for therapeutic approaches. Clinical studies showed that selenium, and carotene supplementation increased serum GSH levels. Dual vitamin C and E supplementation reduced plasma LPO and oxidative stress in HIV-1 patients. Supplementation with -tocopherol or selenium also decreased plasma viral load and

These clinical findings suggest that vascular endothelial cells are exposed to ROS in the form of LPO products, pro-inflammatory cytokines, activated monocytes and phagocytes of the

Nearly 25 antiretroviral drugs have been licensed for the treatment of HIV-1 infected individuals and are divided mechanistically into five classes (reviewed by Estrada & Portilla, 2011): (1) nucleoside reverse transcriptase inhibitor (NRTI), including abacavir (ABC), didanosine (ddI), stavudine (d4T), lamivudine (3TC), tenofovir (TDF), zidovudine (AZT), and emtricitabine (FTC); (2) non-nucleoside reverse transcriptase inhibitor (NNRTI), including nevirapine (NVP), efavirenz (EFV), and etravirine (ETR); (3) protease inhibitor (PI) including atazanavir (ATV), indinavir (IDV), lopinavir (LPV), nelfinavir (NFV), ritonavir (RTV), saquinavir (SQV), darunavir (DRV), fosamprenavir (FPV), and amprenavir (APV); (4) fusion inhibitor, entry inhibitor (chemokine receptor CCR5 inhibitor) including enfuvirtide and maraviroc (MVC); and (5) integrase inhibitor, including raltegravir (RAL). The HAART for management of HIV-1 infection that includes an association of the two

improved T-cell numbers and viability (Suresh et al, 2009; Stehbens, 2004).

NRTIs plus NNRTI and/or PI has been effective to suppress HIV-1 replication.

**5. Oxidative stress associated with antiretroviral therapy** 

suggesting a role of ROS in HV-1-induced endothelial cells dysfunction. Evidence from experimental, observational, and clinical studies suggests that HIV-1 infection itself and the associated pro-inflammatory response can increase the risk of cardiovascular disease.

Multiple mechanisms, both specific and overlapping ways, are proposed to explain how HIV-1 proteins damage the endothelium, considering that viral genome contains nine main genes *(gag, pol, env, tat, rev, vpu, vpr, vif,* and *nef*) and encodes for approximately 15 mature HIV-1 proteins that may interact with any number of unique targets. Proteolytic cleavage of the Gag-Pol precursor protein yields the major structural components of the viral core including matrix p17, capsid p24, nucleocapsides p9 and p6, reverse transcriptase (RT), protease and integrase. Proteolytic cleavage of Env produces the important envelope glycoprotein (gp) gp120 and gp41. The remaining genes encode for the regulatory proteins Tat and Rev, and the accessory proteins Vpu, Vpr, Vif, and Nef (Greene, 1991).

The gp120, Tat, Vpu, and Nef proteins exert some important effects on endothelial cell homeostasis (reviewed by Kline & Sutliff, 2008). HIV-1 proteins can activate several inflammatory pathways in the vascular wall with cytokines release and expression of endothelial molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), and E-selectin (Seigneur et al., 1997; Greenwood et al., 1998; Wolf et al. 2002). The gp120 increases the expression of ICAM-1, but not VCAM-1 or Eselectin, in human coronary artery, lung, brain, umbilical vein, and dermal microvascular endothelial cells. The gp120 also induces the apoptosis in human coronary endothelial cells, the adhesion of monocytes and lymphocytes to the endothelium; gp120 increases the endothelium permeability through cytoskeletal rearrangement, downregulation of tight junction proteins, and increases ROS production. The gp120 negatively affects endothelium function through the production of potent vasoconstrictors. The nonstructural Tat protein contains 86-101 amino acids that are formed from two exons. The first exon contributes to the first 72 amino acids and acts as a transacting nuclear regulatory protein actively secreted by infected cells that is essential for viral replication.

Similar to gp120, Tat protein can promote the apoptosis, monocyte chemoattraction and adhesion, endothelium permeability, proliferation, angiogenesis, and an increase in the expression of matrix metalloproteinases and ROS. It has been demonstrated that viral Tat protein liberated by HIV-1 infected cells interferes with calcium homeostasis, activates caspases and induces mitochondrial generation and accumulation of ROS, all being important events in the apoptotic cascade of several cell types. When activated, peripheral blood T lymphocytes are induced to express Fas/APO-1/CD95 receptor that mediates apoptosis when binding to Fas ligand. CD4+ T cell subset depletion in HIV-1/AIDS patients is the most dramatic effect of apoptosis mediated by redox abnormalities and induction of Fas/APO-1/CD95 receptor expression (Westendorp et al., 1995; Kruman et al., 1998; Jaworowski & Crowe, 1999).

In patients with uncontrolled HIV-1 infection, vasculitis are also observed in small blood vessels, aneurysms in medium and large arteries, significantly decreased levels of highdensity lipoprotein (HDL) cholesterol, and elevated plasma levels of von Willebrand factor, plasminogen activator inhibitor-1 (PAI-1) antigen, and tissue-type plasminogen activator (tPA). Although HIV-1 is likely not vasculotropic, the virus affects endothelium homeostasis and function in important ways (Kline & Sutliff, 2008).

suggesting a role of ROS in HV-1-induced endothelial cells dysfunction. Evidence from experimental, observational, and clinical studies suggests that HIV-1 infection itself and the associated pro-inflammatory response can increase the risk of cardiovascular disease. Multiple mechanisms, both specific and overlapping ways, are proposed to explain how HIV-1 proteins damage the endothelium, considering that viral genome contains nine main genes *(gag, pol, env, tat, rev, vpu, vpr, vif,* and *nef*) and encodes for approximately 15 mature HIV-1 proteins that may interact with any number of unique targets. Proteolytic cleavage of the Gag-Pol precursor protein yields the major structural components of the viral core including matrix p17, capsid p24, nucleocapsides p9 and p6, reverse transcriptase (RT), protease and integrase. Proteolytic cleavage of Env produces the important envelope glycoprotein (gp) gp120 and gp41. The remaining genes encode for the regulatory proteins Tat and Rev, and the accessory proteins Vpu, Vpr, Vif, and Nef

The gp120, Tat, Vpu, and Nef proteins exert some important effects on endothelial cell homeostasis (reviewed by Kline & Sutliff, 2008). HIV-1 proteins can activate several inflammatory pathways in the vascular wall with cytokines release and expression of endothelial molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), and E-selectin (Seigneur et al., 1997; Greenwood et al., 1998; Wolf et al. 2002). The gp120 increases the expression of ICAM-1, but not VCAM-1 or Eselectin, in human coronary artery, lung, brain, umbilical vein, and dermal microvascular endothelial cells. The gp120 also induces the apoptosis in human coronary endothelial cells, the adhesion of monocytes and lymphocytes to the endothelium; gp120 increases the endothelium permeability through cytoskeletal rearrangement, downregulation of tight junction proteins, and increases ROS production. The gp120 negatively affects endothelium function through the production of potent vasoconstrictors. The nonstructural Tat protein contains 86-101 amino acids that are formed from two exons. The first exon contributes to the first 72 amino acids and acts as a transacting nuclear regulatory protein actively secreted

Similar to gp120, Tat protein can promote the apoptosis, monocyte chemoattraction and adhesion, endothelium permeability, proliferation, angiogenesis, and an increase in the expression of matrix metalloproteinases and ROS. It has been demonstrated that viral Tat protein liberated by HIV-1 infected cells interferes with calcium homeostasis, activates caspases and induces mitochondrial generation and accumulation of ROS, all being important events in the apoptotic cascade of several cell types. When activated, peripheral blood T lymphocytes are induced to express Fas/APO-1/CD95 receptor that mediates apoptosis when binding to Fas ligand. CD4+ T cell subset depletion in HIV-1/AIDS patients is the most dramatic effect of apoptosis mediated by redox abnormalities and induction of Fas/APO-1/CD95 receptor expression (Westendorp et al., 1995; Kruman et al., 1998;

In patients with uncontrolled HIV-1 infection, vasculitis are also observed in small blood vessels, aneurysms in medium and large arteries, significantly decreased levels of highdensity lipoprotein (HDL) cholesterol, and elevated plasma levels of von Willebrand factor, plasminogen activator inhibitor-1 (PAI-1) antigen, and tissue-type plasminogen activator (tPA). Although HIV-1 is likely not vasculotropic, the virus affects endothelium homeostasis

(Greene, 1991).

by infected cells that is essential for viral replication.

and function in important ways (Kline & Sutliff, 2008).

Jaworowski & Crowe, 1999).

The vascular endothelium is exposed continually to a number of viral stimuli in the bloodstream. These stimuli include: a) HIV-1 infected CD4+ T cells, monocytes, and macrophages; b) freely circulating HIV-1 viruses; c) HIV-1 proteins released upon host cell lysis; d) actively secreted proteins (Tat and gp120); and e) viral-induced pro-inflammatory cytokines. HIV-1-induced cytokines may also activate the endothelium, leading to enhanced production of ROS, and the release of chemoattractant at localized areas of vascular inflammation. HIV-1-infected individuals have higher plasma levels of hydroperoxides and MDA compared with uninfected individuals, indicating enhanced ROS-mediated LPO. HIV-1-induced ROS likely contribute to endothelium dysfunction through direct effects on the endothelium and/or indirectly through monocytes and macrophages contacting the vessel wall.

Elevated ROS in HIV-1 infection could play a role in various signaling pathways, among which are the mitogen-activated protein kinases (MAPKs). MAPKs are serine and threonine protein kinases, which have three major classes, including extracellular signal-regulated kinase 1 and 2 (ERK1/2) and BMK1, c-Jun N-terminal protein kinases (JNKs) and p38. ROS may mediate activation of MAPKs in a variety of cells, leading to changes in gene expression (Blenis, 1993), downregulation of eNOS, and alteration of other gene expression involved in the endothelium dysfunction. Taken together, these data indicate that oxidative stress activating MAPKs, may be one of the major mechanisms in HIV-1-induced endothelium dysfunction.

HIV-1 infected patients have low circulating levels of the antioxidant vitamin C, cysteine, and GSH, a situation that can lead to increased oxidative stress. Serum GSH levels and GSH peroxidase activity are decreased in HIV-1 patients, while the LPO product MDA, DNA fragmentation in lymphocytes, and total hydroperoxides are increased. These observations have important implications for therapeutic approaches. Clinical studies showed that selenium, and carotene supplementation increased serum GSH levels. Dual vitamin C and E supplementation reduced plasma LPO and oxidative stress in HIV-1 patients. Supplementation with -tocopherol or selenium also decreased plasma viral load and improved T-cell numbers and viability (Suresh et al, 2009; Stehbens, 2004).

These clinical findings suggest that vascular endothelial cells are exposed to ROS in the form of LPO products, pro-inflammatory cytokines, activated monocytes and phagocytes of the immune system.

## **5. Oxidative stress associated with antiretroviral therapy**

Nearly 25 antiretroviral drugs have been licensed for the treatment of HIV-1 infected individuals and are divided mechanistically into five classes (reviewed by Estrada & Portilla, 2011): (1) nucleoside reverse transcriptase inhibitor (NRTI), including abacavir (ABC), didanosine (ddI), stavudine (d4T), lamivudine (3TC), tenofovir (TDF), zidovudine (AZT), and emtricitabine (FTC); (2) non-nucleoside reverse transcriptase inhibitor (NNRTI), including nevirapine (NVP), efavirenz (EFV), and etravirine (ETR); (3) protease inhibitor (PI) including atazanavir (ATV), indinavir (IDV), lopinavir (LPV), nelfinavir (NFV), ritonavir (RTV), saquinavir (SQV), darunavir (DRV), fosamprenavir (FPV), and amprenavir (APV); (4) fusion inhibitor, entry inhibitor (chemokine receptor CCR5 inhibitor) including enfuvirtide and maraviroc (MVC); and (5) integrase inhibitor, including raltegravir (RAL). The HAART for management of HIV-1 infection that includes an association of the two NRTIs plus NNRTI and/or PI has been effective to suppress HIV-1 replication.

The Role of Human Immunodeficiency Virus Type 1 (HIV-1)

associated with a harmful effect in many systems.

levels (Mondal et al., 2004).

tumor necrosis factor alpha;

proteins and PIs.

Proteins and Antiretroviral Drug Therapy in HIV-1-Induced Oxidative Stress 51

evaluated, NFV, specifically, decreased mRNA and protein levels of the LDLR and LRP, which, in turn, decreased the functional activity of these two receptors. One study showed that exposure of IDV or NFV, combined with AZT and EFV, increased ICAM-1 gene expression and that concomitant exposure to TNF- further increased ICAM-1 gene expression, VCAM-1, and endothelial-leukocyte adhesion molecule cell surface protein

The Figure 1 shows the production of NO induced by HIV-1 and its viral proteins (mainly gp120 and Tat proteins), and by PIs in promoting beneficial and deleterious effects in the host cells. The NO is synthesized via MAPK signaling pathway when the macrophage is activated by pro-inflammatory and inflammatory cytokines as result of the HIV-1 infection. Despite the protective effect of NO in the host defense against this pathogen, NO has been

The Figure 2 summarizes some of the effects of ROS and RNS that are induced by HIV-1

Fig. 1. Mechanisms of nitric oxide (NO) production induced by human immunodeficiency virus type 1 (HIV-1) and the viral proteins (mainly gp120 and Tat proteins), and by protease inhibitors (PIs) in promoting beneficial and deleterious effects in the host cells. TNF-:

TNF-R: tumor necrosis factor alpha receptor; IL-1: interleukin 1; IL-1R: interleukin 1 receptor; IFN-: interferon gamma; IFN-R: interferon gamma receptor; IL-6: interleukin 6; IL-6R: interleukin 6 receptor; IL-10: interleukin 10; IL-4: interleukin 4; TGF-: transforming growth factor beta; IFN-/: interferon alpha and interferon beta; MAPK: mitogen-activated protein kinase; IkB: kinase inhibitor nuclear factor-kB; transcriptional nuclear factor kappa

In addition to HIV-1 proteins, the HAART has been related with endothelium dysfunction. Experimental evidence shows that NRTIs are associated with endothelial cell toxicity. NRTIs induce oxidative stress, particularly mitochondrial ROS and seem to play an important role in cell culture and animal models of endothelial cell toxicity. However, clinical evidence for NRTIs-induced vascular/endothelium toxicity is indirect and difficult to define because NRTIs are not prescribed as monotherapy, and cardiovascular effects are often attributed to other components of HAART, such as PIs.

NNRTIs show, in general, the best lipid profile of all anti –HIV-1 drugs because they are associated with an increase in HDL cholesterol and a significant reduction in cholesterol total/HDL ratio. NNRTIs have been associated with a lower risk of myocardial infarction (Worm et al., 2010) that could hypothetically be associated with this good lipid profile. As regard NVP, the mechanism of HDL elevation may be an increase in the production of apolipoprotein-A1 (Franssen et al., 2009).

Among the PIs, lopinavir/ritonavir (LVP/r), DRV/r and ATV alone or with RTV (ATV/r) are the most extensively used PIs at present. PIs-associated dyslipidemia is a frequent classrelated event and can limit their use especially in patients with preexisting increase cardiovascular risk. A meta-analysis of major clinical trials performed in 2009 (Hill et al., 2009) showed that patients randomized to LPV/r or FPV/r presented greater elevations of total cholesterol and triglycerides than those assigned to SQV/r, ATV/r, or DRV/r, without differences in low density lipoprotein cholesterol (LDL) or HDL.

The integrase inhibitor RAL is the first drug in this class and shows a remarkable lack of relevant adverse effects (Emery et al., 2010) and patients treated with RAL presented a significantly low frequency of dyslipidemia (Martinez et al., 2010).

Trials with HIV-1 patients treated with chemokine receptor-5 antagonist MVC have shown that it has a very favorable safety profile. MCV was associated with non-significant changes in total cholesterol, LDL, HDL and triglycerides (Cooper et al., 2010).

The LDL receptor (LDLR) plays a critical role in the regulation of plasma LDL levels (Brown & Goldstein, 1986). By controlling LDL catabolism, the number of hepatic LDLR directly governs the plasma LDL concentrations. The expression of LDLR is under metabolic, hormonal, and genetic control. Growth hormone (GH), insulin, estrogen, and dehydroepiandrosterone (DHEA) may stimulate LDLR expression and reduce plasma LDL cholesterol levels (Wade et al., 1989; Pascale et al., 1995; Rudling et al., 1996). As important hormonal modifications occur in HIV-1 infected patients with lipodystrophy, particularly insulin and DHEA changes, the LDLR expression was evaluated in HIV-1 infected patients with or without lipodystrophy. The results revealed that HIV-1-lipodystrophy is associated with a low expression of LDLR and this decreased expression seems independent of DHEA or insulin secretion (Petit et al., 2002). These authors suggested that the decreased expression of the LDLR may be explained by a direct effect of the PIs (Rayes et al., 1996). Other hypothesis is that PIs lead to dyslipidemia by inhibition of LDLR-related protein (LRP), which has homology to the catalytic site of HIV-1 protease, to which all PIs bind (Carr et al., 1998).

The HIV-1 infected patients with lipodystrophy have also an impaired metabolism of DHEA and insulin, all known to regulate LDLR (Meyer et al., 1998; Walli et al., 1998; Christeff et al., 1999; Behrens et al., 1999;). In addition, HIV-1 PIs also can modulate the function of certain LDLR family members. Tran et al., (2003) demonstrated that among six different HIV-1 PIs

In addition to HIV-1 proteins, the HAART has been related with endothelium dysfunction. Experimental evidence shows that NRTIs are associated with endothelial cell toxicity. NRTIs induce oxidative stress, particularly mitochondrial ROS and seem to play an important role in cell culture and animal models of endothelial cell toxicity. However, clinical evidence for NRTIs-induced vascular/endothelium toxicity is indirect and difficult to define because NRTIs are not prescribed as monotherapy, and cardiovascular effects are often attributed to

NNRTIs show, in general, the best lipid profile of all anti –HIV-1 drugs because they are associated with an increase in HDL cholesterol and a significant reduction in cholesterol total/HDL ratio. NNRTIs have been associated with a lower risk of myocardial infarction (Worm et al., 2010) that could hypothetically be associated with this good lipid profile. As regard NVP, the mechanism of HDL elevation may be an increase in the production of

Among the PIs, lopinavir/ritonavir (LVP/r), DRV/r and ATV alone or with RTV (ATV/r) are the most extensively used PIs at present. PIs-associated dyslipidemia is a frequent classrelated event and can limit their use especially in patients with preexisting increase cardiovascular risk. A meta-analysis of major clinical trials performed in 2009 (Hill et al., 2009) showed that patients randomized to LPV/r or FPV/r presented greater elevations of total cholesterol and triglycerides than those assigned to SQV/r, ATV/r, or DRV/r, without

The integrase inhibitor RAL is the first drug in this class and shows a remarkable lack of relevant adverse effects (Emery et al., 2010) and patients treated with RAL presented a

Trials with HIV-1 patients treated with chemokine receptor-5 antagonist MVC have shown that it has a very favorable safety profile. MCV was associated with non-significant changes

The LDL receptor (LDLR) plays a critical role in the regulation of plasma LDL levels (Brown & Goldstein, 1986). By controlling LDL catabolism, the number of hepatic LDLR directly governs the plasma LDL concentrations. The expression of LDLR is under metabolic, hormonal, and genetic control. Growth hormone (GH), insulin, estrogen, and dehydroepiandrosterone (DHEA) may stimulate LDLR expression and reduce plasma LDL cholesterol levels (Wade et al., 1989; Pascale et al., 1995; Rudling et al., 1996). As important hormonal modifications occur in HIV-1 infected patients with lipodystrophy, particularly insulin and DHEA changes, the LDLR expression was evaluated in HIV-1 infected patients with or without lipodystrophy. The results revealed that HIV-1-lipodystrophy is associated with a low expression of LDLR and this decreased expression seems independent of DHEA or insulin secretion (Petit et al., 2002). These authors suggested that the decreased expression of the LDLR may be explained by a direct effect of the PIs (Rayes et al., 1996). Other hypothesis is that PIs lead to dyslipidemia by inhibition of LDLR-related protein (LRP), which has homology to the catalytic site of HIV-1 protease, to which all PIs bind (Carr et al.,

The HIV-1 infected patients with lipodystrophy have also an impaired metabolism of DHEA and insulin, all known to regulate LDLR (Meyer et al., 1998; Walli et al., 1998; Christeff et al., 1999; Behrens et al., 1999;). In addition, HIV-1 PIs also can modulate the function of certain LDLR family members. Tran et al., (2003) demonstrated that among six different HIV-1 PIs

other components of HAART, such as PIs.

apolipoprotein-A1 (Franssen et al., 2009).

1998).

differences in low density lipoprotein cholesterol (LDL) or HDL.

significantly low frequency of dyslipidemia (Martinez et al., 2010).

in total cholesterol, LDL, HDL and triglycerides (Cooper et al., 2010).

evaluated, NFV, specifically, decreased mRNA and protein levels of the LDLR and LRP, which, in turn, decreased the functional activity of these two receptors. One study showed that exposure of IDV or NFV, combined with AZT and EFV, increased ICAM-1 gene expression and that concomitant exposure to TNF- further increased ICAM-1 gene expression, VCAM-1, and endothelial-leukocyte adhesion molecule cell surface protein levels (Mondal et al., 2004).

The Figure 1 shows the production of NO induced by HIV-1 and its viral proteins (mainly gp120 and Tat proteins), and by PIs in promoting beneficial and deleterious effects in the host cells. The NO is synthesized via MAPK signaling pathway when the macrophage is activated by pro-inflammatory and inflammatory cytokines as result of the HIV-1 infection. Despite the protective effect of NO in the host defense against this pathogen, NO has been associated with a harmful effect in many systems.

The Figure 2 summarizes some of the effects of ROS and RNS that are induced by HIV-1 proteins and PIs.

Fig. 1. Mechanisms of nitric oxide (NO) production induced by human immunodeficiency virus type 1 (HIV-1) and the viral proteins (mainly gp120 and Tat proteins), and by protease inhibitors (PIs) in promoting beneficial and deleterious effects in the host cells. TNF-: tumor necrosis factor alpha;

TNF-R: tumor necrosis factor alpha receptor; IL-1: interleukin 1; IL-1R: interleukin 1 receptor; IFN-: interferon gamma; IFN-R: interferon gamma receptor; IL-6: interleukin 6; IL-6R: interleukin 6 receptor; IL-10: interleukin 10; IL-4: interleukin 4; TGF-: transforming growth factor beta; IFN-/: interferon alpha and interferon beta; MAPK: mitogen-activated protein kinase; IkB: kinase inhibitor nuclear factor-kB; transcriptional nuclear factor kappa

The Role of Human Immunodeficiency Virus Type 1 (HIV-1)

the treatment of these disorders (Xu et al., 2004) .

manner of the HIV-1 induced ROS.

stress induction.

cells (Fu et al., 2005).

Proteins and Antiretroviral Drug Therapy in HIV-1-Induced Oxidative Stress 53

reduction in endothelium-dependent vasorelaxation of porcine coronary arteries (Conklin et al., 2004). The expression of eNOS was significantly decreased in porcine coronary arteries treated by RTV, SQV, and APV. In parallel, RTV also caused a significant reduction of eNOS messenger RNA (mRNA) and protein levels in cultured human coronary artery endothelial

PIs produces serious mitochondrial disturbances as evidenced by reduced cellular respiration and ATP production, decrease mitochondrial membrane potential, increase mitochondrial production of ROS, and enhanced mitochondrial DNA (mtDNA) damage. PIs also increase endothelial cell permeability and leukocyte adhesion in cell culture models. PIs contribute to cardiovascular risk by dysregulating fat cell homeostasis that may explain the high incidence of lipodystrophy and hyperlipidemia in HIV-1 patients. PIs prevent the differentiation of preadipocytes by decreasing matrix metalloproteinase expression, inhibiting adiponectin secretion, and inhibiting triglyceride and very low-density lipoprotein (VLDL) cholesterol clearance and catabolism (Wang et al., 2007). Evidences point to adipocytes as a complex and active endocrine tissue whose secretory products, including adiponectin, play an important role in the regulation of human metabolic alterations and vascular biology (Hamdy, 2005). Adiponectin accounts for approximately 0.01% of total plasma protein and has been shown to be related to lipodystrophy, metabolic alterations, and HIV-1 PIs use. Unlike other adipocyte products, adiponectin correlates with decreased free fatty acid blood concentrations and reduced body mass index. Adiponectin provides protection from vascular diseases by inhibiting local inflammatory signals, preventing preatherogenic plaque formation, and impeding arterial wall thickening (Schondorf et al., 2005). However, HIV-1 PIs such as RTV selectively decreased expression of adiponectin (Kim et al., 2006) suggesting that hypoadiponectinemia is partially responsible for the metabolic disorders induced by HIV-1 PIs, and adiponectin or its agonists might be used for

HIV-1 PIs may also activate different types of MAPKs in different cell types or different culture conditions (Wang et al., 2007), leading to changes in gene expression in the same

Some studies have showed high oxidative stress among the effects of HAART. Mandas et al. (2009) assessed serum oxidant and antioxidant levels in HIV-1 infected population treated with HAART and compared them with those untreated HIV-1 seropositive and HIV-1 seronegative individuals. Serum oxidant levels were significantly higher in the HIV-1 treated group as compared to untreated and control groups. In addition, a decrease of serum total antioxidant status was observed in HIV-1 treated individuals. An important result obtained is that patients who rigorously followed antiretroviral therapy have significantly higher oxidative status than those who have poor HAART adherence. These results indicate that HAART may affect oxidative stress in HIV-1 infected patients and also suggested that antiretroviral therapy may exert a synergic effect with HIV-1 in the oxidative

Another study (Gil et al., 2010) evaluated the effect of two HAART combinations on redox indicators and on progression markers of disease. A cohort of 84 healthy and 84 HIV-1 seropositive subjects was followed for six months. Fifty-six HIV-1 seropositive subjects were distributed in group I (AZT, 3TC, IND) and group II (d4T, 3TC, NEV) according to drug combination. Biomarkers of oxidative stress were evaluated including peroxidation

beta (NF-kB); iNOS: inducible nitric oxide synthase; mRNA: messenger RNA; NO: nitric oxide.

Fig. 2. Beneficial and deleterious effects of the endogenous reactive oxygen species (ROS) and reactive nitrogen species (RNS) that are induced by both human immunodeficiency virus type 1 (HIV-1) proteins and the antiretroviral therapy with protease inhibitors (PIs). ROS and RNS that are accumulated by the imbalance of oxidants and antioxidants molecules exert effects on DNA, lipids, proteins, signaling pathways, immune system cells, neuronal tissue, and endothelial functions. ERK1/2: extracellular signal-regulated kinase 1 and 2; MAPKs: mitogen-activated protein kinases; transcriptional nuclear factor kappa beta (NF-kB).

## **6. Molecular mechanisms of HIV-1 PI-induced endothelium dysfunction**

It is well-known that the endothelium acts as the first-line defense mechanism against the development of vascular injury. It exerts is protective action through modulation of vascular tone, vascular structure, and the interaction of blood components. Endothelial dysfunction may contribute to the systemic manifestations of many diseases, including atherosclerosis. Several reviews have been focused on metabolic disorders such as systemic insulin resistance, dyslipidemia, and peripheral lipodystrophy associated with endothelial dysfunction (Shankar & Dube, 2003; Koutkin & Grinspoon, 2004).

The molecular mechanisms of PIs toxicity in endothelial cells have been described in greatly detail. The effect of PIs on endothelium-depending vasorelaxation was first suggested by the significant reduction of flow-mediated vasodilatation of the brachial artery in HIV-1 infected patients receiving PIs as compared with the patients without PIs treatment (Stein et al., 2001). Other study showed that RTV, APV, or SQV individually caused a significant

beta (NF-kB); iNOS: inducible nitric oxide synthase; mRNA: messenger RNA; NO: nitric

Fig. 2. Beneficial and deleterious effects of the endogenous reactive oxygen species (ROS) and reactive nitrogen species (RNS) that are induced by both human immunodeficiency virus type 1 (HIV-1) proteins and the antiretroviral therapy with protease inhibitors (PIs).

molecules exert effects on DNA, lipids, proteins, signaling pathways, immune system cells, neuronal tissue, and endothelial functions. ERK1/2: extracellular signal-regulated kinase 1 and 2; MAPKs: mitogen-activated protein kinases; transcriptional nuclear factor kappa beta

It is well-known that the endothelium acts as the first-line defense mechanism against the development of vascular injury. It exerts is protective action through modulation of vascular tone, vascular structure, and the interaction of blood components. Endothelial dysfunction may contribute to the systemic manifestations of many diseases, including atherosclerosis. Several reviews have been focused on metabolic disorders such as systemic insulin resistance, dyslipidemia, and peripheral lipodystrophy associated with endothelial

The molecular mechanisms of PIs toxicity in endothelial cells have been described in greatly detail. The effect of PIs on endothelium-depending vasorelaxation was first suggested by the significant reduction of flow-mediated vasodilatation of the brachial artery in HIV-1 infected patients receiving PIs as compared with the patients without PIs treatment (Stein et al., 2001). Other study showed that RTV, APV, or SQV individually caused a significant

ROS and RNS that are accumulated by the imbalance of oxidants and antioxidants

**6. Molecular mechanisms of HIV-1 PI-induced endothelium dysfunction** 

dysfunction (Shankar & Dube, 2003; Koutkin & Grinspoon, 2004).

oxide.

(NF-kB).

reduction in endothelium-dependent vasorelaxation of porcine coronary arteries (Conklin et al., 2004). The expression of eNOS was significantly decreased in porcine coronary arteries treated by RTV, SQV, and APV. In parallel, RTV also caused a significant reduction of eNOS messenger RNA (mRNA) and protein levels in cultured human coronary artery endothelial cells (Fu et al., 2005).

PIs produces serious mitochondrial disturbances as evidenced by reduced cellular respiration and ATP production, decrease mitochondrial membrane potential, increase mitochondrial production of ROS, and enhanced mitochondrial DNA (mtDNA) damage. PIs also increase endothelial cell permeability and leukocyte adhesion in cell culture models. PIs contribute to cardiovascular risk by dysregulating fat cell homeostasis that may explain the high incidence of lipodystrophy and hyperlipidemia in HIV-1 patients. PIs prevent the differentiation of preadipocytes by decreasing matrix metalloproteinase expression, inhibiting adiponectin secretion, and inhibiting triglyceride and very low-density lipoprotein (VLDL) cholesterol clearance and catabolism (Wang et al., 2007). Evidences point to adipocytes as a complex and active endocrine tissue whose secretory products, including adiponectin, play an important role in the regulation of human metabolic alterations and vascular biology (Hamdy, 2005). Adiponectin accounts for approximately 0.01% of total plasma protein and has been shown to be related to lipodystrophy, metabolic alterations, and HIV-1 PIs use. Unlike other adipocyte products, adiponectin correlates with decreased free fatty acid blood concentrations and reduced body mass index. Adiponectin provides protection from vascular diseases by inhibiting local inflammatory signals, preventing preatherogenic plaque formation, and impeding arterial wall thickening (Schondorf et al., 2005). However, HIV-1 PIs such as RTV selectively decreased expression of adiponectin (Kim et al., 2006) suggesting that hypoadiponectinemia is partially responsible for the metabolic disorders induced by HIV-1 PIs, and adiponectin or its agonists might be used for the treatment of these disorders (Xu et al., 2004) .

HIV-1 PIs may also activate different types of MAPKs in different cell types or different culture conditions (Wang et al., 2007), leading to changes in gene expression in the same manner of the HIV-1 induced ROS.

Some studies have showed high oxidative stress among the effects of HAART. Mandas et al. (2009) assessed serum oxidant and antioxidant levels in HIV-1 infected population treated with HAART and compared them with those untreated HIV-1 seropositive and HIV-1 seronegative individuals. Serum oxidant levels were significantly higher in the HIV-1 treated group as compared to untreated and control groups. In addition, a decrease of serum total antioxidant status was observed in HIV-1 treated individuals. An important result obtained is that patients who rigorously followed antiretroviral therapy have significantly higher oxidative status than those who have poor HAART adherence. These results indicate that HAART may affect oxidative stress in HIV-1 infected patients and also suggested that antiretroviral therapy may exert a synergic effect with HIV-1 in the oxidative stress induction.

Another study (Gil et al., 2010) evaluated the effect of two HAART combinations on redox indicators and on progression markers of disease. A cohort of 84 healthy and 84 HIV-1 seropositive subjects was followed for six months. Fifty-six HIV-1 seropositive subjects were distributed in group I (AZT, 3TC, IND) and group II (d4T, 3TC, NEV) according to drug combination. Biomarkers of oxidative stress were evaluated including peroxidation

The Role of Human Immunodeficiency Virus Type 1 (HIV-1)

cytokine, endotoxin, and soluble antigens in the CSN.

dentate gyrus, and the CA3 region of the hippocampus.

administration of GSH to HV-1-infected patients decreases mortality.

be decreased during HIV-1 infection (Yano et al., 1998).

Puttfarcken, 1993).

Proteins and Antiretroviral Drug Therapy in HIV-1-Induced Oxidative Stress 55

is produced by myeloid-monocytic cell lines following HIV-1 infection and the production of this molecule results in subsequent changes in the antioxidant status of these cells because SOD, a superoxide anion scavenger, is generated. Neurofilament, a protein that provides structural stability to neurons, is one of the target proteins of peroxynitrite and the resulting nitration results in disrupted neurofilament assembly and thus neuronal damage (Coyle &

Neurotoxic levels of ROS and RNS are especially produced by the macrophages recruited to the CNS as well as by astrocytes and glial cells activated following different stimuli such as

*In vitro* studies show that gp120 and Tat HIV-1 proteins can be directly toxic to human endothelial cells, compromises BBB integrity by reducing tight junction (occludin) protein expression and enhances monocytes migration across BBB. Protein oxidation was increased in the CSF of HIV-1 patients with mild and severe dementia compared to non-dement HIV-1 patients. Nitrated tyrosine residues, evidence of peroxynitrite reaction with proteins, are increased in brain of HIVD patients. Activation of cytokine receptors and oxidative stress can induce the production of ceramide from membrane sphingomyelin, and recent findings suggest that ceramide is an important mediator of apoptosis. The HIV-1 Tat protein can also induce increase of ceramide and sphingomyelin in culture neurons. Tat can be transported efficiently across the intact BBB. In HIV-1 infected astrocytes, the regulatory gene *tat* is overexpressed, and mRNA levels for Tat protein are elevated in brain extracts from individuals with HIVD. The Tat sequences from brains of patients with HAD are mutated with glutamate substitutions in the second exon, which may decrease its ability to be taken up by cells, thus increasing its extracellular concentrations and possibly neurotoxicity effects in the cell. Brain regions particularly susceptible to Tat toxicity are striatum, hippocampal

Tat has been hypothesized by many studies as a potential contributor to HIVD by many mechanisms (reviewed by Pocernich et al., 2005). Tat protein released by astrocytes produces trimming of neuritis, mitochondrial dysfunction, and cell death in neurons. Tatinduced neurotoxicity is though to be mediated through excitotoxic mechanisms involving calcium. Tat can also induce markers of oxidative stress such as protein and LPO in synaptosomal membranes and neuronal cell cultures. To neutralize the oxidative stress, the GSH protects neurons against ROS directly and indirectly, and binds LPO products. GSH is the major cellular thiol participating in the maintenance of cellular redox status of the neuron and neuronal mitochondria. The biosynthesis of GSH may be compromised by Tat protein. It was hypothesized that the chronic inflammation of CSN by HIV-1, the activation of microglia, and increased lipid and protein oxidation, all observed in HIV-1 infected patients, can lead to the decrease of GSH serum levels and potentially HIVD. Low serum level of GSH is associated with poor survival in HIV-1-infected patients, while

The production of superoxide anions by HIV-1 infected cells is counteracted by SOD, which, in turn, generates hydrogen peroxide (H2O2). Under basal conditions this is scavenged by catalase. To date, clear evidence exists that catalase activity is modified in brain tissue of AIDS patients. However, it has recently been reported that catalase is diminished in CD8+ T lymphocytes from HIV-1 positive individuals, suggesting the H2O2 scavenger activity might

potential (PP), MDA, total hydroperoxides (HPO), AOPP and, percent of DNA fragmentation (% FDNA). There were also evaluated biomarkers of antioxidant status, including catalase, SOD and GSH at baseline and six months after HAART started. In this study, the concentration of antioxidants was low at baseline, and LPO index and DNA fragmentation were increased. After HAART had been started, catalase values for both groups receiving treatment showed no significant difference. For group II, all other parameters of oxidative stress were significantly higher than those for group I and the HIV-1 positive not treated, except for GSH values in group II which was lower than group I values. These data suggest poor prognostic for group II. The findings suggest that increased oxidative stress occurs additionally to persistent redox imbalance associated to HIV-1 infection during apparently successfully HAART.

HAART may increase chemically reactive species in circulation, possibly by producing more oxidized metabolites derived from the interaction between ROS and infected-cell biomolecules. This is supported by several biochemical mechanisms, such as mitochondrial interference, following treatment with HAART-NRTI and activation of the P450 hepatic system by HAART, when comprising PIs (La Asuncion et al., 1998; Kumar et al., 1999; Hulgan et al., 2003; Lewis, 2003; Cossarizza & Moyle, 2004; Day & Lewis, 2004).

## **7. Oxidative stress in HIV-1 infection associated with neurological disorders**

The mechanisms by which HIV-1 first enters in the central nervous system (CNS) remain obscure. However, loss of blood-brain-barrier (BBB) integrity may be an important part of some of the tissue damage that accompanies HIV-1 infection of the brain, and may facilitate viral entry into the CNS. The active replication of HIV-1 into macrophages and microglia represents a reservoir for the virus and an important step for the neuropathogenesis of HIV-1 infection. This process leading to the production of inflammatory products and, in turn, to the production of an excess formation of free radical species, is involved in the subsequent increased permeability of the BBB and has been suggested to play a key role in the neuropathogenesis of HIV-1 infection. The combination of BBB damage and elevated plasma viral load is associated with neurocognitive impairment and an increased risk for development of HIV-1-induced dementia (HIVD). In addition, oxidative stress has been demonstrated in the brain and cerebrospinal fluid (CSF) from HIV-1-infected individuals and it is proposed to be a key event in the pathophysiology of HIVD.

One of the neurotoxins that is suggested to be involved in neuronal damage is NO. NO is a nitrogen free radical generates in many tissues, including the CNS, via bioconversion of Larginine into L-citrulline by nNOS (Lamas et al., 1998). It can be released constitutively by neurons in response to many neurochemical stimuli, including excitatory neurotransmission and changes of Ca2+ influx (Moncada et al., 1991). NO release has been induced *in vitro* from glial cells following the addition of inflammatory cytokines and soluble antigens such as the HIV-1 coating gp120 glycoprotein (Dawson et al., 1993; Mollace & Nistico, 1995). Proinflammatory cytokines including IL-1, TNF-, and IFN- which are released in HIV-1 infected brain tissue have been shown to upregulate the iNOS. To modulate this response, the NO formation is downregulated by the cytokines tissue grown factor beta (TGF-), and IFN alpha/beta (IFN-/), according to Hua et al., (1998).

Evidences show that although the direct neurotoxic effects of NO are modest, they are greatly enhanced by reacting with superoxide anion to form peroxynitrite. Superoxide anion

potential (PP), MDA, total hydroperoxides (HPO), AOPP and, percent of DNA fragmentation (% FDNA). There were also evaluated biomarkers of antioxidant status, including catalase, SOD and GSH at baseline and six months after HAART started. In this study, the concentration of antioxidants was low at baseline, and LPO index and DNA fragmentation were increased. After HAART had been started, catalase values for both groups receiving treatment showed no significant difference. For group II, all other parameters of oxidative stress were significantly higher than those for group I and the HIV-1 positive not treated, except for GSH values in group II which was lower than group I values. These data suggest poor prognostic for group II. The findings suggest that increased oxidative stress occurs additionally to persistent redox imbalance associated to HIV-1

HAART may increase chemically reactive species in circulation, possibly by producing more oxidized metabolites derived from the interaction between ROS and infected-cell biomolecules. This is supported by several biochemical mechanisms, such as mitochondrial interference, following treatment with HAART-NRTI and activation of the P450 hepatic system by HAART, when comprising PIs (La Asuncion et al., 1998; Kumar et al., 1999;

**7. Oxidative stress in HIV-1 infection associated with neurological disorders**  The mechanisms by which HIV-1 first enters in the central nervous system (CNS) remain obscure. However, loss of blood-brain-barrier (BBB) integrity may be an important part of some of the tissue damage that accompanies HIV-1 infection of the brain, and may facilitate viral entry into the CNS. The active replication of HIV-1 into macrophages and microglia represents a reservoir for the virus and an important step for the neuropathogenesis of HIV-1 infection. This process leading to the production of inflammatory products and, in turn, to the production of an excess formation of free radical species, is involved in the subsequent increased permeability of the BBB and has been suggested to play a key role in the neuropathogenesis of HIV-1 infection. The combination of BBB damage and elevated plasma viral load is associated with neurocognitive impairment and an increased risk for development of HIV-1-induced dementia (HIVD). In addition, oxidative stress has been demonstrated in the brain and cerebrospinal fluid (CSF) from HIV-1-infected individuals

One of the neurotoxins that is suggested to be involved in neuronal damage is NO. NO is a nitrogen free radical generates in many tissues, including the CNS, via bioconversion of Larginine into L-citrulline by nNOS (Lamas et al., 1998). It can be released constitutively by neurons in response to many neurochemical stimuli, including excitatory neurotransmission and changes of Ca2+ influx (Moncada et al., 1991). NO release has been induced *in vitro* from glial cells following the addition of inflammatory cytokines and soluble antigens such as the HIV-1 coating gp120 glycoprotein (Dawson et al., 1993; Mollace & Nistico, 1995). Proinflammatory cytokines including IL-1, TNF-, and IFN- which are released in HIV-1 infected brain tissue have been shown to upregulate the iNOS. To modulate this response, the NO formation is downregulated by the cytokines tissue grown factor beta (TGF-), and

Evidences show that although the direct neurotoxic effects of NO are modest, they are greatly enhanced by reacting with superoxide anion to form peroxynitrite. Superoxide anion

Hulgan et al., 2003; Lewis, 2003; Cossarizza & Moyle, 2004; Day & Lewis, 2004).

and it is proposed to be a key event in the pathophysiology of HIVD.

IFN alpha/beta (IFN-/), according to Hua et al., (1998).

infection during apparently successfully HAART.

is produced by myeloid-monocytic cell lines following HIV-1 infection and the production of this molecule results in subsequent changes in the antioxidant status of these cells because SOD, a superoxide anion scavenger, is generated. Neurofilament, a protein that provides structural stability to neurons, is one of the target proteins of peroxynitrite and the resulting nitration results in disrupted neurofilament assembly and thus neuronal damage (Coyle & Puttfarcken, 1993).

Neurotoxic levels of ROS and RNS are especially produced by the macrophages recruited to the CNS as well as by astrocytes and glial cells activated following different stimuli such as cytokine, endotoxin, and soluble antigens in the CSN.

*In vitro* studies show that gp120 and Tat HIV-1 proteins can be directly toxic to human endothelial cells, compromises BBB integrity by reducing tight junction (occludin) protein expression and enhances monocytes migration across BBB. Protein oxidation was increased in the CSF of HIV-1 patients with mild and severe dementia compared to non-dement HIV-1 patients. Nitrated tyrosine residues, evidence of peroxynitrite reaction with proteins, are increased in brain of HIVD patients. Activation of cytokine receptors and oxidative stress can induce the production of ceramide from membrane sphingomyelin, and recent findings suggest that ceramide is an important mediator of apoptosis. The HIV-1 Tat protein can also induce increase of ceramide and sphingomyelin in culture neurons. Tat can be transported efficiently across the intact BBB. In HIV-1 infected astrocytes, the regulatory gene *tat* is overexpressed, and mRNA levels for Tat protein are elevated in brain extracts from individuals with HIVD. The Tat sequences from brains of patients with HAD are mutated with glutamate substitutions in the second exon, which may decrease its ability to be taken up by cells, thus increasing its extracellular concentrations and possibly neurotoxicity effects in the cell. Brain regions particularly susceptible to Tat toxicity are striatum, hippocampal dentate gyrus, and the CA3 region of the hippocampus.

Tat has been hypothesized by many studies as a potential contributor to HIVD by many mechanisms (reviewed by Pocernich et al., 2005). Tat protein released by astrocytes produces trimming of neuritis, mitochondrial dysfunction, and cell death in neurons. Tatinduced neurotoxicity is though to be mediated through excitotoxic mechanisms involving calcium. Tat can also induce markers of oxidative stress such as protein and LPO in synaptosomal membranes and neuronal cell cultures. To neutralize the oxidative stress, the GSH protects neurons against ROS directly and indirectly, and binds LPO products. GSH is the major cellular thiol participating in the maintenance of cellular redox status of the neuron and neuronal mitochondria. The biosynthesis of GSH may be compromised by Tat protein. It was hypothesized that the chronic inflammation of CSN by HIV-1, the activation of microglia, and increased lipid and protein oxidation, all observed in HIV-1 infected patients, can lead to the decrease of GSH serum levels and potentially HIVD. Low serum level of GSH is associated with poor survival in HIV-1-infected patients, while administration of GSH to HV-1-infected patients decreases mortality.

The production of superoxide anions by HIV-1 infected cells is counteracted by SOD, which, in turn, generates hydrogen peroxide (H2O2). Under basal conditions this is scavenged by catalase. To date, clear evidence exists that catalase activity is modified in brain tissue of AIDS patients. However, it has recently been reported that catalase is diminished in CD8+ T lymphocytes from HIV-1 positive individuals, suggesting the H2O2 scavenger activity might be decreased during HIV-1 infection (Yano et al., 1998).

The Role of Human Immunodeficiency Virus Type 1 (HIV-1)

micronutrients (Stehbens, 2004).

component of green tee.

both groups (Allard et al., 1998).

of the disease to AIDS (Ogunro et al., 2005).

concentration.

Proteins and Antiretroviral Drug Therapy in HIV-1-Induced Oxidative Stress 57

cells lytic activity, DNA repair, the antibodies formation, and macrophage and neuthrophil function. In experimental and human models, the zinc deficiency caused an imbalance between Th1 and Th2 functions resulting in decreased production of IFN-. These specific effects on T cell proliferation and function are not duplicated by other

Selenium deficiency diminishes cell-mediated immunity and depresses B-cell function, and it is associated with the occurrence, virulence, and disease progression to overt AIDS (Stehbens, 2004). Apoptosis of the cells is fundamental to progression of the disease that correlates with the decrease in plasma zinc, selenium, and vitamin E (Farvier et al., 1994). Many antioxidants have been tried for AIDS therapy including selenium, vitamin C, vitamin E, lipoic acid, carotene, whey proteins, and the epigallocatechin gallate (EGCG), the major

However, there are conflicting reports in the values of antioxidant vitamin E and vitamin C and SOD enzyme activity among HIV-1 infected patients in various stages in the literature (Allard et al., 1998; Stambullian et al., 2007). Suresh et al. (2009) showed that vitamin E, vitamin C, SOD, and TAC levels are decreased in HIV-1 patients, and the depletion was pronounced in HIV-1 symptomatic compared to HIV-1 asymptomatic individuals, in contrast to previous studies where were no significant differences in antioxidant vitamins in

McDermid et al. (2002) investigated the relation between dietary antioxidant intake and oxidative stress in clinically stable HIV-1 positive and HIV-1 negative adults. The results suggested dietary selenium intake was strongly and inversely associated with plasma MDA, but dietary antioxidant intakes were not related to peripheral blood mononuclear cell GSH

Total antioxidant status has been reduced in HIV-1 infected patients, probably due depletion of antioxidant molecules when they are consumed in the process of protecting cells against ROS induced oxidative damage in a magnitude that is related to advancement

Endothelial dysfunction induced by HIV-1 PIs may possible be reversed by antioxidants, including ginsenosides, selenium, curcumin (Chai et al., 2005a; Chai et al., 2005b), and resveratrol (Touzet & Philips, 2010). Therefore, it has been proposed by some researches that the oxidative stress and antioxidant status of HIV-1 seropositive patients could be monitored periodically during the disease progression. The possibility of counteracting oxidative stress by a pool of proper antioxidant plus an appropriate diet, mainly in patients whose blood antioxidant deficiencies can be easily rebalanced may have real health benefit

A new class of non-peptidic macrocyclic (MnII) complexes that possesses SOD enzymatic activity has been synthesized, which has the same activity as native SOD but can significantly cross the BBB (Salvemini et al., 1999). A SOD mimetic complex has been shown to significantly protect against the apoptotic cell death that occurs in astroglia that was incubated with supernatants of HIV-1 infected human macrophages. This effect was accompanied by a reduction of MDA concentration in astroglial cells and by a reduction of nitrotyrosine staining in these cells, showing that the effect of this mimetic complex occurred via reduction of ROS formation, and in turn, could reduce the neurodegenerative

and represent a promising way of inhibiting the progression of disease.

processes that occur in neuroAIDS (Mollace et al., 2001).

## **8. Antioxidant status in HIV-1 infection**

Although the concentration of plasma antioxidant components can be measured individually, these measurements may be time- and cost-consuming and labor intensive. In addition, it may not accurately reflect the total antioxidant status (Wayner et al., 1987). Total antioxidant capacity considers the cumulative effect of all oxidants present in blood and any fluids (Nagy et al., 2006) and it could be evaluated by several assays including total peroxyl radical trapping antioxidant parameter (TRAP), total antioxidant capacity (TAC), ferric reducing ability (FRAP), and their variations.

It has been previously shown that the HIV-1 infected individuals are oxidative stressed and have significantly lower antioxidant concentrations than HIV-1 seronegative individuals.

There is experimental evidence that different metabolic events that occur as consequence of HIV-1 infection directly influence the consumption of antioxidant components thus contributing to the increase of oxidative stress. Studies have found impaired antioxidant defense in HIV-1 infected patients and the antioxidant depletion indicates a decrease in immune function. Cells of immune system generally require a higher antioxidant concentration than other cells to retain redox balance, and preserve integrity and function (De La Fuente et al., 2002).

There are numerous studies reporting GSH deficiency in HIV-1 infection. The concentration of GSH is low in plasma, lung epithelial lining fluid, and peripheral blood mononuclear cells of HIV-1 infected individuals (Buhl et al., 1989; Roederer et al., 1993). Studies *in vitro* have shown that low GSH levels impair T cell function and also promote HIV-1 expression, suggesting a link between GSH deficiency and progression of HIV-1 disease (Kalebic et al., 1991; Roederer et al., 1993). Poor survival rates of HIV-1 seropositive individuals with low GSH levels and improved survival when GSH was replenished were also reported (Herzenberg et al., 1997). Taken together, these data can proposed that a persistent oxidative stress leads an accelerated rate of consumption of GSH that is not matched by an equal in the rate of synthesis of the tripeptide.

Gil et al. (2003) showed both a reduction of GSH levels and an increased in MDA and total hydroperoxides levels were detected in the plasma of HIV-1 seropositive individuals. These patients also showed an increase of DNA fragmentation in lymphocytes, reduction of glutathione peroxidase, and an increase in SOD activity in erythrocytes. There are several studies of disturbed GSH metabolism in HIV-1 infected patients. Arkrust et al. (2003) showed that, during HAART, the decrease in virus load and the increase in CD4+ T cell count are accompanied by both an improvement in the abnormal GSH-redox status and an increase in the subnormal levels of antioxidant vitamins. They have shown that HIV-1 infected patients are characterized by a decrease in both reduced GSH and vitamin C, the two most important hydrophilic antioxidants.

HIV-1 infection results in considerably reduced α-tocopherol concentrations and very low plasma zinc and selenium levels. Zinc and copper ions inhibit intracellular HIV-1 replication (Sprietsma, 1997). The precise mechanism by which the antioxidant effects of zinc is accomplished stems from its involvement in SOD and other enzymatic process. In humans, marked zinc deficiency strongly compromises the immune function and often enhances vulnerability to fatal opportunistic infections. It decreases CD4+ T helper cell function, CD8+ T cell cytotoxic activity, serum thymulin activity, and the interleukin-2 (IL-2) production by peripheral blood mononuclear cells. It also reduces the natural killer

Although the concentration of plasma antioxidant components can be measured individually, these measurements may be time- and cost-consuming and labor intensive. In addition, it may not accurately reflect the total antioxidant status (Wayner et al., 1987). Total antioxidant capacity considers the cumulative effect of all oxidants present in blood and any fluids (Nagy et al., 2006) and it could be evaluated by several assays including total peroxyl radical trapping antioxidant parameter (TRAP), total antioxidant capacity (TAC), ferric

It has been previously shown that the HIV-1 infected individuals are oxidative stressed and have significantly lower antioxidant concentrations than HIV-1 seronegative individuals. There is experimental evidence that different metabolic events that occur as consequence of HIV-1 infection directly influence the consumption of antioxidant components thus contributing to the increase of oxidative stress. Studies have found impaired antioxidant defense in HIV-1 infected patients and the antioxidant depletion indicates a decrease in immune function. Cells of immune system generally require a higher antioxidant concentration than other cells to retain redox balance, and preserve integrity and function

There are numerous studies reporting GSH deficiency in HIV-1 infection. The concentration of GSH is low in plasma, lung epithelial lining fluid, and peripheral blood mononuclear cells of HIV-1 infected individuals (Buhl et al., 1989; Roederer et al., 1993). Studies *in vitro* have shown that low GSH levels impair T cell function and also promote HIV-1 expression, suggesting a link between GSH deficiency and progression of HIV-1 disease (Kalebic et al., 1991; Roederer et al., 1993). Poor survival rates of HIV-1 seropositive individuals with low GSH levels and improved survival when GSH was replenished were also reported (Herzenberg et al., 1997). Taken together, these data can proposed that a persistent oxidative stress leads an accelerated rate of consumption of GSH that is not matched by an equal in

Gil et al. (2003) showed both a reduction of GSH levels and an increased in MDA and total hydroperoxides levels were detected in the plasma of HIV-1 seropositive individuals. These patients also showed an increase of DNA fragmentation in lymphocytes, reduction of glutathione peroxidase, and an increase in SOD activity in erythrocytes. There are several studies of disturbed GSH metabolism in HIV-1 infected patients. Arkrust et al. (2003) showed that, during HAART, the decrease in virus load and the increase in CD4+ T cell count are accompanied by both an improvement in the abnormal GSH-redox status and an increase in the subnormal levels of antioxidant vitamins. They have shown that HIV-1 infected patients are characterized by a decrease in both reduced GSH and vitamin C, the

HIV-1 infection results in considerably reduced α-tocopherol concentrations and very low plasma zinc and selenium levels. Zinc and copper ions inhibit intracellular HIV-1 replication (Sprietsma, 1997). The precise mechanism by which the antioxidant effects of zinc is accomplished stems from its involvement in SOD and other enzymatic process. In humans, marked zinc deficiency strongly compromises the immune function and often enhances vulnerability to fatal opportunistic infections. It decreases CD4+ T helper cell function, CD8+ T cell cytotoxic activity, serum thymulin activity, and the interleukin-2 (IL-2) production by peripheral blood mononuclear cells. It also reduces the natural killer

**8. Antioxidant status in HIV-1 infection** 

reducing ability (FRAP), and their variations.

(De La Fuente et al., 2002).

the rate of synthesis of the tripeptide.

two most important hydrophilic antioxidants.

cells lytic activity, DNA repair, the antibodies formation, and macrophage and neuthrophil function. In experimental and human models, the zinc deficiency caused an imbalance between Th1 and Th2 functions resulting in decreased production of IFN-. These specific effects on T cell proliferation and function are not duplicated by other micronutrients (Stehbens, 2004).

Selenium deficiency diminishes cell-mediated immunity and depresses B-cell function, and it is associated with the occurrence, virulence, and disease progression to overt AIDS (Stehbens, 2004). Apoptosis of the cells is fundamental to progression of the disease that correlates with the decrease in plasma zinc, selenium, and vitamin E (Farvier et al., 1994).

Many antioxidants have been tried for AIDS therapy including selenium, vitamin C, vitamin E, lipoic acid, carotene, whey proteins, and the epigallocatechin gallate (EGCG), the major component of green tee.

However, there are conflicting reports in the values of antioxidant vitamin E and vitamin C and SOD enzyme activity among HIV-1 infected patients in various stages in the literature (Allard et al., 1998; Stambullian et al., 2007). Suresh et al. (2009) showed that vitamin E, vitamin C, SOD, and TAC levels are decreased in HIV-1 patients, and the depletion was pronounced in HIV-1 symptomatic compared to HIV-1 asymptomatic individuals, in contrast to previous studies where were no significant differences in antioxidant vitamins in both groups (Allard et al., 1998).

McDermid et al. (2002) investigated the relation between dietary antioxidant intake and oxidative stress in clinically stable HIV-1 positive and HIV-1 negative adults. The results suggested dietary selenium intake was strongly and inversely associated with plasma MDA, but dietary antioxidant intakes were not related to peripheral blood mononuclear cell GSH concentration.

Total antioxidant status has been reduced in HIV-1 infected patients, probably due depletion of antioxidant molecules when they are consumed in the process of protecting cells against ROS induced oxidative damage in a magnitude that is related to advancement of the disease to AIDS (Ogunro et al., 2005).

Endothelial dysfunction induced by HIV-1 PIs may possible be reversed by antioxidants, including ginsenosides, selenium, curcumin (Chai et al., 2005a; Chai et al., 2005b), and resveratrol (Touzet & Philips, 2010). Therefore, it has been proposed by some researches that the oxidative stress and antioxidant status of HIV-1 seropositive patients could be monitored periodically during the disease progression. The possibility of counteracting oxidative stress by a pool of proper antioxidant plus an appropriate diet, mainly in patients whose blood antioxidant deficiencies can be easily rebalanced may have real health benefit and represent a promising way of inhibiting the progression of disease.

A new class of non-peptidic macrocyclic (MnII) complexes that possesses SOD enzymatic activity has been synthesized, which has the same activity as native SOD but can significantly cross the BBB (Salvemini et al., 1999). A SOD mimetic complex has been shown to significantly protect against the apoptotic cell death that occurs in astroglia that was incubated with supernatants of HIV-1 infected human macrophages. This effect was accompanied by a reduction of MDA concentration in astroglial cells and by a reduction of nitrotyrosine staining in these cells, showing that the effect of this mimetic complex occurred via reduction of ROS formation, and in turn, could reduce the neurodegenerative processes that occur in neuroAIDS (Mollace et al., 2001).

The Role of Human Immunodeficiency Virus Type 1 (HIV-1)

macrophages, adipocytes, and in neuronal cells.

disorders in HIV-1 infected individuals.

pp. 1917-1921, ISSN 0036-8075.

Vol.108, pp. 331-337, ISSN 0039-6060.

**10. References** 

**9. Conclusion** 

further investigation in individuals with diverse disease status.

Proteins and Antiretroviral Drug Therapy in HIV-1-Induced Oxidative Stress 59

D, zinc, and selenium) are required to build the evidence base. The long-term clinical benefits, adverse effects, and optimal formulation of multiple micronutrient supplements require

The exogenous supply of antioxidants using novel and more-specific molecules that scavenge free radical might allow further advances in understanding the processes that underlie the pathogenesis of HIV-1 infection and thus might represent the basis for novel and potentially efficient strategies in the complementary treatment of neurological,

There is clear evidence that the gp120 and Tat HIV-1 proteins and antiretroviral drugs directly and indirectly induce oxidative stress. Damage-induced by oxidative stress in endothelial cells and neurons may be correlated with an increase in the risk of cardiovascular disease and dementia, respectively, in HIV-1 infected patients. Although differences may exists to the relative contribution and mechanisms of toxicity, the preponderance of clinical and experimental data suggest roles for both of these factors in the context of HIV-1 infection. In assessing cardiovascular risk, it is important to take into account potential contributions from both infection and therapy. To various degrees, multiple HIV-1 viral proteins and antiretroviral drugs activate cell signaling cascades, induce oxidative stress, disturb mitochondrial function, alter gene expression, and impair lipid metabolism. These changes occur in endothelial cells, in vascular muscle cells,

The main changes that have been reported by *in vivo* and *in vitro* studies are the increase of the LPO, protein oxidation, and NO metabolites, decrease in the individual antioxidants defenses such as vitamin C, vitamin E, GSH, catalase, selenium, and zinc. In addition, the total status antioxidant is also impaired in HIV-1 infected individuals. NO cannot be rigidly classified as an anti-inflammatory or pro-inflammatory molecule, but it can be considered a true inflammatory mediator. It has been also reported that oxidative stress in HIV-1 infected individuals is associated with increase of DNA fragmentation in lymphocytes, reduction of

The better knowledge of the ways in which HIV-1 proteins and antiretroviral drugs interact with each other and with the host cells, mainly the endothelial, the neuronal, and immune system cells, may contribute to discover new approaches to be associated with the antiretroviral therapies in order to prevent cardiovascular diseases and neurological

Adamson, D.C.; Wildemann, B.; Sasaki, M.; Glass, J.D.; McArthur, J.C.; Christov, V.I.;

Barbul, A.; Lazzarou, S.A.; Efron, D.T.; Wasserkrug, H.L. & Efron, G. (1990). Arginine

Dawson, T.M. & Dawson, V.L. (1996). Immunologic NO synthase: elevation in severe AIDS dementia and induction by HIV-1 gp 41. *Science*, Vol. 274, No. 5294,

enhances would healing and lymphocyte immune responses in humans. *Surgery*,

glutathione peroxidase, and an increase in SOD activity in erythrocytes.

endothelium, and cardiovascular diseases associated with the HIV-1 infection.

Many clinical trials on HIV-1 dementia have centered on drugs that block receptors or are antagonists to the neurotoxic chemokines and cytokines released from activated microglia, macrophages, and astrocytes. These drugs, including nimodipine (L-type calcium channel antagonist), peptide T (possible chemokine receptor blocker), selegiline and deprenyl (monoamine oxidase-B inhibitors), lexipafant (platelet-activating factor antagonist), and CPI-1189 (TNF antagonist), indirectly act as antioxidants by blocking the downstream effects of these neurotoxic agents that usually result in an increase of ROS, RNS, and neuronal death (Turchan et al., 2003).

The importance of micronutrients in the prevention and treatment of childhood infections is well known, and evidence is emerging that micronutrient interventions may also affect HIV-1 transmission and progression. To clarify this issue, Friis (2006) reviewed evidences on the role of micronutrient supplementation in HIV-1 transmission and progression. The author concluded that interventions to improve micronutrient intake and status could contribute to a reduction in the magnitude and impact of the global HIV-1 epidemic. However, more research is needed before specific recommendations can be made. Fawzi et al (2005) underscored that poor nutrition and HIV-1 related adverse health outcomes contribute to a vicious cycle that may be slowed down by using nutritional interventions, including vitamins and minerals. Among children, periodic supplementation with vitamin A starting at six months of age has been shown to be beneficial in reducing mortality and morbidity among both HIV-1-infected and uninfected children. Limited data exist on the role of other nutrient supplements among children. Among HIV-1 infected adults, the safety and the efficacy of vitamin A supplements need further study, although adequate dietary intake of this essential nutrient is recommended. Multivitamin supplements were efficacious in reducing adverse pregnancy outcomes and early childhood infections, and is currently provided to HIV-1 infected pregnant women in many programs. The efficacy of such supplements among HIV-1 negative pregnant women needs further study. Daily multivitamin supplements were found to reduce HIV-1 disease progression among men and women and could be provided to adults in early stages of HIV-1 disease to prolong the time before antiretroviral therapy.

In order to assess whether micronutrient supplements are effective and safe in reducing mortality and morbidity in adults and children with HIV-1 infection, 30 randomized controlled trials were selected that compared the effects of micronutrient supplements (vitamins, trace elements, and combinations of these) with other supplements, placebo or no treatment on mortality, morbidity, pregnancy outcomes, immunologic indicators, and anthropometric measures in HIV-1 infected adults and children (Irlam et al, 2005, 2010). Any adverse effects of supplementation were recorded in 30 trials involving 22,120 participants: 20 trials of single supplements (vitamin A, vitamin D, zinc, selenium) and 10 of multiple micronutrients. Eight trials were undertaken in child populations. The results of this metaanalysis showed that multiple micronutrient supplements reduced morbidity and mortality in HIV-1 infected pregnant women and their offspring and also improved early child growth in one large randomized controlled trial in Africa. Additional research is needed to determine if these are generalisable findings. Vitamin A supplementation is beneficial and safe in HIV-1 infected children, but further evidence is needed to establish if supplementation confers similar benefits in HIV-1 infected adults. Zinc is safe in HIV-1 infected adults and children. It may have similar benefits in HIV-1 infected children and adults, and uninfected children with diarrhea, as it does in HIV-1 uninfected children. Further trials of single supplements (vitamin D, zinc, and selenium) are required to build the evidence base. The long-term clinical benefits, adverse effects, and optimal formulation of multiple micronutrient supplements require further investigation in individuals with diverse disease status.

The exogenous supply of antioxidants using novel and more-specific molecules that scavenge free radical might allow further advances in understanding the processes that underlie the pathogenesis of HIV-1 infection and thus might represent the basis for novel and potentially efficient strategies in the complementary treatment of neurological, endothelium, and cardiovascular diseases associated with the HIV-1 infection.

## **9. Conclusion**

58 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Many clinical trials on HIV-1 dementia have centered on drugs that block receptors or are antagonists to the neurotoxic chemokines and cytokines released from activated microglia, macrophages, and astrocytes. These drugs, including nimodipine (L-type calcium channel antagonist), peptide T (possible chemokine receptor blocker), selegiline and deprenyl (monoamine oxidase-B inhibitors), lexipafant (platelet-activating factor antagonist), and CPI-1189 (TNF antagonist), indirectly act as antioxidants by blocking the downstream effects of these neurotoxic agents that usually result in an increase of ROS, RNS, and

The importance of micronutrients in the prevention and treatment of childhood infections is well known, and evidence is emerging that micronutrient interventions may also affect HIV-1 transmission and progression. To clarify this issue, Friis (2006) reviewed evidences on the role of micronutrient supplementation in HIV-1 transmission and progression. The author concluded that interventions to improve micronutrient intake and status could contribute to a reduction in the magnitude and impact of the global HIV-1 epidemic. However, more research is needed before specific recommendations can be made. Fawzi et al (2005) underscored that poor nutrition and HIV-1 related adverse health outcomes contribute to a vicious cycle that may be slowed down by using nutritional interventions, including vitamins and minerals. Among children, periodic supplementation with vitamin A starting at six months of age has been shown to be beneficial in reducing mortality and morbidity among both HIV-1-infected and uninfected children. Limited data exist on the role of other nutrient supplements among children. Among HIV-1 infected adults, the safety and the efficacy of vitamin A supplements need further study, although adequate dietary intake of this essential nutrient is recommended. Multivitamin supplements were efficacious in reducing adverse pregnancy outcomes and early childhood infections, and is currently provided to HIV-1 infected pregnant women in many programs. The efficacy of such supplements among HIV-1 negative pregnant women needs further study. Daily multivitamin supplements were found to reduce HIV-1 disease progression among men and women and could be provided to adults in early stages of HIV-1 disease to prolong the time

In order to assess whether micronutrient supplements are effective and safe in reducing mortality and morbidity in adults and children with HIV-1 infection, 30 randomized controlled trials were selected that compared the effects of micronutrient supplements (vitamins, trace elements, and combinations of these) with other supplements, placebo or no treatment on mortality, morbidity, pregnancy outcomes, immunologic indicators, and anthropometric measures in HIV-1 infected adults and children (Irlam et al, 2005, 2010). Any adverse effects of supplementation were recorded in 30 trials involving 22,120 participants: 20 trials of single supplements (vitamin A, vitamin D, zinc, selenium) and 10 of multiple micronutrients. Eight trials were undertaken in child populations. The results of this metaanalysis showed that multiple micronutrient supplements reduced morbidity and mortality in HIV-1 infected pregnant women and their offspring and also improved early child growth in one large randomized controlled trial in Africa. Additional research is needed to determine if these are generalisable findings. Vitamin A supplementation is beneficial and safe in HIV-1 infected children, but further evidence is needed to establish if supplementation confers similar benefits in HIV-1 infected adults. Zinc is safe in HIV-1 infected adults and children. It may have similar benefits in HIV-1 infected children and adults, and uninfected children with diarrhea, as it does in HIV-1 uninfected children. Further trials of single supplements (vitamin

neuronal death (Turchan et al., 2003).

before antiretroviral therapy.

There is clear evidence that the gp120 and Tat HIV-1 proteins and antiretroviral drugs directly and indirectly induce oxidative stress. Damage-induced by oxidative stress in endothelial cells and neurons may be correlated with an increase in the risk of cardiovascular disease and dementia, respectively, in HIV-1 infected patients. Although differences may exists to the relative contribution and mechanisms of toxicity, the preponderance of clinical and experimental data suggest roles for both of these factors in the context of HIV-1 infection. In assessing cardiovascular risk, it is important to take into account potential contributions from both infection and therapy. To various degrees, multiple HIV-1 viral proteins and antiretroviral drugs activate cell signaling cascades, induce oxidative stress, disturb mitochondrial function, alter gene expression, and impair lipid metabolism. These changes occur in endothelial cells, in vascular muscle cells, macrophages, adipocytes, and in neuronal cells.

The main changes that have been reported by *in vivo* and *in vitro* studies are the increase of the LPO, protein oxidation, and NO metabolites, decrease in the individual antioxidants defenses such as vitamin C, vitamin E, GSH, catalase, selenium, and zinc. In addition, the total status antioxidant is also impaired in HIV-1 infected individuals. NO cannot be rigidly classified as an anti-inflammatory or pro-inflammatory molecule, but it can be considered a true inflammatory mediator. It has been also reported that oxidative stress in HIV-1 infected individuals is associated with increase of DNA fragmentation in lymphocytes, reduction of glutathione peroxidase, and an increase in SOD activity in erythrocytes.

The better knowledge of the ways in which HIV-1 proteins and antiretroviral drugs interact with each other and with the host cells, mainly the endothelial, the neuronal, and immune system cells, may contribute to discover new approaches to be associated with the antiretroviral therapies in order to prevent cardiovascular diseases and neurological disorders in HIV-1 infected individuals.

## **10. References**


The Role of Human Immunodeficiency Virus Type 1 (HIV-1)

ISSN 0008-6363.

813, ISSN 0022-1899.

ISSN 0027-8424.

Proteins and Antiretroviral Drug Therapy in HIV-1-Induced Oxidative Stress 61

Concklin, B.S.; Fu, W.; Lin, P.H.; Lumsden, A.B.; Yao, Q. & Chen, C. (2004). HIV protease

Coopper, D.; Heera, J.; Goodrich, J.; Tawadrous, M.; Saag, M.; Dejesus, E.; Clumeck, N.;

Cossarizza, A. & Moyle, G. (2004). Antiretroviral nucleoside and nucleotide analogues and

Coyle, J.T. & Putfarcken, P. (1993). Oxidative stress, glutamate, and neurodegenerative

Das, U.N.; Podma, M.; Sogar, P.S.; Ramesh, G. & Koratkar, R. (1990). Stimulation of free

Dawson, V.L;. Dawson, T.M.; Uhl, G.R. & Snyder, S.H. (1993). Human immunodeficiency

Day, B.J; & Lewis, W. (2004). Oxidative stress in NRTI-induced toxicity: evidence from

De La Asunción, J.; Del Olmo, M.L.; Sastre, J.; Millán, A.; Pellín, A.; Pallardó, F.V. & Viña, J.

De La Fuente, M.; Miquel, J.; Catalán, M.P.; Victor, V.M. & Guayerbas, N. (2002). The

Droge, W.; Eck, H.P.; Gmunder, H. & Mihm, S. (1991). Modulation of lymphocytes functions

Estrada, V & Portilla KJ. (2011). Dyslipidemia related to antiretroviral therapy. *AIDS* 

*Medicine Supplements*, Vol. 91, No.3C, pp. 140S-144S, ISSN 0002-9343. Emery, S. & Winston, A. (2009). Raltegravir: a new choice in HIV and new chance for

radical generation in human leucocytes by various agents including tumor necrosis factor is a calmodulin-dependent process. *Biochemical and Biophysical Research* 

virus type 1 coat protein neurotoxicity mediated by nitric oxide in primary cortical cultures. *Proceedings of National Academy of Sciences USA*, Vol.90, pp. 3256-3259,

clinical experience and experiments *in vitro* and *in vivo*. *Cardiovascular Toxicology*,

(1998). AZT treatment induces molecular and ultrastructural oxidative damage to muscle mitochondria. Prevention by antioxidant vitamins. *The Journal of Clinical* 

amount of thiolic antioxidant ingestion related to improve several immune functions is higher in aged than in adult mice. *Free Radical Biology & Medicine,* Vol.

and immune responses by cysteine and cysteine derivatives. *The American Journal of* 

mitochondria. *AIDS*, Vol. 18, No. 2, pp. 137-151, ISSN 0269-9370.

*Communications*, Vol. 167, No.3, pp. 1030–1036, ISSN 0006-291X.

disorders. *Science*, Vol. 262, pp. 689-695, ISSN 0036-8075.

Vol. 4, No. 3, pp. 207-216, ISSN 1530-7905.

36, pp. 119-126, ISSN 0891-5849.

*Investigation,* Vol*.* 102, No. 1, pp. 4-9, ISSN 0021-9738.

research. *Lancet*, Vol.374, pp. 764-766, ISSN 0099-5355.

*Reviews*, Vol.13, pp. 49-56, ISSN 0269-9370.

alterations. *AIDS*, Vo.13, pp. 2251-2260, ISSN 0269-9370.

antiretroviral therapy: correlation between dyslipidemia and steroid hormone

inhibitor ritonavir decreases endothelium-dependent vasorelaxation and increases superoxide in porcine arteries. *Cardiovascular Research*, Vol. 63, No.1, pp.168-175,

Walmsley, S.; Ting, N.; Coakley, E.; Reeves, J.D.; Reyes-Teran, G.; Westby, M.; Van Der Ryst, E.; Ive, P.; Mohapi, L.; Mingrone, H.; Horban, A.; Hackman, F.; Sullivan, J. & Mayer, H. (2010). Maraviroc versus efavirenz, both in combination with zidovudine-lamivudine, for the treatment of antiretroviral-naïve subjects with CCR5-tropic HIV-1 infection. *Journal of Infectious Diseases*, Vo. 201, No.6, pp. 803-


Beckman, J.S. & Koppenol, W.H. (1996). Nitric oxide, superoxide, and peroxynitrite: the

Beherns, G.; Dejam, A.; Schmidt, H.; Balks, H.J.; Braasbant, G. & Körner, T. (1999). Impaired

Blenis, J. (1993). Signal transduction via the MAP kinases: proceed at your own RSK.

Blond, D.; Raoul, H.; Le Grand, R. & Dormont, D. (2000). Nitric oxide synthesis enhances

Bodgan, C. (2001). Nitric oxide and the immune response. *Nature Immunology*, Vol.2, pp. 907-

Buhl, R.; Jaffe, H.A.; Holroyd, K.J.; Wells, F.B.; Mastrangeli, A.; Saltini, C.; Cantin, A.M. &

Butterfield, D.A.; Castegna, A.; Lauderback, C.M. & Drake, J. (2002). Evidence that amyloid

Butterfield, D.A. & Stadman, E. R. (1997). Protein oxidation processes in aging brain. *Advances in Cell Aging and Gerontology*, Vol. 2, pp.161-191, ISSN 1566-3125. Cairoli, E.; Scott-Algara, D.; Pritsch, O.; Dighiero, G. & Cayota, A. (2008). HIV-1 induced

Carr, A.; Samaras, K.; Chilsom, D.J. & Cooper, D.A. (1998). Pathogenesis of HIV-1 protease

Chai, H.; Zhou, W.; Lin, P.; Lumsden, A.; Yao, Q. & Chen, C. (2005a). Ginsenosides block

Chai, H.; Yan, S.; Lin, P.; Lumsden, A.B.; Yao, Q. & Chen, C. (2005b). Curcumin blocks HIV

Christeff, N.; Melchior, J.C.; de Truchis, P.; Perronne, C.; Nunez, E.A. & Gougeon, M.L.

resistance. *The Lancet*, Vol.351, pp. 1881-1883, ISSN 0099-5355.

No.6, pp. H2965-2971, ISSN 0363-6135.

*Journal of Virology*, Vol.74, No.19, pp. 8904-9812, ISSN 0022-528X.

C1437, ISSNJ 0002-9513.

916, ISSN 1529-2908.

8424.

5355.

0197-4580.

1521-6616.

ISSN 1072-7515.

*Nutrition*, Vol.9, pp.287-302, ISSN 0199-9885.

good, the bad, ad ugly. *The American Journal of Physiology*, Vol.271, pp. C1424-

glucose tolerance, beta cell function and lipid metabolism in HIV patients under treatment with protease inhibitors. *AIDS*, Vo.13, pp. F63-F67, ISSN 0269-9370. Beutler, E. (1989). Nutritional and metabolic aspects of glutathione. *Annual Review of* 

*Proceedings of National Academy of Sciences USA*, Vol. 90, pp. 5889-5892, ISSN 0027-

human immunodeficiency virus replication in primary human macrophages.

Crystal, R.G. (1989). Systemic glutathione deficiency in symptom-free HIVseropositive individuals. *The Lancet*, Vol. 2, No. 8675, pp. 1294-1298, ISSN 0099-

beta-peptide-induced lipid peroxidation and its sequelae in Alzheimer's disease brain contribute to neuronal death. *Neurobiology of Aging*, Vol.32, pp.655-664, ISSN

decrease of nitric oxide production and inducible nitric oxide synthase expression during *in vivo* and *in vitro* infection. *Clinical Immunology*, Vol.127, pp. 26-33, ISSN

inhibitor-associated peripheral lipodystrophy, hyperlipidemia, and insulin

HIV protease inhibitor ritonavir-induced vascular dysfunction of porcine coronary arteries. *American Journal of Physiology - Heart and Circulatory Physiology*, Vol. 288,

protease inhibitor ritonavir-induced vascular dysfunction in porcine coronary arteries. *Journal of the American College of Surgeons,* Vol. 200, No. 6, pp. 820-830,

(1999). Lipodystrophy defined by a clinical score in HIV-infected men on highly

antiretroviral therapy: correlation between dyslipidemia and steroid hormone alterations. *AIDS*, Vo.13, pp. 2251-2260, ISSN 0269-9370.


The Role of Human Immunodeficiency Virus Type 1 (HIV-1)

No.5, pp. 1967-1972. ISSN 0027-8424.

*Trials*, Vol. 10, pp. 1-12, ISSN 1528-4336.

177, No.2, pp. 397-407, ISSN 0022-1007.

Vol. 12, pp*.* CD003650, ISSN 1469-493X.

Vol.42, pp. 1472-1476, ISSN 0028-3878.

No.1, pp.11-15, ISSN 0041-8781.

pp. CD003650, ISSN 1469-493X.

682X.

0022-538X.

Proteins and Antiretroviral Drug Therapy in HIV-1-Induced Oxidative Stress 63

Halliwell, B. & Gutteridge JMC. (1999). Detection of free radicals and others reactive species:

Hamdy, O. Lifestyle modification and endothelial function in obese subjects. (2005). *Expert* 

Herzenberg, L.A.; De Rosa, S.C.; Dubs, J.G.; Roederer M, Anderson MT, Ela SW, Deresinski

Hill, A.; Sawyer, W. & Gazzard, B. (2009). Effects of first-line use of nucleoside analogues,

Holt, P.G.; Oliver, J.; Bilyk, N. McMenamin C, McMenamin PG, Kraal G, Thepen T. (1993).

Hua, L.L.; Liu, J.S.H.; Brosnan, C.F. & Lee, C.C. (1998). Selective inhibition of human glial

Irlam, J.H.; Visser, M.E.; Rollins, N. & Siegfried, N. (2005). Micronutrient supplementation in

Israel, N. & Gougerot-Pocidalo, M.A. (1997). Oxidative stress in human immunodeficiency

Janssen, R.S.; Nwanyamwu, O.C.; Selk, R.M. & Stehr-Green, J.K. (1992). Epidemiology of

Jimenez, J.L.; Gonzalez-Nicolas, J.; Alvarez, S. Fresno M, Muñoz-Fernández MA. (2001).

Jordão Júnior, AA; Figueiredo JF, Silveira S; Junqueira-Franco MV & Vannucchi H. (1998).

sclerosis. *Annals of Neurology*, Vol. 43, No.3, pp. 384-387, ISSN 1531-8249. Hulgan T, Morrow J, D'Aquila RT, Raffanti S, Morgan M, Rebeiro P, Haas DW. (2004).

Oxford, UK, ISBN 0198500459 (Hbk),. ISBN 0198500440 (Pbk).

*Review of Cardiovascular Therapy*, Vol.3, pp.231-241, ISSN 1477-9072.

trapping and fingerprinting. In: *Free Radical in Biology and Medicine*. Halliwell, B. & Gutteridge, J.M.C. (editors), pp 351-429, Oxford University Press 3th ed, ISBN,

SC, Herzenberg LA. (1997). Glutathione deficiency is associated with impaired survival in HIV disease. *Proceedings of National Academy of Science USA*, Vol. 94,

efavirenz, and ritonavir boosted protease inhibitors on lipid levels. *HIV Clinical* 

Downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells *in vivo* by resident alveolar macrophages. *Journal of Experimental Medicine*, Vol.

inducible nitric oxide synthase by interferon-beta implications for multiple

Oxidative stress is increased during treatment of human immunodeficiency virus infection. *Clinical Infectious Diseases,* Vol. 4, No. 3, pp. 207-216, ISSN 1058-4838. Irlam, J.H; Visser, M.M.; Rollins, N.N. & Siegfried, N. (2010). Micronutrient supplementation

in children and adults with HIV infection. *Cochrane Database Systematic Reviews,* 

children and adults with HIV infection. *Cochrane Database Systematic Reviews,* Vol. 4,

virus infection. *Cellular and Molecular Life Sciences*, Vol. 53, pp. 864-870, ISSN1420-

human immunodeficiency virus encephalopathy in the United States. *Neurology*,

Regulation of human immunodeficiency virus type 1 replication in human T lymphocytes by nitric oxide. *The Journal of Virology*, Vol. 75, pp. 4655-4663, ISSN

Urinary excretion of vitamin A and thiobarbituric acid reactive substance in AIDS patients. *Revista do Hospital das Clínicas da Faculdade de Medicina de São Paulo*, Vol. 53,


Evans, T.G; Rasmussen, K.; Wiebke, G. & Hibbs, J.B. (1994) Nitric oxide synthesis in patients

Favier, A.; Sappey, C.; Leclerc, P.; Faure, P. & Micoud, M. (1994). Antioxidant status and

Fawzi, W.; Msamanga, G.; Spiegelman, D. & Hunter, D.J. (2005). Studies of vitamins and

Fu, W.;Chai, H.; Yao, Q. & Chen, C. (2005). Effects of HIV protease inhibitor ritonavir on

Gil, L.; Martínez, G.; González, I.; Tarinas, A.; Álvarez, A.; Giuliani, A.; Molina, R.; Tápanes,

Greene, W.C. (1991). The molecular biology of human immunodeficiency virus type 1

Greenspan, H.C. & Aruoma, O. (1994). Could oxidative stress initiate programmed cell

Greenwood, A.J.; Highes, J.; Wallace, G.; Seed, P.; Stanford, M.R. & Graham, E.M. (1998).

Groeneveld, P.H.P.; Kroon, F.P.; Nibbering, P.H.; Bruisten, S.M.; Van Swieten, P. & Van

Guevara, I.; Iwanejko, J.; Dembińska-Kieć, A.; Pankiewicz, J.; Wanat, A.; Anna, P.; Gołabek,

Gunnett, C.A.; Heistad, D.D. & Faraci, F.M. (2003). Gene-targeted mice reveal a critical role

*Journal of Infectious Diseases*, Vol.28, pp. 341-345, ISSN 0036-5548.

*Chimica Acta,* Vol. 274, No.2, pp. 177-188, ISSN 0009-8981.

Vol. 34, pp. 2970-2974, ISSN 0039-2499.

*Biomedicine and Pharmacotherapy,* (in press), ISSN 0753-3322.

86, ISSN 0009-9104.

6618.

4793.

2797.

pp.713-714, ISSN 0956-4624.

Vol. 91, pp. 165-180, ISSN 0009-2797.

Vol. 135, N*o.* 4, pp*.* 938-944, ISSN 0022-3166.

with advanced HIV infection. *Clinical and Experimental Immunology*, Vol. 97, pp. 83-

lipid peroxidation in patients infected with HIV. *Chemico-Biological Interactions,* 

minerals and HIV transmission and disease progression. *The Journal of Nutrition,* 

vasomotor function and endothelial nitric oxide synthase expression. *Journal of Acquired immune Deficiency Syndromes*, Vol.39, No.2, pp. 152-158, ISSN 1525-4135. Gil, L.; Tarinas, A.; Hernádez, D.; Riverón, B.V.; Pérez, D.; Tápanes, R.; Capo, V. & Pérez, J.

(2010). Altered oxidative stress indexes related to disease progression marker in human immunodeficiency virus infected patients with antiretroviral therapy.

R.; Pérez, J. & León, O.S. (2003). Contribution to characterization of oxidative stress in HIV/AIDS patients. *Pharmacological Research*, Vol.47, pp.217-224, ISSN 1043-

infection. *The New England Journal of Medicine*, Vol. 324, pp. 308-317, ISSN 0028-

death in HIV infection? A role from plant derived metabolites having synergistic antioxidant activity. *Chemico-Biological Interactions*, Vol.143, pp.145-148, ISSN 0009-

Soluble intercellular adhesion molecule-1 (sICAM-1) and vascular cell adhesion molecule -1 (sVCAM-1) in patients with HIV/AIDS does not appear to correlate with cytomegalovirus retinitis. *International Journal of STD & AIDS*, Vol.9, No.11,

Furth, R. (1996). Increased production of nitric oxide correlates with viral load and activation of mononuclear phagocytes in HIV-infected patients. *Scandinavian* 

I.; Bartuś, S.; Malczewska-Malec, M. & Szczudlik, A. (1998). Determination of nitrite/nitrate in human biological material by the simple Griess reaction. *Clinica* 

for inducible nitric oxide synthase in vascular dysfunction during diabetes. *Stroke*,


The Role of Human Immunodeficiency Virus Type 1 (HIV-1)

421-430, ISSN 0960-5428.

ISSN 0009-9147.

ISSN 0270-9139.

pp221-225, ISSN 0309-3913.

3636.

Proteins and Antiretroviral Drug Therapy in HIV-1-Induced Oxidative Stress 65

Meyer, L.; Rabaud, C.; Ziegler, O.; May, T. & Drouin, P. (1998). Protease inhibitors, diabetes

Mollace, V. & Nistico, G. (1995). Release of nitric oxide from astroglial cells: a key

Moncada, S. et al. (1991). Nitric oxide: Physiology, pathophysiology and pharmacology.

Mondal, D.; Pradhan, I.; Ali, M. & Agrawal, K.C. (2004). HAART drugs induce oxidative

Nathan, C. (1007). Inducible nitric oxide synthase: what difference does it make? *The Journal* 

Navarro-Gonzalvez,J.A.; Garcia-Benayas, C. & Arenas, J. (1998). Semi-automated

Ngondi, J.L.; Oben, J.; Etame, L.H.; Forkah, D.M. & Mbanya, D. (2006). The effect of different

Ogunro, P.S.; Ogungbamigbe, T.O.; Ajala, M.O. & Egbewale, B.E. (2005). Total antioxidant

Petit, J.M.; Doung, M.; Duvillard, L.; Florentin, E.; Portier, H.; Lizard, G.; Brun, J.M.;

*European Journal of Clinical Investigation*, Vol.32, pp. 354-359, ISSN 0014-2972. Pietraforte, D.; Tritarelli, E.; Testa, U. & Minetti, M. (1994). Gp 120 HIV envelope

Radi, R. (2004). Nitric oxide, oxidants, and protein tyrosine nitration. *Proceedings of the National Academy of Sciences USA*, Vol. 101, pp. 4003-4008, ISSN 0027-8424. Rayyes, O.A.; Wallmark, A. & Floren, C.H. (1996). Cyclosporine inhibits catabolism of low-

*of Clinical Investigation,* Vol. 100, pp. 2417-2423, ISSN 0021-9738.

Cameroon. *AIDS Research Therapy*, Vol.3, pp.19, ISSN 1742-6405.

*Brain Research Reviews*, Vol. 50, pp.14-26,ISSN 0165-0173.

*Deficiency Syndrome*, Vol. 29, No. 2, pp. 158-164, ISSN 1525-4135.

*Pharmacological Reviews*, Vol. 43, pp. 109-142, ISSN 0031-6997.

*Medicine*, Vol. 5, pp. 812-819, ISSN 1076-1551.

seropositive and HIV-seronegative men and women. *Journal of Acquired Immune* 

mellitus and blood lipids. *Diabetes & Metabolism*, Vol.24, pp. 547-549, ISSN 1262-

mechanism in neuroimmune disorders. *Advances in Neuroimmunology*, Vol. 5, pp.

stress in human endothelial cells and increase endothelial recruitment of mononuclear cells: exacerbation by inflammatory cytokines and amelioration by antioxidants. *Cardiovascular Toxicology*, Vol. 4, No. 3, pp. 287-302, ISSN 1530-7905. Mossalayi, M.D.; Becherel, P.A. & Debre, P. (1999). Critical role of nitric oxide during the

apoptosis of peripheral blood leukocytes from patients with AIDS. *Molecular* 

measurement of nitrate in biological fluids. *Clinical Chemistry,* Vol.44, pp. 679-681,

combination therapies on oxidative stress markers in HIV infected patients in

status and lipid peroxidation in HIV-1 infected patients in a rural area of south western Nigeria. *African Journal of Medicine and Medical Sciences*, Vol. 34, No.3,

Gambert, P. & Verges, B. (2002). LDL-receptors expression in HIV-infected patients: relations to antiretroviral therapy, hormonal status, and presence of lypodystrophy.

glycoprotein increases the production of nitric oxide in human monocyte-derived macrophages. *Journal of Leukocyte Biology*, Vol. 55, pp.175-182, ISSN 0741-5400. Pocernich, C.B.; Sultana, R.; Mohmmad-Abdul, H.; Nath, A. & Butterfield, A. (2005). HIV-

dementia, Tat-induced oxidative stress, and antioxidant therapeutic considerations.

density lipoprotein in HEPG2 cells by about 25%. *Hepatology*, Vol.24, pp. 613-619,


Kalebic, T.; Kinter, A.; pol, G.; Anderson, M.E.; Meister, A. & Fauci, A.S. (1991). Suppression

Kim, R.J.; Wilson,C.G.; Wabitsch, M.; Lazar MA, Steppan CM. (2006). HIV protease inhibitor

Kline, E.R. & Sutliff, R.L. (2008). The roles of HIV-1 proteins and antiretroviral drug therapy

Koutkia, P. & Grinspoon, S. (2003). HIV-associated lipodystrophy: pathogenesis,

Kumar, G.N.; Dykstra, J.; Roberts. E;M.; Jayanti, V.K.; Hickman, D.; Uchic, J.; Yao, Y.; Surber,

Lamas, S.; Pérez-Sala, D. & Moncada, S. (1998). Nitric oxide: from discovery to the

Lander, H.M.; Sehaipal, P.; Levine, D.M. & Novogrodsky, A. (1993). Activation of human

Lewis W. (2003). Mitochondrial dysfunction and nucleoside reverse transcriptase inhibitor

Li, R.; Wang, W.Q.; Zhang, H.; Yang, X.; Fan, Q.; Christopher, T.A.; Lopez, B.L.; Tao, L.;

Mandas, A.; Iorio, E.L.; Congiu, M.G.; Balestrieri, C.; Mereu, A.; Cau, D.; Dessi, S. & Curreli,

Mannick, J.B. (1995). The antiviral role of nitric oxide. *Research Immunology*, Vol.146, pp. 693-

Martinez, E.; Larrouse, M.; Libre, J. Gutiérrez F, Saumoy M, Antela A, Knobel H, Murillas J,

McDermid, J.M.; Lalonde, R.G.; Gray-Donald, K.; Baruchel, S. & Kubow, S. (2002).

SPIRAL study. *AIDS*, Vol. 24, No.11, pp. 1697-1707, ISSN 0269-9370

819, ISSN 0269-9370.

pp.303-317, ISSN.

6147.

ISSN 1110-7243.

697, ISSN 0923-2494.

Vol.14, No.6, pp. 949-1002, ISSN 1930-7381.

*Disposition*, Vol. 27, No. 8, pp. 902-908, ISSN 0056-9556.

*Journal of Immunology*, Vol.150, pp.1509-1516, ISSN 0022-1767.

*Endocrinology and Metabolism,* Vol.293, No.6, pp. E1703-E1708.

*Research*, Vol.58, No. 3, pp.189-197, ISSN0166-3542.

56, No. 5, pp. 752-769, ISSN 1081-5589.

of human immunodeficiency virus expression in chronically infected monocytic cells by glutathione, glutathione ester, and N-acetylcysteine. *AIDS*, Vol. 6, pp. 815-

specific alterations in human adipocyte differentiation and metabolism. *Obesity*,

in HIV-1-associated endothelial dysfunction. *Journal of Investigative Medicine*, Vol.

prognosis, treatment, and controversies. *Annual Review of Medicine,* Vol. 55,

B.; Thomas, S. & Granneman, G.R. (1999). Potent inhibition of the cytochrome P450 3A-mediated human liver microsomal metabolism of a novel HIV protease inhibitor by ritonavir: a positive drug-drug interaction. *Drug Metabolism and* 

clinic. *Trends in Pharmacological Sciences*, Vol. 19, No.11, pp. 436-438, ISSN 0165-

peripheral blood mononuclear cells by nitric oxide-generating compounds. *The*

therapy: experimental clarifications and persistent clinical question. *Antiviral* 

Goldstein, B.J.; Gao, F. & Ma, X.L. (2007). Adiponectin improves endothelial function in hyperlipidemic rats by reducing oxidative/nitrative stress and differential regulation of eNOS/iNOS activity. *American Journal of Physiology,* 

N. (2009). Oxidative imbalance in HIV-1 infected patients treated with antiretroviral therapy. *Journal of Biomedicine & Biotechnology* , Vol. 2009, pp.749575,

Berenguer J, Pich J, Pérez I, Gatell JM & SPIRAL Study Group. (2010). Substitution of raltegravir for ritonavir-boosted protease inhibitors in HIV-infected patients: the

Association between dietary antioxidant intake and oxidative stress in HIV-

seropositive and HIV-seronegative men and women. *Journal of Acquired Immune Deficiency Syndrome*, Vol. 29, No. 2, pp. 158-164, ISSN 1525-4135.


The Role of Human Immunodeficiency Virus Type 1 (HIV-1)

No.10, pp. 1859-1969, ISSN 0022-2275.

December, 2007. Available in :

. Access in January 30, 2008.

9370.

0028-0836.

2524-2532, ISSN 0022-1767.

Proteins and Antiretroviral Drug Therapy in HIV-1-Induced Oxidative Stress 67

Torre, D.; Pugliese, A. & Speranza, F. (2002). Role of nitric oxide in HIV-1 infection: friend or

Touzet, O. & Philips, A. (2010). Resveratrol protects against protease inhibitor-induced

Tran, H.; Robinson, S.; Mikhailenko, I. & Strickland, D.K. (2003). Modulation of the LDL

Turchan, J.; Sactor, N.; Wojna, V.; Conant, K. & Nath, A. (2003). Neuroprotective therapy for HIV dementia. *Current HIV Research*, Vol.1, pp. 373-383, ISSN 1570-162X. UNAIDS - Joint United Nations Programme on HIV/AIDS. AIDS epidemic update.

Walli, R.; Herfort, O.; Michl, G.M.;Demant , T. ; Jägr, H. & Dietele, C. (1998) Treatment with

Wanchu, A.; Rana, S.V.; Pallikkuth, S. & Sachdeva, R.K. (2009). Oxidative Stress in HIV-

Wang, X.; Chai, H.; Yao, Q. & Chen, C. (2007). Molecular mechanisms of HIV protease

Westendorp, M.O.; Frank, R.; Ochsenbauer, C.; Stricker, K.; Dhein, J.; Walczak, H,

Witko-Sarsat, V.; Friedlander, M.; Nguyen-Khoa, T.; Capeillere-Blandin, C.; Nguyen, A.T.;

Wolf, K.; Tsakiris, D.A.; Weber, R.; Erb, P.; Battegay, M. & Swiss HIV Cohort Study. (2002).

Worm, S.; Sabin, C.; Weber, R. , Reiss, P.; El-Sadr, W.; Dabis, F.; De Wit, S.; Law, M.;

*Retroviruses*, Vol.25, No.12, pp.1307-1311, ISSN 0889-2229.

*Syndromes*, Vol.44, No.5, pp.493-499, ISSN 1525-4135.

*Diseases*, Vol. 185, No.4, pp. 456-462, ISSN 0022-1899

*Infectious Diseases*, Vol. 201, No.3, pp. 318-330, ISSN 0022-1899.

reactive oxygen species production, reticulum stress and lipid raft perturbation.

receptor and LPR levels by HIV protease inhibitors. *Journal of Lipid Research*, Vol. 44,

<http://www.unaids.org/hivaidsinfo/statistics/fact\_sheets/pdfs/brazil\_em.pdf>

protease inhibitors associated with peripheral insulin resistance and impaired oral glucose tolerance in HIV-1 infected patients. *AIDS*, Vol.12, F167-F173, ISSN 0269-

Infected Individuals: A Cross-Sectional Study. *AIDS Research and Human* 

inhibitor-induced endothelial dysfunction. *Journal of Acquired Immune Deficiency* 

Debatin, K.M. & Krammer, P.H. (1995). Sensitization of T cells to CD95 mediated apoptosis by HIV-1 Tat and gp 120. *Nature*, Vol.375, pp. 497-500, ISSN

Canteloup, S.; Dayer, J.M.; Jungers, P.; Drüeke, T. & Descamps-Latscha, B. (1998). Advanced oxidation protein products as novel mediators of inflammation and monocyte activation in chronic renal failure. *The Journal of Immunology*, Vol. 161, pp.

Antiretroviral therapy reduces markers of endothelial and coagulation activation in patients infected with human immunodeficiency virus type 1. *Journal of Infectious* 

Monforte, A.D.; Friis-Møller, N.; Kirk, O.; Fontas, E.; Weller, I.; Phillips, A. & Lundgren, J. (2010). Risk of myocardial infarction in patients with HIV infection exposed to specific individuals antiretroviral drugs from the 3 major drug classes: the data collection on adverse events of anti-HIV drugs (D.A.D) study. *Journal of* 

foe. *Lancet Infectious Diseases*, Vol. 2, pp.273-280, ISSN 1473-3099.

*AIDS*, Vol. 24, No. 10, pp. 1437-1447, ISSN 0269-9370.


Repetto, M.; Reides, C.; Gomez, M.L.; Costa, M.; Griemberg, G. & Llesuy, S. (1006).

Roederer, M.F.; Staal, J.T.; Anderson, M.; Rabin, R.; Raju, P.A.; Herzenberg, L.A. &

Schöndorf, T.; Maiworm, A.; Emmison, N.; Forst, T. & Pfützner, A. (2005). Biological

cardiovascular risk. *Clinical Laboratory,* Vol.51, pp. 489-494, ISSN 1433-6510. Schwarz, K.B. (1996). Oxidative stress during viral infection: a review. *Free Radical Biology &* 

Seigneur, M.; Constans, J.; Blann A.; Renard, M.; Pellegrin, J.L.; Amiral, J.; Boisseau, M. &

Shankar, S.S. & Dube, M.P. (2004). Clinical aspects of endothelial dysfunction associated

Smyth, M.J. (1991). Glutathione modulates activation-dependent proliferation of

Sprietsma, J.E. (1997). Zinc-controlled Th1/Th2 switch significantly

Stehbens, W.E. (2004). Oxidative stress in viral hepatitis and AIDS. *Experimental and* 

Stein, J.H.; Klein, M.A.; Bellehumeur, J.L.; McBride, P.E.; Wiebe, D.A.; Otvos, J.D. & Sosman

Suresh, D.R.; Annam, V.;, Pratibha, K. & Prasad, B.V.M. (2009). Total antioxidant capacity –

Tao, L.; Gao, E.; Jiao, X.; Yuan, Y.; Li, S.; Christopher, T.A.; Lopez, B.L.; Koch, W.; Chan, L.;

Torre, D.; Ferrario, G.; Speranza, F.; Martegani, R. & Zeroli, C. (1996a). Increased levels of

Torre, D.; Ferrario, G.; Speranza, F.; Orani, A.; Fiori, G.P. & Zeroli, C. (1996b). Serum

*New York Academy of Sciences*, Vol.677, pp. 113-125, ISSN 0077-8923.

*Medicine*, Vol. 21, No. 5, pp.641-649, ISSN 0891-5849.

*Cardiovascular Toxicology*, No.4, pp.261-269, ISSN 1530-7905.

*Molecular Pathology,* Vol.77, pp.121-132, ISSN 0014-4800.

*Circulation*, Vol.104, No.3, pp.257-262, ISSN 0009-7322.

*Journal of Biomedical Science,* Vol.16, pp.61-64, ISSN 1021-7770

*Circulation,* Vol.115, No.11, pp.1408-1416, ISSN 0009-7322.

*Pathology*, Vol.49, pp.574-576, ISSN 0021-9746.

*Clinical Infectious Diseases*, Vol. 22, pp.650-653, ISSN 1058-4838.

117, ISSN 0009-8981.

6245.

0022-1767.

0306-9877.

Oxidative stress in blood of HIV patients. *Clinical Chimica Acta*, Vol. 255, pp. 107-

Herzenberg, L.A. (1993). Dysregulation of leukocytes glutathione in AIDS. *Annals of* 

background and role of adiponectin as marker for insulin resistance and

Conri, C. (1997). Soluble adhesion molecules and endothelial cell damage in HIV infected patients. *Thrombosis and Haemostasis*, Vol.77, No.4, pp. 646-649, ISSN 0340-

with human immunodeficiency virus infection and antiretroviral agents.

human peripheral blood lymphocyte populations without regulating their activated function. *The Journal of Immunology*, Vol.146, pp. 1921-1927, ISSN

determines development of disease. *Medical Hypotheses*, Vol. 49, pp.1-14, ISSN

JM. (2001). Use of human immunodeficiency virus-1 protease inhibitors is associated with atherogenic lipoprotein changes and endothelial dysfunction.

a novel early biochemical marker of oxidative stress in HIV infected individuals.

Goldstein, B.J. & Ma, X.L. (2007). Adiponectin cardioprotection after myocardial ischemia/reperfusion involves the reduction of oxidative/nitrative stress.

nitrite in the sera of children infected with human immunodeficiency virus type 1.

concentrations of nitrite in patients with HIV-1 infection. *Journal of Clinical* 


**3** 

*México* 

**HIV Toxins: Gp120 as an** 

Leonor Huerta and César N. Cortés Rubio

**Independent Modulator of Cell Function** 

*Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México* 

Current notions on the pathogenesis of the HIV-induced disease sustain that progressive immunodeficiency results from a combination of the cell cytotoxicity produced by infection and replication of the virus in the target cells, mainly immune system cells, and of the indirect, harmful effects mediated by two main mechanisms: a sustained, chronic activation of the immune system that turns into immune dysfunction with the progressive degradation of lymphoid tissues, and the immunoregulatory and toxic properties of extracellular viral proteins on bystander cells (Choudhary et al., 2007; Moir et al., 2011). Bystander, non infected cells that show altered function and death, include cells with null or low expression of the CD4

receptor, such as CD8+ T and B lymphocytes, dendritic cells, neurons and tumor cells.

literature illustrating the diversity of the effects induced by this molecule.

The HIV Env protein is synthesized in the form of the gp160 precursor, which processing and folding occur through what is known as the secretory pathway: the Env precursor

**2. Structure-function and evolutionary considerations** 

The HIV-1 Gp120 protein has properties that maintain resemblance with animal toxins. Active forms of free Gp120 can be found at nanomolar concentrations in the plasma of a considerable proportion of HIV infected individuals (Rychert et al., 2010; Gilbert et al., 1991; Santosuosso et al., 2009). A number of in vitro and in vivo activities have been described for the extracellular form of this molecule, indicating that it may contribute to deregulation of immune function and damage to several tissues during HIV infection. Activation, apoptosis, chemotaxis and impaired cellular function are the most frequently reported effects of Gp120 in the absence of HIV infection. Gp120 interacts with chemokine receptors (mainly CXCR4 and CCR5) which are expressed by different cells and tissues, besides the immune system, thus providing a range of possible target cells for toxic effects. However, the high structural variability of Gp120, absorption by host's glycan-binding compounds, and the complexity of the regulation processes involved in chemokine receptor function, have made difficult to asses the actual significance of the free form of this molecule for AIDS pathogenesis. On the other hand, soluble Gp120 or peptides derived of active portions of the molecule may be considered as potential therapeutic agents to target undesirable cells, i.e., tumor cells. This article provides a review of the main factors influencing the biological outcome of the interaction of the soluble form of Gp120 with cells and tissues, and a selection of recent

**1. Introduction** 


## **HIV Toxins: Gp120 as an Independent Modulator of Cell Function**

Leonor Huerta and César N. Cortés Rubio

*Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México México* 

## **1. Introduction**

68 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Xu, A.; Yin, S.; Wong, I.; Chan, K.W. & Lam, K.S. (2004). Adiponectin

Yamaguchi, Y.; Yoshikawa, N.; Kagota, S.; Nakamura, K.; Haginaka, J. & Kunitomo, M.

Yano, S.;Yano, N.; Rodriguez, N.; Baek, J.H.; Que, X.; Yamamura, Y. & Kim, S.J. (1998).

Zangerle, R.; Fuchs, D.; Reibnegger, G.; Werner-Felmayer, G.; Gallati, H.; Wachter, H. &

ISSN 0013-7227.

0171-2985.

pp. 380-386, ISSN 1089-8603.

24, No.2, pp. 349-359, ISSN 0891-5849.

ameliorated dyslipidemia induced by the human immunodeficiency virus protease inhibitor ritonavir in mice. *Endocrinology*, Vol. 145, No.2, pp. 487-494,

(2006). Elevated circulating levels of markers of oxidative-nitrative stress and inflammation in a genetic rat model of metabolic syndrome. *Nitric Oxide*, Vol.15,

Suppression of intracellular hydrogen peroxide generation and catalase levels in CD8+ T lymphocytes from HIV+ individuals. *Free Radical Biology & Medicine*, Vol.

Werner, E.R. (1995). Serum nitrite plus nitrate in infection with human immunodeficiency virus type 1. *Immunobiology*, Vol. 193, No.1, pp. 59-70, ISSN

Current notions on the pathogenesis of the HIV-induced disease sustain that progressive immunodeficiency results from a combination of the cell cytotoxicity produced by infection and replication of the virus in the target cells, mainly immune system cells, and of the indirect, harmful effects mediated by two main mechanisms: a sustained, chronic activation of the immune system that turns into immune dysfunction with the progressive degradation of lymphoid tissues, and the immunoregulatory and toxic properties of extracellular viral proteins on bystander cells (Choudhary et al., 2007; Moir et al., 2011). Bystander, non infected cells that show altered function and death, include cells with null or low expression of the CD4 receptor, such as CD8+ T and B lymphocytes, dendritic cells, neurons and tumor cells.

The HIV-1 Gp120 protein has properties that maintain resemblance with animal toxins. Active forms of free Gp120 can be found at nanomolar concentrations in the plasma of a considerable proportion of HIV infected individuals (Rychert et al., 2010; Gilbert et al., 1991; Santosuosso et al., 2009). A number of in vitro and in vivo activities have been described for the extracellular form of this molecule, indicating that it may contribute to deregulation of immune function and damage to several tissues during HIV infection. Activation, apoptosis, chemotaxis and impaired cellular function are the most frequently reported effects of Gp120 in the absence of HIV infection. Gp120 interacts with chemokine receptors (mainly CXCR4 and CCR5) which are expressed by different cells and tissues, besides the immune system, thus providing a range of possible target cells for toxic effects. However, the high structural variability of Gp120, absorption by host's glycan-binding compounds, and the complexity of the regulation processes involved in chemokine receptor function, have made difficult to asses the actual significance of the free form of this molecule for AIDS pathogenesis. On the other hand, soluble Gp120 or peptides derived of active portions of the molecule may be considered as potential therapeutic agents to target undesirable cells, i.e., tumor cells.

This article provides a review of the main factors influencing the biological outcome of the interaction of the soluble form of Gp120 with cells and tissues, and a selection of recent literature illustrating the diversity of the effects induced by this molecule.

## **2. Structure-function and evolutionary considerations**

The HIV Env protein is synthesized in the form of the gp160 precursor, which processing and folding occur through what is known as the secretory pathway: the Env precursor

HIV Toxins: Gp120 as an Independent Modulator of Cell Function 71

of the Env complex and that of the pore-forming toxins such as the actinoporins, the sea anemone toxins: 1) attachment of toxin to the cell surface through recognition of specific cellular membrane components; 2) transfer of the N-terminal segment to the lipid-water interface; 3) oligomerization of the toxin on the cell surface followed by the insertion of multiple -helices monomeres into the membrane to form an ion conductive channel (Kristan et al., 2009; Edwards & Hessinger 2000; Butzke & Luch, 2010). As in the case of Env, the N-terminal portion of the toxin is essential for the final pore formation step (Kristan 2009). Finally, membrane-binding and pore-forming functions relay on different domains in

Early works reported sequence homology between a short portion of Gp120 and the putative actives sites of the snake neurotoxin alpha-bungarotoxin and the rabies virus glycoprotein (Neri et al., 1990; Bracci & Neri, 1995), which interact with the mammals' nicotinic acetylcholine receptor, a member of the ligand-gated ion channel proteins. Later, it was found that Gp120 can bind to the acetylcholine binding site of the nicotinic receptor and the binding can be inhibited by an albumin-conjugated peptide encompassing the 160-170 amino acids of Gp120 (Bracci et al., 1997), which belong to a relatively conserved region of the Gp120 V2 loop. Gp120 can act as competitive antagonist of the nicotinic acetycholine receptors. Although the overall structure of the snake neurotoxins consists of a low molecular weight protein with three beta-strands with finger-like loops (Pawlak et al., 2006; Ackermann et al., 1998), and thus it is quite different to that of Gp120 and the rabies glycoprotein (both belonging to the Class I fusion proteins), the homologous sequence was located in a loop structure in both viral proteins and the snake neurotoxin, suggesting an evolutionary convergence towards the appropriate acetylcholine receptor binding structure.

In principle, the biological effects of the interaction of Gp120 with CXCR4 and CCR5 may be conditioned by events similar to those regulating the coreceptor activity after interaction with their corresponding natural ligands. This section presents a general review of the characteristics of these receptors and the main extracellular events regulating their function. Comprehensive reviews of the regulatory pathways involved in CXCR4 and CCR5 signaling can be found elsewhere (Busillo & Benovic 2007; Kucia et al., 2005; Oppermann, 2004; Wu &

CXCR4 and CCR5 belong to the super family of the seven-transmembrane G-protein coupled receptors (GPCRs). CXCR4 has SDF-1 as its sole natural ligand, whereas CCR5 can interact with several chemokines, mainly CCL5, CCL3, CCL4, CCL8 and CCL14 (RANTES, MIP-1alpha, MIP-1beta, MCP-2, and CC-1, respectively). Ligand binding triggers phosphorylation at various sites of the intracellular domains, which act as signals for migration, activation and transcription. Like for others GPCRs, ligand binding induces receptor desensitization and internalization to avoid prolonged activation, followed by degradation or recycling (Marchese et al., 2008). In addition, chemokine receptors can also be subjected to "heterologous desensitization", i.e., inhibition of receptor function by signaling processes triggered by ligand binding to an unrelated GPCR. Thus, cross heterologous desensitization of T cell functions can be induced by CCR5 and CXCR4 ligands, resulting in mutual interference with cellular signaling, adhesion and chemotaxis (Hecht et al., 2003). In another example, it has been shown that activation of toll-like receptor 2 (TLR2) negatively regulates CCR5 on human blood monocytes, inhibiting

both Env and animal pore-forming toxins.

**3. The CXCR4 and CCR5 chemokine receptors** 

Yoder, 2009).

protein (gp160) is co-translationally translocated into the endoplasmic reticulum (ER), where 10 disulfide bonds are formed and the molecule starts to fold. Glycosylation of most of the approximately 30 potential N-linked glycosylation sites, with around 25 of them located in the Gp120 region (Zhang et al., 2004), also occurs co-translationally in the ER. Disulfide bond formation and glycosylation, along with interaction with the lectin chaperones calnexin and calreticulin, allows the proper final folding of the gp160 precursor (Earl et al., 1991; Otteken et al., 1996). Then, the molecule forms trimers and is transported to the Golgi complex, where the cleavage into the surface (Gp120) and transmembrane (Gp41) subunits is carried out. Cleavage of the precursor by host proteases generates the N-terminal hydrophobic fusion peptide of Gp41. Gp120 and Gp41 are kept joined by non-covalent interactions on the surface of infected cells and virions.

Binding to cell membranes and disruption of the lipid bilayer integrity are the basic functional properties of the HIV Env complex. Env mediates the fusion of biological membranes that allows the entry of the virus nucleocapsid into target cells, as well as the fusion of infected with non-infected cells. Env-mediated membrane fusion is involved in virus entry, cell-to-cell transmission of virus particles, and syncytia formation. Membrane fusion is a multi-step process which is conducted by Gp120/Gp41 heterotrimers and involves: a) binding of Gp120 to the CD4 receptor on the cell surface, an interaction that is favored by adhesion molecules (Cantin et al., 1997, Bastiani et al., 1997); b) conformational rearrangements allowing Gp120 to interact with a coreceptor molecule, mainly CCR5 and CXCR4; c) projection of a trimer formed by the extended chains of the Gp41 ectodomain; d) insertion of the Gp41 amino-terminal hydrophobic ends, the fusion peptides, into the target membrane and the subsequent packing of the Gp41 molecule into a 6-helix bundle, a structure which formation provides the free energy necessary for membrane fusion (Jones et al., 1998; Melikyan et al., 2000; Sattentau & Moore, 1991; Sullivan et al., 1998; Trkola et al., 1996; Wu et al., 1996; Wyatt & Sodroski, 1998).

Gp120 oligosaccharide moieties greatly influence Gp120 folding, processing, and intracellular transport (Stansell & Desrosiers, 2010), and the ability of the virus to escape from host neutralizing antibodies. N-linked glycosylation sites are main targets of neutralizing antibodies, which exert selective pressure on the viral surface. Thus, it has been postulated that the evolving glycan shield is a mechanism to avoid elimination of the infection by the humoral immune response (Wei et al., 2003; Canducci et al, 2009). Instead, it has been frequently observed that the enzymatic removal of Gp120 oligosaccharides does not greatly affect the interaction of Gp120 with CD4 (Bahraoui et al., 1992; Fenouillet et al., 1989). However, glycosylation is necessary for the acquisition of the proper folding of Gp120 in the ER required for interaction with CD4 (Li et al., 1993). On the other hand, glycans play an important role in the usage of CXCR4 and CCR5 (Polzer et al., 2002; Ogert et al., 2001; Bandres et al., 1998).

Env share a number of structural and biological characteristics with pore-forming protein toxins from widely separated phyla such as bacteria, plants, cnidaria and mammals (Iacovache, et al., 2008): they undergo extensive post-translational modifications, are specific for susceptible structures (acceptor sites), have an hetero-oligomer structure, they tend to aggregate, show variable toxic efficiency among different cell types, act through their poreforming activity (in conjunction with Gp41), have neurotoxic effects, and present considerable and continuous genetic variation (Butzke & Luch, 2010; Suput, 2009; Kristan et al., 2009). Particularly, a striking similitude exists among the mechanism of pore formation

protein (gp160) is co-translationally translocated into the endoplasmic reticulum (ER), where 10 disulfide bonds are formed and the molecule starts to fold. Glycosylation of most of the approximately 30 potential N-linked glycosylation sites, with around 25 of them located in the Gp120 region (Zhang et al., 2004), also occurs co-translationally in the ER. Disulfide bond formation and glycosylation, along with interaction with the lectin chaperones calnexin and calreticulin, allows the proper final folding of the gp160 precursor (Earl et al., 1991; Otteken et al., 1996). Then, the molecule forms trimers and is transported to the Golgi complex, where the cleavage into the surface (Gp120) and transmembrane (Gp41) subunits is carried out. Cleavage of the precursor by host proteases generates the N-terminal hydrophobic fusion peptide of Gp41. Gp120 and Gp41 are kept joined by non-covalent

Binding to cell membranes and disruption of the lipid bilayer integrity are the basic functional properties of the HIV Env complex. Env mediates the fusion of biological membranes that allows the entry of the virus nucleocapsid into target cells, as well as the fusion of infected with non-infected cells. Env-mediated membrane fusion is involved in virus entry, cell-to-cell transmission of virus particles, and syncytia formation. Membrane fusion is a multi-step process which is conducted by Gp120/Gp41 heterotrimers and involves: a) binding of Gp120 to the CD4 receptor on the cell surface, an interaction that is favored by adhesion molecules (Cantin et al., 1997, Bastiani et al., 1997); b) conformational rearrangements allowing Gp120 to interact with a coreceptor molecule, mainly CCR5 and CXCR4; c) projection of a trimer formed by the extended chains of the Gp41 ectodomain; d) insertion of the Gp41 amino-terminal hydrophobic ends, the fusion peptides, into the target membrane and the subsequent packing of the Gp41 molecule into a 6-helix bundle, a structure which formation provides the free energy necessary for membrane fusion (Jones et al., 1998; Melikyan et al., 2000; Sattentau & Moore, 1991; Sullivan et al., 1998; Trkola et al.,

Gp120 oligosaccharide moieties greatly influence Gp120 folding, processing, and intracellular transport (Stansell & Desrosiers, 2010), and the ability of the virus to escape from host neutralizing antibodies. N-linked glycosylation sites are main targets of neutralizing antibodies, which exert selective pressure on the viral surface. Thus, it has been postulated that the evolving glycan shield is a mechanism to avoid elimination of the infection by the humoral immune response (Wei et al., 2003; Canducci et al, 2009). Instead, it has been frequently observed that the enzymatic removal of Gp120 oligosaccharides does not greatly affect the interaction of Gp120 with CD4 (Bahraoui et al., 1992; Fenouillet et al., 1989). However, glycosylation is necessary for the acquisition of the proper folding of Gp120 in the ER required for interaction with CD4 (Li et al., 1993). On the other hand, glycans play an important role in the usage of CXCR4 and CCR5 (Polzer et al., 2002; Ogert et al., 2001;

Env share a number of structural and biological characteristics with pore-forming protein toxins from widely separated phyla such as bacteria, plants, cnidaria and mammals (Iacovache, et al., 2008): they undergo extensive post-translational modifications, are specific for susceptible structures (acceptor sites), have an hetero-oligomer structure, they tend to aggregate, show variable toxic efficiency among different cell types, act through their poreforming activity (in conjunction with Gp41), have neurotoxic effects, and present considerable and continuous genetic variation (Butzke & Luch, 2010; Suput, 2009; Kristan et al., 2009). Particularly, a striking similitude exists among the mechanism of pore formation

interactions on the surface of infected cells and virions.

1996; Wu et al., 1996; Wyatt & Sodroski, 1998).

Bandres et al., 1998).

of the Env complex and that of the pore-forming toxins such as the actinoporins, the sea anemone toxins: 1) attachment of toxin to the cell surface through recognition of specific cellular membrane components; 2) transfer of the N-terminal segment to the lipid-water interface; 3) oligomerization of the toxin on the cell surface followed by the insertion of multiple -helices monomeres into the membrane to form an ion conductive channel (Kristan et al., 2009; Edwards & Hessinger 2000; Butzke & Luch, 2010). As in the case of Env, the N-terminal portion of the toxin is essential for the final pore formation step (Kristan 2009). Finally, membrane-binding and pore-forming functions relay on different domains in both Env and animal pore-forming toxins.

Early works reported sequence homology between a short portion of Gp120 and the putative actives sites of the snake neurotoxin alpha-bungarotoxin and the rabies virus glycoprotein (Neri et al., 1990; Bracci & Neri, 1995), which interact with the mammals' nicotinic acetylcholine receptor, a member of the ligand-gated ion channel proteins. Later, it was found that Gp120 can bind to the acetylcholine binding site of the nicotinic receptor and the binding can be inhibited by an albumin-conjugated peptide encompassing the 160-170 amino acids of Gp120 (Bracci et al., 1997), which belong to a relatively conserved region of the Gp120 V2 loop. Gp120 can act as competitive antagonist of the nicotinic acetycholine receptors. Although the overall structure of the snake neurotoxins consists of a low molecular weight protein with three beta-strands with finger-like loops (Pawlak et al., 2006; Ackermann et al., 1998), and thus it is quite different to that of Gp120 and the rabies glycoprotein (both belonging to the Class I fusion proteins), the homologous sequence was located in a loop structure in both viral proteins and the snake neurotoxin, suggesting an evolutionary convergence towards the appropriate acetylcholine receptor binding structure.

## **3. The CXCR4 and CCR5 chemokine receptors**

In principle, the biological effects of the interaction of Gp120 with CXCR4 and CCR5 may be conditioned by events similar to those regulating the coreceptor activity after interaction with their corresponding natural ligands. This section presents a general review of the characteristics of these receptors and the main extracellular events regulating their function. Comprehensive reviews of the regulatory pathways involved in CXCR4 and CCR5 signaling can be found elsewhere (Busillo & Benovic 2007; Kucia et al., 2005; Oppermann, 2004; Wu & Yoder, 2009).

CXCR4 and CCR5 belong to the super family of the seven-transmembrane G-protein coupled receptors (GPCRs). CXCR4 has SDF-1 as its sole natural ligand, whereas CCR5 can interact with several chemokines, mainly CCL5, CCL3, CCL4, CCL8 and CCL14 (RANTES, MIP-1alpha, MIP-1beta, MCP-2, and CC-1, respectively). Ligand binding triggers phosphorylation at various sites of the intracellular domains, which act as signals for migration, activation and transcription. Like for others GPCRs, ligand binding induces receptor desensitization and internalization to avoid prolonged activation, followed by degradation or recycling (Marchese et al., 2008). In addition, chemokine receptors can also be subjected to "heterologous desensitization", i.e., inhibition of receptor function by signaling processes triggered by ligand binding to an unrelated GPCR. Thus, cross heterologous desensitization of T cell functions can be induced by CCR5 and CXCR4 ligands, resulting in mutual interference with cellular signaling, adhesion and chemotaxis (Hecht et al., 2003). In another example, it has been shown that activation of toll-like receptor 2 (TLR2) negatively regulates CCR5 on human blood monocytes, inhibiting

HIV Toxins: Gp120 as an Independent Modulator of Cell Function 73

Wieczorek et al., 2000; Spiegel et al., 2004). Studies using RNA interference (RNAi) to reduce the expression of CXCR4 in animal models, have found that this treatment readily reduce growth and inhibits metastasis in a number of tumors, like breast and prostate cancer (Liang et al., 2005; Wang et al., 2011), melanoma (Kim et al., 2010), and neuroblastoma (Wang et al.,

Although Gp120 interaction with CD4 can induce signaling events in many cell types, a number of effects that were originally attributed to the Gp120-CD4 interaction have been recently found to be explained by binding and signaling events mediated mainly by CXCR4 and CCR5. It should be noted, however, that although signaling intermediates recruited by Gp120 and the natural chemokine receptor ligands are usually the same, the ability of Gp120 to activate those signal transduction pathways may depend on the cell activation status. In general, activated cells are more sensitive to the activity of Gp120 than resting cells (Kinet et

Gp120 signaling through CXCR4 triggers intracellular events facilitating infection by the HIV. Recently, it was found that Gp120 increases the dynamics of actin by activating cofilin, an actin-depolymerizing factor, which promotes the movement of the viral preintegration complex toward the centre of the cytoplasm. CXCR4-mediated actin rearrangement markedly facilitates viral infection of resting T cells (Yoder et al., 2008). Similarly, CXCR4 signaling after interaction with Gp120 is involved in a variety of other activation events in T cells and macrophages (Table 1). Likewise, it is well known that Gp120 exerts chemotactic effects on T, dendritic cells (DC), and monocyte/macrophages (Table 1). Conversely, it has been also reported that Gp120 can inhibit migration of T (Trushin et al., 2010) and B cells (Badr et al., 2005). It has been suggested that reprogramming of the CD4+ T-cell migration behavior induced by Gp120 may provides a mechanism for lymphadenopathy during HIV

A study using oligonucleotide microarrays showed that tropism of Gp120 for the CCR5 and CXCR4 receptors, along with the cell activation status, are related to the Gp120 biological activity. R5 and X4 HIV envelopes (CCR5 and CXCR4-tropic Gp120, respectively) were found to induce distinct gene expression profiles in primary peripheral blood mononuclear cells (Cicala et al., 2006a). In this study, both R5 and X4 Gp120 activated genes associated with cell proliferation and protein tyrosine kinases, although R5 envelopes were more pronounced in their capacity to activate the p38 mitogen-activated protein kinase (p38 MAPK) cascade. In addition, R5 Gp120 exclusively activated a subset of genes in the resting CD4+ T cell population derived from viremic individuals. p38 is activated in macrophages, neutrophils, and T cells by numerous extracellular mediators of inflammation, including chemoattractants, cytokines, chemokines, and bacterial lipopolysaccharide. Functional responses involving p38 include respiratory burst activity, chemotaxis, granular exocytosis, adherence and apoptosis (Ono & Han, 2000). Activation of p38 kinase has also been associated with HIV replication (Muthumani et al., 2004) and thus, it is proposed that R5 envelopes induce genes that may facilitate replication of virus in resting CD4+ T cells, contributing to the establishment and/or maintenance of viral reservoirs, and the productive infection at mucosal surfaces, favoring transmission (Cicala et al., 2006a). Other studies also shown that R5 and X4 Gp120 can activate NFATs and induce their translocation into the nucleus. Translocation of NFATs is an important signal for HIV transcription, given

**4. In vitro and in vivo effects of extracellular Gp120 on cell function** 

al., 2002; Weissman et al., 1997; Schols & De Clercq, 1996).

infection (Green et al., 2009).

2006).

monocyte migration after pathogen recognition (Fox et al., 2011). On the other hand, it is clear that that signaling through CD4 by the CD4 ligand interleukin-16 (IL-16) desensitizes the chemokine receptors CCR5, CXCR4, and CXCR3 (Rahangdale et al., 2006; Van Drenth et al., 2000).

CXCR4 is expressed by many tissues and cell types, such as T leukocytes, progenitor cells in the bone morrow, endothelial (Murdoch et al., 1999) and epithelial cells (lung, retina, intestine), and tumor cells. In the brain, CXCR4 has been found in the endothelial cells forming the blood-brain barrier, microglia, neurons, and astrocytes (Berger et al., 1999; Edinger et al., 1997). CXCR4 is important for lymphocyte trafficking and recruitment of lymphocytes and monocytes at sites of inflammation, and plays a role in cell proliferation, organogenesis and vascularization. On the other hand, CCR5 is expressed on resting T-cells with a memory/effector phenotype, monocytes, macrophages and immature dendritic cells (Blanpain et al., 2002). Differentiation of monocytes to macrophages is accompanied by an increase of the CCR5 expression (Kaufmann et al., 2001). Increased CCR5 expression has been found to be induced by interferon-alpha (IFN-alpha) in thymus implants infected by the R5 HIV (Stoddart et al., 2010). Expression of CCR5 in T CD4+ cells is particularly high in mucosa-associated lymphoid tissues (MALT), where the fraction of CCR5+ CD4+ T cells is >50%. It is known that signaling through CCR5 is significantly involved in the induction of an immunological hyporesponsive state that leads to oral tolerance to high doses of antigen (DePaolo et al., 2004) and prevents uncontrolled postinfarction inflammation of myocardium in mice (Dobaczewski et al., 2010). Anti-inflammatory properties of CCR5+ mononuclear cells have been related to the expression of high levels of IL-10 and their ability to recruit CD4+/foxp3+ regulatory T cells (Tregs) (Dobaczewski et al., 2010).

The expression of CXCR4 on the cell surface is increased by several cytokines (IL-4, IL-2, IL-7, IL-10, IL-5, TGF-1), as well as by fibroblast and vascular growth factors, whereas it is reduced by others, mainly those pro-inflammatory cytokines (TNF-alpha, INF-gamma, and IL-1-beta). However this pattern is not absolute and it is thought that mixed signals regulate de expression of CXCR4 signaling in different circumstances (reviewed in Busillo & Benovic 2007). On the other hand, sensitization of CXCR4 (priming to low concentrations of SDF) through its translocation to lipid rafts during inflammatory responses has also been described (Wysoczynski et al., 2005).

Membrane events participating in the regulation of CXCR4 and CCR5 function include dimerization as well as extensive downregulation by endocytosis and/or macropinocytosis. In addition, proteases released by neutrophils cleavage the N-terminus extracellular portion of CXCR4, avoiding ligand interaction (Hezareh et al., 2004; Lévesque et al., 2003).

In the last decade, the CXCR4-SF-1 axis has been increasingly involved in the generation, progression and metastasis of a variety of tumors, so that the expression of CXCR4 is currently considered an important biomarker for identification of the metastatic potential of primary tumors and a potential therapeutic target (Nimmagadda et al., 2010; Muller et al., 2001). CXCR4 was found to be one of the few genes which elevated over expression and function was associated to high osteolytic bone metastatic activity of human breast cancer cells in immunodeficient mice (Kang et al., 2003). In addition, CXCR4 is expressed on normal tissue-committed stem cells, which are currently considered a potential source of transformed cells. There are evidences that the CXCR4-SDF-1 axis can mediate locomotion, chemotaxis, adhesion, and even proliferation and survival of these cells, as well as the secretion of matrix proteases by different cell types (Fernandis et al., 2004; Janowska-

monocyte migration after pathogen recognition (Fox et al., 2011). On the other hand, it is clear that that signaling through CD4 by the CD4 ligand interleukin-16 (IL-16) desensitizes the chemokine receptors CCR5, CXCR4, and CXCR3 (Rahangdale et al., 2006; Van Drenth et

CXCR4 is expressed by many tissues and cell types, such as T leukocytes, progenitor cells in the bone morrow, endothelial (Murdoch et al., 1999) and epithelial cells (lung, retina, intestine), and tumor cells. In the brain, CXCR4 has been found in the endothelial cells forming the blood-brain barrier, microglia, neurons, and astrocytes (Berger et al., 1999; Edinger et al., 1997). CXCR4 is important for lymphocyte trafficking and recruitment of lymphocytes and monocytes at sites of inflammation, and plays a role in cell proliferation, organogenesis and vascularization. On the other hand, CCR5 is expressed on resting T-cells with a memory/effector phenotype, monocytes, macrophages and immature dendritic cells (Blanpain et al., 2002). Differentiation of monocytes to macrophages is accompanied by an increase of the CCR5 expression (Kaufmann et al., 2001). Increased CCR5 expression has been found to be induced by interferon-alpha (IFN-alpha) in thymus implants infected by the R5 HIV (Stoddart et al., 2010). Expression of CCR5 in T CD4+ cells is particularly high in mucosa-associated lymphoid tissues (MALT), where the fraction of CCR5+ CD4+ T cells is >50%. It is known that signaling through CCR5 is significantly involved in the induction of an immunological hyporesponsive state that leads to oral tolerance to high doses of antigen (DePaolo et al., 2004) and prevents uncontrolled postinfarction inflammation of myocardium in mice (Dobaczewski et al., 2010). Anti-inflammatory properties of CCR5+ mononuclear cells have been related to the expression of high levels of IL-10 and their

ability to recruit CD4+/foxp3+ regulatory T cells (Tregs) (Dobaczewski et al., 2010).

described (Wysoczynski et al., 2005).

The expression of CXCR4 on the cell surface is increased by several cytokines (IL-4, IL-2, IL-7, IL-10, IL-5, TGF-1), as well as by fibroblast and vascular growth factors, whereas it is reduced by others, mainly those pro-inflammatory cytokines (TNF-alpha, INF-gamma, and IL-1-beta). However this pattern is not absolute and it is thought that mixed signals regulate de expression of CXCR4 signaling in different circumstances (reviewed in Busillo & Benovic 2007). On the other hand, sensitization of CXCR4 (priming to low concentrations of SDF) through its translocation to lipid rafts during inflammatory responses has also been

Membrane events participating in the regulation of CXCR4 and CCR5 function include dimerization as well as extensive downregulation by endocytosis and/or macropinocytosis. In addition, proteases released by neutrophils cleavage the N-terminus extracellular portion

In the last decade, the CXCR4-SF-1 axis has been increasingly involved in the generation, progression and metastasis of a variety of tumors, so that the expression of CXCR4 is currently considered an important biomarker for identification of the metastatic potential of primary tumors and a potential therapeutic target (Nimmagadda et al., 2010; Muller et al., 2001). CXCR4 was found to be one of the few genes which elevated over expression and function was associated to high osteolytic bone metastatic activity of human breast cancer cells in immunodeficient mice (Kang et al., 2003). In addition, CXCR4 is expressed on normal tissue-committed stem cells, which are currently considered a potential source of transformed cells. There are evidences that the CXCR4-SDF-1 axis can mediate locomotion, chemotaxis, adhesion, and even proliferation and survival of these cells, as well as the secretion of matrix proteases by different cell types (Fernandis et al., 2004; Janowska-

of CXCR4, avoiding ligand interaction (Hezareh et al., 2004; Lévesque et al., 2003).

al., 2000).

Wieczorek et al., 2000; Spiegel et al., 2004). Studies using RNA interference (RNAi) to reduce the expression of CXCR4 in animal models, have found that this treatment readily reduce growth and inhibits metastasis in a number of tumors, like breast and prostate cancer (Liang et al., 2005; Wang et al., 2011), melanoma (Kim et al., 2010), and neuroblastoma (Wang et al., 2006).

## **4. In vitro and in vivo effects of extracellular Gp120 on cell function**

Although Gp120 interaction with CD4 can induce signaling events in many cell types, a number of effects that were originally attributed to the Gp120-CD4 interaction have been recently found to be explained by binding and signaling events mediated mainly by CXCR4 and CCR5. It should be noted, however, that although signaling intermediates recruited by Gp120 and the natural chemokine receptor ligands are usually the same, the ability of Gp120 to activate those signal transduction pathways may depend on the cell activation status. In general, activated cells are more sensitive to the activity of Gp120 than resting cells (Kinet et al., 2002; Weissman et al., 1997; Schols & De Clercq, 1996).

Gp120 signaling through CXCR4 triggers intracellular events facilitating infection by the HIV. Recently, it was found that Gp120 increases the dynamics of actin by activating cofilin, an actin-depolymerizing factor, which promotes the movement of the viral preintegration complex toward the centre of the cytoplasm. CXCR4-mediated actin rearrangement markedly facilitates viral infection of resting T cells (Yoder et al., 2008). Similarly, CXCR4 signaling after interaction with Gp120 is involved in a variety of other activation events in T cells and macrophages (Table 1). Likewise, it is well known that Gp120 exerts chemotactic effects on T, dendritic cells (DC), and monocyte/macrophages (Table 1). Conversely, it has been also reported that Gp120 can inhibit migration of T (Trushin et al., 2010) and B cells (Badr et al., 2005). It has been suggested that reprogramming of the CD4+ T-cell migration behavior induced by Gp120 may provides a mechanism for lymphadenopathy during HIV infection (Green et al., 2009).

A study using oligonucleotide microarrays showed that tropism of Gp120 for the CCR5 and CXCR4 receptors, along with the cell activation status, are related to the Gp120 biological activity. R5 and X4 HIV envelopes (CCR5 and CXCR4-tropic Gp120, respectively) were found to induce distinct gene expression profiles in primary peripheral blood mononuclear cells (Cicala et al., 2006a). In this study, both R5 and X4 Gp120 activated genes associated with cell proliferation and protein tyrosine kinases, although R5 envelopes were more pronounced in their capacity to activate the p38 mitogen-activated protein kinase (p38 MAPK) cascade. In addition, R5 Gp120 exclusively activated a subset of genes in the resting CD4+ T cell population derived from viremic individuals. p38 is activated in macrophages, neutrophils, and T cells by numerous extracellular mediators of inflammation, including chemoattractants, cytokines, chemokines, and bacterial lipopolysaccharide. Functional responses involving p38 include respiratory burst activity, chemotaxis, granular exocytosis, adherence and apoptosis (Ono & Han, 2000). Activation of p38 kinase has also been associated with HIV replication (Muthumani et al., 2004) and thus, it is proposed that R5 envelopes induce genes that may facilitate replication of virus in resting CD4+ T cells, contributing to the establishment and/or maintenance of viral reservoirs, and the productive infection at mucosal surfaces, favoring transmission (Cicala et al., 2006a). Other studies also shown that R5 and X4 Gp120 can activate NFATs and induce their translocation into the nucleus. Translocation of NFATs is an important signal for HIV transcription, given

HIV Toxins: Gp120 as an Independent Modulator of Cell Function 75

Activation of PKCε and its upstream effector PI3K/Akt, involved in HIV-

CXCR4 in CD4+ and CD8+ T cells. CXCR4 Iyengar et al.,

Enhancing of expression of the cellular Tat cofactors Tat-Sf1 and

Activation of major G proteindependent pathways: calcium mobilization, phosphoinositide-3 kinase, and Erk-1/2 MAPK

Actin cytoskeleton rearrangements

Facilitation of viral replication in

Binding of gp120 to α4β, an integrin mediating migration of lymphocytes to gut-associated lymphoid tissue, activates LFA-1, favoring formation

Lck-dependent phosphorylation and inactivation of cofilin, a cellular depolymerizing factor.

lymphocytes Chemotaxis Chemotaxis CCR5 Weissman

**involved Reference** 

CXCR4 Missè et al., 2005

et al., 1997

et al., 2004

Cicala et al., 2006

Arthos et al., 2008

1999

CXCR4 Balabanian

CD4 Trushin et al., 2010

CXCR4 Yoder et al., 2008

CD4 Hashimoto

CXCR4 Herbein et al., 1998

CD4 Schols et al., 1996

1999

et al., 1997

CD4 CCR5 CXCR4

Integrin α4β7

**Cell Mayor finding Mechanism or concurrent events Receptor**

1 replication.

SPT5.

activation.

resting cells.

nucleous.

CD8+ T cells.

unstimulated PBMC. Reduction of activation and proliferation of CD8+ T cells.

of virological synapses.

Activation of the cellular actin depolymerizing factor cofilin, which promotes the movement of the viral preintegration complex to the

Reduction of the expression of the proto-oncogene Bcl-2 with induction of apoptosis in CD4+ but not in

Activation of macrophages for enhanced expression of TNF and TNFRII. Apoptosis was mediated by the interaction between macrophage TNF-α and the TNFRII on CD8+ T.

Synthesis of high amounts of IL-10 by

Chemotaxis CD4-independent signaling. CXCR4 Iyengar et al.,

lymphocytes Chemotaxis CD4-independent signaling through

Umbilical cord blood CD4+ T lymphocytes

Blood CD4+T

Blood CD4+ T

Blood CD4+ T lymphocytes

Blood CD4+ T lymphocytes

Blood CD4+ T lymphocytes

Blood CD4+ T lymphocytes

Blood resting CD4+ T lymphocytes

Blood CD4+ and

Blood CD8+ T lymphocytes

Blood CD8+ T

Blood CD4+ and

CD8+ T lymphocytes

lymphocytes Anergy

CD8+ T lymphocytes HIV-1 replication in non-dividing

Chemotaxis of unstimulated CD4+ T cells.

Inhibition of SDF-1-induced chemotaxis

NFAT nuclear translocation in resting cells

Activation of LFA-1

Induction of actin dynamics in resting CD4+ T

cells

Apoptosis

Apoptosis dependent of macrophage activation

cells

that the HIV long terminal repeat (LTR) contains NFATs binding sites which are able to enhance transcription of viral genes (Cron et al., 2000; Cicala et al., 2006b; Kinoshita et al., 1998; Williams & Greene, 2007).


that the HIV long terminal repeat (LTR) contains NFATs binding sites which are able to enhance transcription of viral genes (Cron et al., 2000; Cicala et al., 2006b; Kinoshita et al.,

> Protein tyrosine kinase Pyk2 phosphorylation-dependent cell

Translocation of Gp120 and CXCR4

CD4-independent phosphorylation

Inhibition of T cell activation and signaling through the TCR by Gp120/anti- Gp120 complexes, probably by sequestering p56 (lck) to

Caspase 8 dependent NF-kappaB activation and enhanced HIV

perturbation of the transepithelial electrical resistance and decrease of

Activation of GPR15/Bob, presumably in a GalCer-rich membrane subdomain involving **involved Reference** 

CXCR4 Misse et al., 1999

CD4 Goldman

Davis et al., 1997

et al., 1997

Melar et al., 2007

Maresca et al.,

N.D.\* Bren et al., 2009

2003

CXCR4 Vlahakis et al., 2003

2000

2007

al., 2003

CD4 Schols et al., 1996

CXCR4 CCR5

CXCR4 CCR5

GPR15/ Bob GalCer CXCR4

protein CXCR4 Trushin et al.,

signal transduction pathway CD4 Kryworuchko et

**Cell Mayor finding Mechanism or concurrent events Receptor**

growth, survival and differentiation.

into early endosomes.

the cytoskeleton.

Ca2+ fluxing.

replication.

PKC activation. Microtubule disruption,

glucose absorption.

activation.

lymphocytes Apoptosis Activation of the proapoptotic p38

lymphocytes Anergy Dysregulation of the IL-2/IL-2R

Gia protein signaling, and independent of caspase cascade

lymphocytes Apoptosis Activation of caspases 3 and 6 CD4 Cicala et al.,

Diminished production of IL-2 and IL-4 and reduction of the proliferative

responses of stimulated cells Production of high amounts of IL-10, INFγ and TNF-α of unstimulated cells.

of Pyk2.

CHO cell line Ca2+ mobilization Coreceptor and CD4-dependent

1998; Williams & Greene, 2007).

transduction

Gp120-CXCR4 cointernalization, cell signaling, and chemotaxis

Apoptosis and viral replication

Functional alterations resembling HIV enteropathy

Apoptosis

CD4+ T cell lines Signal

CD4+ T cell line Anergy

T cell lines

CD4+ T cells, kidney epithelial cell lines and blood CD4+ lymphocytes

Intestinal cell

line

Human hepatocyte cell lines and primary hepatocytes

Blood CD4+ T

Blood CD4+ T

Blood CD4+ T

Blood CD4+ T

lymphocytes Anergy


HIV Toxins: Gp120 as an Independent Modulator of Cell Function 77

granule cells Neurotoxicity Signaling through CXCR4. CXCR4 Bachis et al.,

Activation of the mitochondrial

Enhancing of outward potassium currents via CXCR4 and cAMPdependent PKA signaling.

Imbalanced glutamine synthetase activity accompanied by generation

Pretreatment with platelet-derived growth factor BB reduced gp120 associated neurotoxicity and rescued

Induction of decreased levels of intracellular GSH (reduced glutathione), GPx (glutathione peroxidase), and GR (glutathione reductase) and increased levels of MDA (malondialdehyde)

Infusion of Gp120 into the brain enhanced tumor metastasis. Blocked

Tumor regression associated with significant decreases in CD44, CD34, and LYVE-1 and increases in caspase

Gp120 also supressed GHRH release

Loss of body weight in chronically

Arrests of cell cycle in G1 trough signaling by the p38 MAPK.

by antagonists of IL-1.

by pituitary cells in vitro.

treated animals.

3 and 9.

Gp120 and Tat induction of phosphorylation of MLK3

cells Apoptosis Caspase-3-mediated apoptosis. CXCR4 Bachis et al.,

**involved Reference** 

2004

CCR5 Melli et al., 2006

CXCR4 Xu et al., 2011

N.D. Ziye et al., 2006

2006

N.D. Peng et al., 2008

N.D. Price et al., 2005

et al., 1998

Mulroney et al., 1998

Okamoto et al., 2007

N.D. Hodgson

CXCR4 Singh et al., 2009

GHRH receptor

CXCR4 CCR5

N.D. Visalli et al., 2007

CXCR4

**Cell Mayor finding Mechanism or concurrent events Receptor**

caspase pathway.

(MAP3K11).

of free radicals.

neurite outgrowth.

Rat cerebellar

Rodent dorsal root ganglia and sensory neurons

Rat and human neurons Human monocytes

Rat cerebellar

Human astroglia

Human neuroblastoma cell line differentiated into neurons. Fetal rat neurons

Rat brain endothelial cell

Rat lung metastasis of mammary adenocarcinoma

cells.

mice

cells

from HIV/gp120 transgenic mice

Prostate cancer tumor in SCID

Rats pituitary

Rat neuronal progenitor cells

line

Rat microglia Neurotoxicity

Neurotoxicity, axonal

degeneration and apoptosis

Neuronal death and monocyte activation

Glutamine metabolism dysfuntion and apoptosis

Apoptosis

Oxidative stress

Tumor retention and enhancing of metastasis

Apoptosis and inhibition of tumor growth

Supression of growth hormone (GH) release

Inhibition of proliferation


Modulation of ~300 genes. Induction of the expression of cytokines, chemokines, kinases, and transcription factors associated with antigen-specific T cell activation but

not cell proliferation.

monocytes Anergy Induction of IL-10 production CD4 Schols et al.,

THP-1 and U937 cells.

pathways

cytokines.

kinase.

apoptosis.

prevented by TGF-β1.

chemoattractants.

Lyn, PI3K and Pyk2.

Enhancement of MCP-1, MIP-1β, and RANTES secretion by primary monocytes/ macrophages but not by

Pertussis toxin-insensitive signal transduction, activation of Ca2+ channels and Pyk2 and MAPK

Secretion of the MIP-1β and MCP-1

Release of pro-inflammatory

Activation of multiple protein kinases like the Src family kinase

Migration of dendritic cells mediated by a novel pathway involving phosphorylation of Pyk2 and activation of the p38 MAP

Gp120-induced cleavage of CD62L by a mechanism dependent on matrix metalloproteinase 1 and 3, CD4, CXCR4, G!i, and p38 MAPK. Increase of CD95-mediated

Activation of NMDA receptors with increase of neuronal calcium concentration. Impairment of neuronal calcium homeostasis was

Dramatic and persistent release of

Increment of intracellular calcium induced increased cyclooxygenase and 5-lipoxygenase activity. Membrane lipoperoxidation and mitochondrial uncoupling.

and apoptosis Activation of JNK N.D. Bodner et al.,

**involved Reference** 

N.D. Cicala et al., 2002

1996

2001

2001

CCR5 Cheung et al., 2008

CCR5 Anand et al., 2009

CCR5 Badr et al., 2005

1999

CXCR4 Maccarrone et al., 2002

2002

Meucci et al., 1996

Fantuzzi et al.,

Del Corno et al.,

CCR5 CXCR4

CCR5 CXCR4

CXCR4

NMDA receptors

calcium from intracellular stores. N.D. Medina et al.,

**Cell Mayor finding Mechanism or concurrent events Receptor**

PBMCs and monocytederived macrophages (MDMs)

Blood

Blood

Blood

MDMs

monocytes and MDMs

monocytes and MDMs

Immature DCs derived from monocytes

Tonsil primary naïve and memory B cells

Rat hippocampal neurons

Rat hippocampal

Rat hippocampal

neurons

Human neuroblastoma

neurons

cells

Transcriptional program conducive to productive HIV infection.

Production of β chemokines

Aberrant activation

Production of pro-inflammatory cytokines

Migration

Apoptosis and inhibition of B cell chemotaxis.

Apoptosis and necrosis

Increase of intracellular calcium concentration.

Neurotoxicity Necrosis

Neurotoxicity


HIV Toxins: Gp120 as an Independent Modulator of Cell Function 79

binding of soluble Gp120 to CD4 facilitate apoptosis of primary human CD4+ T cells, but that it was caused primordially by the Gp120-CXCR4 interaction, since apoptosis was prevented by the CXCR4 inhibitor AMD3100 and by the anti-CXCR4 antibody 12G5 (Trushin et al., 2007). Similarly, soluble Gp120-induced apoptosis mediated by CXCR4 was demonstrated in adult human hepatocytes, which lack CD4 (Vlahakis et al., 2003)**.** Binding of Gp120 to CXCR4 is also able to induce apoptosis of CD8+ T cells by upregulating the expression of TNF and TNF-receptor II on interacting CD8+ T cells and macrophages (Herbein, et al., 1998). Thus, the expression of CXCR4 or CCR5 may restrict the cell

Recent studies have shown that the expression of CXCR4 on cancer cells makes them susceptible to apoptosis induced by the HIV-1 envelope. Endo et al. (2008) observed that apoptosis of breast cancer cell lines induced by HIV-1 particles was dependent on Gp120 and CXCR4 but not CD4. In addition, a Gp120 mutant with low CD4 binding ability induced apoptosis in breast cancer cells but not in T-cells. Importantly, conformational heterogeneity of CXCR4 in breast cancer cells in comparison with CXCR4 in T cells was related to the ability of Gp120 to induce apoptosis mediated by CXCR4 (Endo et al., 2008, 2010). Likewise, it has been shown that the Gp120-CXR4 interaction mediated apoptosis of prostate cancer cell lines but not of normal prostatic epithelial cells (Singh et al., 2009). Anergy is a state of inhibition of proliferation and/or effector functions normally induced in T cells after encounter with antigen; the cell stay alive and functional inactivation is reversible upon antigen removal. It is induced by incomplete stimulation though the TCR and costimulatory molecules, and by the normal stimulation in the presence of IL-10 (Schwartz, 2003). Studies addressing the anergic effect of Gp120 use activation with anti-CD3 or mitogenactivation to simulate the effect of antigen stimulation. The contribution of anergy to the reduced immune function induced by X4 Gp120 in peripheral blood lymphocytes (PBMC) was early described by Schols and De Clercq (Schols & De Clercq, 1996). The addition of low concentrations of Gp120 was able to inhibit the proliferative response and the production of interleukin-2 (IL-2) and interleukin-4 (IL-4) in PBMC previously stimulated with an anti-CD3 antibody and concanavalin-A. In contrast, Gp120 induced the production of high amounts of IL-10, gamma interferon (IFN-g), and tumor necrosis factor alpha (TNF-a) in unstimulated PBMC. The induction of IL-10 by Gp120 was found to be important for the inhibitory effect of Gp120 on PBMC proliferation. Thus, X4 Gp120 can reduce the function of T lymphocytes by directly inducing anergy or by stimulation of the production of anergy-inducer immunosupressive cytokines. Importantly, the activation status played an important role in

sensitivity to Gp120 and explain the differential response of T cells subsets.

the cytokine pattern induced by Gp120 in PBMC (Schols & De Clercq, 1996).

CXCR4 by SDF-1 (Masci et al., 2003).

Evidence of the participation of chemokine receptors in the induction of anergy by Gp120 has been obtained in studies of the long-lasting hypo-responsiveness to antigen stimulation caused by Gp120 in naive T lymphocytes. Gp120 was found to induce anergy by stimulating the activity of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA), which causes the progressive accumulation of the phosphorylated form of the cAMP-responsive element binding, a pathway which is also activated by the ligation of

It should be noted that although there is an association between circulating Gp120 and the induction of proinflammatory and immunoregulatory cytokines like IL-6, IL-10, and TNFalpha in some HIV infected individuals (Rychert et al., 2010), this effect can not be necessarily induced by direct cell interaction with Gp120, since cytokines could be induced


\* Not determined

Table 1. *In vitro* and *in vivo e*ffects of soluble gp120

In addition to activation for proliferation, Gp120 can exert a diversity of potent effects in immune system cells in vitro, being apoptosis the most frequently reported, although anergy, and induction of proinflammatory cytokine production are also well known effects. An early review of the influence of Gp120 on the immune system was carried out by Chirmule and Pahwa in 1996 (see reference). Table 1 shows recent studies about the effect of Gp120 on immune system cells, confirming early findings and adding new effects, particularly those related to the induction or inhibition of chemotaxis, and the role of Gp120/anti-Gp120 immune complexes on depletion of bystander lymphocytes. Table 1 also shows the receptor implicated in each case.

Apoptosis is the event more frequently attributed to the interaction of Gp120 with CD4 and coreceptor molecules. The importance of Gp120-mediated apoptosis for AIDS pathogenesis was assessed in an early study performed on lymph-node cell suspensions prepared from three HIV-positive patients. Free Gp120 colabeled with both apoptotic and normal CD4+ T lymphocytes, although it was more often identified on apoptotic than on normal CD4+ T lymphocytes but not on CD8+ T lymphocytes or B cells. HIV particles were not found associated either with normal or apoptotic lymphocytes. This study pointed out that free Gp120 can bind to CD4+ T cells in lymph nodes of HIV-infected individuals and potentially mark them for premature death by apoptosis (Sunila et al., 1997).

Holm and cols., demonstrated that the affinity of native, virion-associated Gp120, for the CD4 and CXCR4 or CCR5 receptors was important for induction of apoptosis on primary human CD4+ T cells with an activated phenotype. In this study, virions expressing a mutant Gp120 defective for CD4 binding induced apoptosis, whereas mutants defective for CXCR4 binding did not. These observations indicated that the Gp120-CD4 interaction did not induce apoptosis, but seems to promote it by enhancing the exposure of the CXCR4 binding site on Gp120 (Holm et al., 2004). Gp120 expressed by *env*-transfected, non-infected cells, also induced CXCR4-dependent apoptosis in umbical cord CD4+ CXCR4+ cells; apoptosis was inhibited by SDF-1 (Roggero et al., 2001).

As for virion-associated Gp120, studies performed with recombinant Gp120 showed that Gp120 induced apoptosis through Fas-dependent and Fas-independent mechanisms and that not all lymphocytes were equally sensitive (reviwed in Cicala et al., 2000). Induction of apoptosis by soluble Gp120 was characterized by Thrushin and cols., whose shown that

Reduction of the expression of ICAM-1- and laminin. Lipidperoxidation.

Enhancing of sensitivity to CCL20 and CCL21 and inhibition of migration in response to

sphingosine-1-phosphate. Increased accumulation of cells in lymph nodes with a reciprocal decrease in

In addition to activation for proliferation, Gp120 can exert a diversity of potent effects in immune system cells in vitro, being apoptosis the most frequently reported, although anergy, and induction of proinflammatory cytokine production are also well known effects. An early review of the influence of Gp120 on the immune system was carried out by Chirmule and Pahwa in 1996 (see reference). Table 1 shows recent studies about the effect of Gp120 on immune system cells, confirming early findings and adding new effects, particularly those related to the induction or inhibition of chemotaxis, and the role of Gp120/anti-Gp120 immune complexes on depletion of bystander lymphocytes. Table 1 also

Apoptosis is the event more frequently attributed to the interaction of Gp120 with CD4 and coreceptor molecules. The importance of Gp120-mediated apoptosis for AIDS pathogenesis was assessed in an early study performed on lymph-node cell suspensions prepared from three HIV-positive patients. Free Gp120 colabeled with both apoptotic and normal CD4+ T lymphocytes, although it was more often identified on apoptotic than on normal CD4+ T lymphocytes but not on CD8+ T lymphocytes or B cells. HIV particles were not found associated either with normal or apoptotic lymphocytes. This study pointed out that free Gp120 can bind to CD4+ T cells in lymph nodes of HIV-infected individuals and potentially

Holm and cols., demonstrated that the affinity of native, virion-associated Gp120, for the CD4 and CXCR4 or CCR5 receptors was important for induction of apoptosis on primary human CD4+ T cells with an activated phenotype. In this study, virions expressing a mutant Gp120 defective for CD4 binding induced apoptosis, whereas mutants defective for CXCR4 binding did not. These observations indicated that the Gp120-CD4 interaction did not induce apoptosis, but seems to promote it by enhancing the exposure of the CXCR4 binding site on Gp120 (Holm et al., 2004). Gp120 expressed by *env*-transfected, non-infected cells, also induced CXCR4-dependent apoptosis in umbical cord CD4+ CXCR4+ cells; apoptosis

As for virion-associated Gp120, studies performed with recombinant Gp120 showed that Gp120 induced apoptosis through Fas-dependent and Fas-independent mechanisms and that not all lymphocytes were equally sensitive (reviwed in Cicala et al., 2000). Induction of apoptosis by soluble Gp120 was characterized by Thrushin and cols., whose shown that

**involved Reference** 

et al., 2010

N.D. Louboutin

CD4 Green et al., 2009

**Cell Mayor finding Mechanism or concurrent events Receptor**

blood and spleen.

Rat brain

mice

PBMCs in SCID

\* Not determined

endothelial cells Cytotoxicity

Reprogramming of the CD4+ T-cell migratory behavior

Table 1. *In vitro* and *in vivo e*ffects of soluble gp120

shows the receptor implicated in each case.

was inhibited by SDF-1 (Roggero et al., 2001).

mark them for premature death by apoptosis (Sunila et al., 1997).

binding of soluble Gp120 to CD4 facilitate apoptosis of primary human CD4+ T cells, but that it was caused primordially by the Gp120-CXCR4 interaction, since apoptosis was prevented by the CXCR4 inhibitor AMD3100 and by the anti-CXCR4 antibody 12G5 (Trushin et al., 2007). Similarly, soluble Gp120-induced apoptosis mediated by CXCR4 was demonstrated in adult human hepatocytes, which lack CD4 (Vlahakis et al., 2003)**.** Binding of Gp120 to CXCR4 is also able to induce apoptosis of CD8+ T cells by upregulating the expression of TNF and TNF-receptor II on interacting CD8+ T cells and macrophages (Herbein, et al., 1998). Thus, the expression of CXCR4 or CCR5 may restrict the cell sensitivity to Gp120 and explain the differential response of T cells subsets.

Recent studies have shown that the expression of CXCR4 on cancer cells makes them susceptible to apoptosis induced by the HIV-1 envelope. Endo et al. (2008) observed that apoptosis of breast cancer cell lines induced by HIV-1 particles was dependent on Gp120 and CXCR4 but not CD4. In addition, a Gp120 mutant with low CD4 binding ability induced apoptosis in breast cancer cells but not in T-cells. Importantly, conformational heterogeneity of CXCR4 in breast cancer cells in comparison with CXCR4 in T cells was related to the ability of Gp120 to induce apoptosis mediated by CXCR4 (Endo et al., 2008, 2010). Likewise, it has been shown that the Gp120-CXR4 interaction mediated apoptosis of prostate cancer cell lines but not of normal prostatic epithelial cells (Singh et al., 2009).

Anergy is a state of inhibition of proliferation and/or effector functions normally induced in T cells after encounter with antigen; the cell stay alive and functional inactivation is reversible upon antigen removal. It is induced by incomplete stimulation though the TCR and costimulatory molecules, and by the normal stimulation in the presence of IL-10 (Schwartz, 2003). Studies addressing the anergic effect of Gp120 use activation with anti-CD3 or mitogenactivation to simulate the effect of antigen stimulation. The contribution of anergy to the reduced immune function induced by X4 Gp120 in peripheral blood lymphocytes (PBMC) was early described by Schols and De Clercq (Schols & De Clercq, 1996). The addition of low concentrations of Gp120 was able to inhibit the proliferative response and the production of interleukin-2 (IL-2) and interleukin-4 (IL-4) in PBMC previously stimulated with an anti-CD3 antibody and concanavalin-A. In contrast, Gp120 induced the production of high amounts of IL-10, gamma interferon (IFN-g), and tumor necrosis factor alpha (TNF-a) in unstimulated PBMC. The induction of IL-10 by Gp120 was found to be important for the inhibitory effect of Gp120 on PBMC proliferation. Thus, X4 Gp120 can reduce the function of T lymphocytes by directly inducing anergy or by stimulation of the production of anergy-inducer immunosupressive cytokines. Importantly, the activation status played an important role in the cytokine pattern induced by Gp120 in PBMC (Schols & De Clercq, 1996).

Evidence of the participation of chemokine receptors in the induction of anergy by Gp120 has been obtained in studies of the long-lasting hypo-responsiveness to antigen stimulation caused by Gp120 in naive T lymphocytes. Gp120 was found to induce anergy by stimulating the activity of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA), which causes the progressive accumulation of the phosphorylated form of the cAMP-responsive element binding, a pathway which is also activated by the ligation of CXCR4 by SDF-1 (Masci et al., 2003).

It should be noted that although there is an association between circulating Gp120 and the induction of proinflammatory and immunoregulatory cytokines like IL-6, IL-10, and TNFalpha in some HIV infected individuals (Rychert et al., 2010), this effect can not be necessarily induced by direct cell interaction with Gp120, since cytokines could be induced

HIV Toxins: Gp120 as an Independent Modulator of Cell Function 81

et al., 2005). The progressive increase in the immune activation with increased expression of cytokines is suggested to cause neuropathological changes and neuronal and axonal damage. A recent report shows that Gp120 is able to activate rat microglia and cause neurotoxicity by inducing an increase in the expression of the voltage-gated K+ channels (KV), enhancing the cell outward K+ currents. The Gp120-associated enhancement of K+ current was blocked by a CXCR4 receptor antagonist or a specific protein kinase A (PKA) inhibitor. This data suggest that interaction of Gp120 with CXCR4 may underlay the microglia activation leading to neurotoxin production and neuronal apoptosis (Xu et al., 2011). In other study, Gp120-mediated neurotoxicity was found to involve signaling trough the p38 MAPK in macrophages, microglia and neuronal cells. Gp120-mediated p38 MAPK activation and neuronal death was prevented by CCL4 (MIP-1beta), one of the CCR5 ligands (Medders et al., 2010). On the other hand, soluble Tat is able to cross the BBB and to induce the production of chemoattractive factors by astrocytes and monocytes (mainly MCP-1, which is considered one of the most important chemokines in HIV infection and HAND),

It is known that molecular diversity produce a variety of ligand-receptor interactions, which in turn, induce signaling events that diverge from the optimal agonist effect (Edwards & Evavold, 2011). Thus, an important issue to be considered in the studies of the biological activity of Gp120 is its extreme heterogeneity at the amino acid sequence and glycosylation levels. A survey of the HIV sequences contained in Los Alamos database in the year 2000 showed that, of 566 full-length Gp120 protein sequences, protein lengths varied from 484 to 543 amino acids because of the insertions and deletions found in hypervariable regions. Main factors contributing to Env variation are: base-substitution due a lack of proofreading during the reverse transcription of the HIV genome, large insertions and deletions, and recombination. These processes are accelerated by the viral high replication rate, the rapid viral turnover and the pressure to change imposed for the immune response of the HIV infected individuals (Korber et al., 2001). Many of substitutions at the hypervariable regions of Gp120, as well as insertions and deletions involve glycosylation sites, so that the number

Another source of Gp120 molecular variation is the non-uniform content of carbohydrate units. The addition of oligosaccharides and oligomerization of the Gp160 precursor are both co-translational events that take place in the ER (reviewed by Land & Braakman 2001). It is known that incomplete or "immature" glycosylation is present in trimeric Gp120, due to steric limitations imposed to the glycan-modifying enzymes in the Golgi apparatus. Numerous Gp120 glycosylation variants can be produced even within a single cell population, as has been shown in the H9 lymphoblastoid cell line (Pal et al., 1993; Mizuochi et al., 1990). Instead, recombinant monomeric Gp120 is believed to contain fully mature glycans (Eggink et al., 2010; Binley et al., 2010). Thus, monomeric and native, trimeric Gp120 derived from virus and infected cells, may differ in their pattern of glycosylation (Means & Desrosiers, 2000; Mizuochi 1990). A recent study of the expression of a model oligomeric Gp120 showed that N-glycosylation of varied depending on the cell type used for expression (Raska et al., 2010). Cell-dependent addition of oligosaccharides may explain the observation that HIV laboratory strains exposed a higher proportion of high-mannose

and the expression of CCR5 on monocytes (Weiss et al., 1999).

**5. Relevance of extracellular Gp120 to HIV pathogenesis** 

of N-linked glycosylation sites ranges from 18 to 33 (Korber et al., 2001).

glycans that HIV primary isolates (Astoul et al., 2000).

also by deposition of Gp120-anti-Gp120 immune complexes, which has been associated with disease progression (Daniel et al., 2001; Gerencer et al., 1998).

Evidences indicate that Gp120, Tat and Nef may be largely involved in the events allowing the initial entry of HIV into the brain and in the injury and apoptosis of neurons. HIV gains entry into the brain at the asymptomatic stage of the infection trough infected circulating monocytes or as free virus. It is thought that entry is favored by a subclinical, early loss of the functional integrity of tight junctions of the brain endothelium, the brain blood barrier (BBB) (Strazza et al., 2011; Annunziata, 2003). Once in the brain, monocytes can repopulate the resident macrophage population and become a productive source of virus, extending the infection to microglia, astrocytes and endothelial cells, where it can establish a protected reservoir and give rise to the production of cytokines and chemokines (An et al., 1999). Inflammatory soluble factors like IL-1 and TNF-alpha, along with high amounts of viral proteins like Gp120, Tat and Nef, likely released by a particular kind of monocytes (CD14lowCD16+) (Thieblemont et al., 1995), may cause a continuous activation of the brain endothelium, leading to the attraction and diapedesis of more virus and activated cells. Increased numbers of CD14lowCD16+ monocytes in the circulation associates with HIVassociated neurocognitive disorders (HAND) (Thieblemont et al., 1995; reviewed in Gras & Kaul, 2010) and are abundant in brain autopsies from patients with HIV encephalitis (Fisher-Smith et al, 2001).

Perturbation of the brain blood barrier (BBB) may be induced by the HIV non-productive infection of brain endothelial cells by micropinocytosis or adsorptive endocytosis of the virus mediated by Gp120 (Banks et al., 2001). The transit of free virions by a paracellular route favored by TNF-alpha has been also observed (Fiala et al., 1997). Another explanation is the increase of BBB permeability by the activity of viral proteins. It has been found that soluble forms of Tat, Nef and Gp120 proteins, which circulate in the blood of HIV infected patients, alter the expression of cell junction proteins and thus disrupt the integrity of the BBB (reviewed in Toborek et al., 2005; Kanmogne et al., 2005). Gp120 is also able to increase monocyte migration through a brain microendothelial cells monolayer and to reduce the transendothelial electric resistance (Kanmogne et al., 2007). The presence of functional CD4 and chemokine receptors on discrete regions of brain microvessels derived from children has been demonstrated (Stins et al., 2004). In the presence of interferon (IFN)-gamma, children brain microvessels, but not adult brain microvessels, suffer cytotoxicity induced by Gp120. The effect associated with an increase of the expression levels of CCR3 and CCR5 induced by IFN-gamma. Several Gp120 peptides and RANTES, but not SDF-1, inhibited the Gp120 cytotoxic effect. Authors also showed that Gp120-mediated endothelial cell cytotoxicity involved the p38 MAPK pathway. Thus, a blood-brain barrier dysfunction induced by Gp120 in the brain of HIV-1-infected children may explain the higher incidence of HAND in this population (Khan et al., 2007).

Besides its potential role in BBB damage, chemokine receptors have been involved in direct and indirect Gp120-induced neuronal damage. Macrophages and microglia, the resident immunocompetent phagocytic cells in the brain, are the main cellular reservoirs of HIV in the central nervous system. Activated microglia produces free radicals and proinflammatory cytokines and chemokines which can damage neurons. Gp120 and Tat activates human fetal microglia in vitro, the resident phagocytes of the brain, to induce the expression of CD40 and MHC class II, and the secretion of inflammatory mediators, like cytokines, chemokines, and neurotoxins favoring the recruitment of cell from the circulation (reviwed in D'Aversa

also by deposition of Gp120-anti-Gp120 immune complexes, which has been associated with

Evidences indicate that Gp120, Tat and Nef may be largely involved in the events allowing the initial entry of HIV into the brain and in the injury and apoptosis of neurons. HIV gains entry into the brain at the asymptomatic stage of the infection trough infected circulating monocytes or as free virus. It is thought that entry is favored by a subclinical, early loss of the functional integrity of tight junctions of the brain endothelium, the brain blood barrier (BBB) (Strazza et al., 2011; Annunziata, 2003). Once in the brain, monocytes can repopulate the resident macrophage population and become a productive source of virus, extending the infection to microglia, astrocytes and endothelial cells, where it can establish a protected reservoir and give rise to the production of cytokines and chemokines (An et al., 1999). Inflammatory soluble factors like IL-1 and TNF-alpha, along with high amounts of viral proteins like Gp120, Tat and Nef, likely released by a particular kind of monocytes (CD14lowCD16+) (Thieblemont et al., 1995), may cause a continuous activation of the brain endothelium, leading to the attraction and diapedesis of more virus and activated cells. Increased numbers of CD14lowCD16+ monocytes in the circulation associates with HIVassociated neurocognitive disorders (HAND) (Thieblemont et al., 1995; reviewed in Gras & Kaul, 2010) and are abundant in brain autopsies from patients with HIV encephalitis

Perturbation of the brain blood barrier (BBB) may be induced by the HIV non-productive infection of brain endothelial cells by micropinocytosis or adsorptive endocytosis of the virus mediated by Gp120 (Banks et al., 2001). The transit of free virions by a paracellular route favored by TNF-alpha has been also observed (Fiala et al., 1997). Another explanation is the increase of BBB permeability by the activity of viral proteins. It has been found that soluble forms of Tat, Nef and Gp120 proteins, which circulate in the blood of HIV infected patients, alter the expression of cell junction proteins and thus disrupt the integrity of the BBB (reviewed in Toborek et al., 2005; Kanmogne et al., 2005). Gp120 is also able to increase monocyte migration through a brain microendothelial cells monolayer and to reduce the transendothelial electric resistance (Kanmogne et al., 2007). The presence of functional CD4 and chemokine receptors on discrete regions of brain microvessels derived from children has been demonstrated (Stins et al., 2004). In the presence of interferon (IFN)-gamma, children brain microvessels, but not adult brain microvessels, suffer cytotoxicity induced by Gp120. The effect associated with an increase of the expression levels of CCR3 and CCR5 induced by IFN-gamma. Several Gp120 peptides and RANTES, but not SDF-1, inhibited the Gp120 cytotoxic effect. Authors also showed that Gp120-mediated endothelial cell cytotoxicity involved the p38 MAPK pathway. Thus, a blood-brain barrier dysfunction induced by Gp120 in the brain of HIV-1-infected children may explain the higher incidence

Besides its potential role in BBB damage, chemokine receptors have been involved in direct and indirect Gp120-induced neuronal damage. Macrophages and microglia, the resident immunocompetent phagocytic cells in the brain, are the main cellular reservoirs of HIV in the central nervous system. Activated microglia produces free radicals and proinflammatory cytokines and chemokines which can damage neurons. Gp120 and Tat activates human fetal microglia in vitro, the resident phagocytes of the brain, to induce the expression of CD40 and MHC class II, and the secretion of inflammatory mediators, like cytokines, chemokines, and neurotoxins favoring the recruitment of cell from the circulation (reviwed in D'Aversa

disease progression (Daniel et al., 2001; Gerencer et al., 1998).

(Fisher-Smith et al, 2001).

of HAND in this population (Khan et al., 2007).

et al., 2005). The progressive increase in the immune activation with increased expression of cytokines is suggested to cause neuropathological changes and neuronal and axonal damage. A recent report shows that Gp120 is able to activate rat microglia and cause neurotoxicity by inducing an increase in the expression of the voltage-gated K+ channels (KV), enhancing the cell outward K+ currents. The Gp120-associated enhancement of K+ current was blocked by a CXCR4 receptor antagonist or a specific protein kinase A (PKA) inhibitor. This data suggest that interaction of Gp120 with CXCR4 may underlay the microglia activation leading to neurotoxin production and neuronal apoptosis (Xu et al., 2011). In other study, Gp120-mediated neurotoxicity was found to involve signaling trough the p38 MAPK in macrophages, microglia and neuronal cells. Gp120-mediated p38 MAPK activation and neuronal death was prevented by CCL4 (MIP-1beta), one of the CCR5 ligands (Medders et al., 2010). On the other hand, soluble Tat is able to cross the BBB and to induce the production of chemoattractive factors by astrocytes and monocytes (mainly MCP-1, which is considered one of the most important chemokines in HIV infection and HAND), and the expression of CCR5 on monocytes (Weiss et al., 1999).

## **5. Relevance of extracellular Gp120 to HIV pathogenesis**

It is known that molecular diversity produce a variety of ligand-receptor interactions, which in turn, induce signaling events that diverge from the optimal agonist effect (Edwards & Evavold, 2011). Thus, an important issue to be considered in the studies of the biological activity of Gp120 is its extreme heterogeneity at the amino acid sequence and glycosylation levels. A survey of the HIV sequences contained in Los Alamos database in the year 2000 showed that, of 566 full-length Gp120 protein sequences, protein lengths varied from 484 to 543 amino acids because of the insertions and deletions found in hypervariable regions. Main factors contributing to Env variation are: base-substitution due a lack of proofreading during the reverse transcription of the HIV genome, large insertions and deletions, and recombination. These processes are accelerated by the viral high replication rate, the rapid viral turnover and the pressure to change imposed for the immune response of the HIV infected individuals (Korber et al., 2001). Many of substitutions at the hypervariable regions of Gp120, as well as insertions and deletions involve glycosylation sites, so that the number of N-linked glycosylation sites ranges from 18 to 33 (Korber et al., 2001).

Another source of Gp120 molecular variation is the non-uniform content of carbohydrate units. The addition of oligosaccharides and oligomerization of the Gp160 precursor are both co-translational events that take place in the ER (reviewed by Land & Braakman 2001). It is known that incomplete or "immature" glycosylation is present in trimeric Gp120, due to steric limitations imposed to the glycan-modifying enzymes in the Golgi apparatus. Numerous Gp120 glycosylation variants can be produced even within a single cell population, as has been shown in the H9 lymphoblastoid cell line (Pal et al., 1993; Mizuochi et al., 1990). Instead, recombinant monomeric Gp120 is believed to contain fully mature glycans (Eggink et al., 2010; Binley et al., 2010). Thus, monomeric and native, trimeric Gp120 derived from virus and infected cells, may differ in their pattern of glycosylation (Means & Desrosiers, 2000; Mizuochi 1990). A recent study of the expression of a model oligomeric Gp120 showed that N-glycosylation of varied depending on the cell type used for expression (Raska et al., 2010). Cell-dependent addition of oligosaccharides may explain the observation that HIV laboratory strains exposed a higher proportion of high-mannose glycans that HIV primary isolates (Astoul et al., 2000).

HIV Toxins: Gp120 as an Independent Modulator of Cell Function 83

In the recent years, the interaction of Gp120 with chemokine receptors, enabled by the initial interaction with CD4, as been identified as the origin of many of the effects of Gp120 on the function of immune system cells and other tissues. By reviewing the recent literature, a general picture emerges that indicate that properties of Gp120 strongly associate with its chemokine receptor specificity and the activation status of the target cells. R5 Gp120 is able to activate resting cells, where X4 Gp120 seems to induce mainly anergy and apoptosis. On the other hand, X4 Gp120 is able to enhance the activation phenotype in cells that have been previously stimulated. A proper understanding of the influence of cell status on the effect of particular forms of Gp120 on cell viability and function is necessary to get an integrated

Several studies indicate that CD4 is not required for apoptosis of tumor cells induced by Gp120, since it can be mediated by CXCR4. In particular, the importance of CXCR4 expression for development and metastasis of breast cancer cells has been demonstrated, as well as for the Gp120-mediated apoptosis of these cells. Interestingly, HIV infected individuals do not present an increased incidence of this type of tumor, whereas they develop others (Amir et al., 2000; Herida et al., 2003; Pantanowitz & Dezube, 2001). Although other factors may determine this effect, participation of Gp120 can not be discarded. The complex structure and variability of Gp120 provides a substrate for the

Ackermann, E., Ang, E., Kanter, J., Tsigelny, I., & Taylor, P. (1998). Identification of pairwise

Amir, H., Kaaya, E., Kwesigabo, G., & Kiitinya, J. (2000). Breast cancer before and during the

An, S., Groves, M., Gray, F., & Scaravilli, F. (1999). Early entry and widespread cellular

Anand, A., Prasad, A., Bradley, R., Deol, Y., Nagaraja, T., Ren, X., Terwilliger, E., & Ganju, R.

Annunziata, P. (2003). Blood-brain barrier changes during invasion of the central nervous

Arthos, J., Cicala, C., Martinelli, E., Macleod, K., Van Ryk, D., Wei, D., Xiao, Z., Veenstra, T.,

interactions in the alpha-neurotoxin-nicotinic acetylcholine receptor complex through double mutant cycles. *The Journal of Biological Chemistry*, Vol. 273, No. 18,

AIDS epidemic in women and men: a study of Tanzanian Cancer Registry Data 1968 to 1996. *Journal of the National Medical Association,* Vol.96, No. 6, (Jun 2000), pp.

involvement of HIV-1 DNA in brains of HIV-1 positive asymptomatic individuals. *Journal of Neuropathology & Experimental Neurology*, Vol. 58, No.11, (Nov 1999), pp.

(2009). HIV-1 gp120-induced migration of dendritic cells is regulated by a novel kinase cascade involving Pyk2, p38 MAPkinase, and LSP1. *Blood*, Vol. 114, No. 17,

system by HIV-1. Old and new insights into the mechanism. *Journal of Neurology*,

Conrad, T., Lempicki, R., McLaughlin, S., Pascuccio, M., Gopaul, R., McNally, J., Cruz, C., Censoplano, N., Chung, E., Reitano, K., Kottilil, S., Goode, D., & Fauci, A. (2008). HIV-1 envelope protein binds to and signals through integrin α4β7, the gut mucosal homing receptor for peripheral T cells. *Nature Immunology,* Vol. 9, No. 3,

search of active molecules targeting chemokine receptor-expressing tumor cells.

view of the significance of free Gp120 for the HIV disease.

(May 1998), pp.10958-10964, ISSN 0021-9258

301-3055, ISSN 0027-9684

1156-1162, ISSN 0022-3069

(2009), pp. 3588-3600, ISSN 0006-4971

(March 2008), pp. 301–309, ISSN 1529-2908

Vol. 250, No. 8, (Aug 2003), pp.901-906, ISSN 0340-5354

**7. References** 

The actual amount of Gp120 in tissues and fluids of the HIV infected individual is another important consideration regarding the role of free Gp120 in AIDS pathogenesis. The Gp120 Env subunit can shed from viral particles and infected cells in vitro to adopt a water-soluble form (McKeating et al., 1991; Smith-Franklin et al., 2002; Layne et al., 1992; Schneider et al., 1986). As described in the previous section of this review, a myriad of biological activities has been described for soluble Gp120, and thus the potential of this molecule to account for a significant portion of the physiological dysfunction observed during the HIV-1 infection is considerable. However, few studies have estimated the extension of the presence of Gp120 in the organism of HIV-infected subjects. Gp120 has been detected in the circulation of about on third of HIV-infected subjects at concentrations of 4-130 pM (Rychert et al., 2010) and 2- 20 pM (Gilbert et al., 1991) in early and chronic HIV-infected subjects, respectively. A different study by Oh and cols. (1992) reported a higher concentration in plasma, although the methodology used has been questioned (Klasse & Moore, 2004). The amount of Gp120 bound to tissues can be relevant to the understanding of the dynamics of this molecule in the body. A recent study by Santosuosso and cols. (2009) showed that concentration of Gp120 in secondary lymphoid tissues obtained from autopsies of HIV-infected subjects can be high (up to 9007 pg/ml, or 75 pM), even when Gp120 is not detected in plasma. Although a distinction among the amount of soluble Gp120 and virus or cell-associated Gp120 was not clear in this study, it was shown that Gp120 can accumulate in lymphoid tissues early in the HIV infection, and that levels of viral protein in these tissues can exceed significantly those found in plasma.

The presence of physiologically significant amounts of soluble Gp120 in vivo is still a matter of debate. Klasse and More (2004) have discussed several factors that may limit the effective concentration of Gp120 in fluids and tissues, like the capture of Gp120 by antibodies and serum lectins (Daniel et al., 1998), and the absorption of Gp120 by proteoglycans on cell surfaces (Mbemba et al., 1999;). The soluble mannose binding lectin (MBL), a innate immunity molecule present in the human serum, is able to capture HIV particles probably through the high-mannose glycosylation sites of the Gp120/Gp41 complex (Saifuddin et al., 2000). It has been proposed that MBL can participate in the clearance of HIV, since it activates complement and opzonise particles for binding to phagocytic cells (Mass et al., 1998; Pastinen et al., 1998). Soluble Gp120 could be also cleared or inactivated by MBL.

#### **6. Conclusion**

Gp120 is a molecule with remarkable properties, some of which are related to a probable evolutionary relationship with animal toxins, and others to its interaction and adaptation to the human immunological and physiological environment. The Gp120 primary role in viral entry using CD4 and chemokine receptors, allow it to induce signaling events which final outcome depends on the particular cell physiological status, thus leading to activation, altered function or death. The high mutation rate of the *env* gene, combined with a rapid replication and viral turnover rates and the pressure to change imposed for the immune response, allow HIV (and the secreted Gp120 molecule) to extent its range of functional capabilities and cellular tropism. Along with other viral proteins such as Nef and Tat, which also have a spectrum of biological effects as soluble proteins, Gp120 may be an important mediators of the bystander CD4+-T-cell death and chronic inflammation that are hallmark of the disease leading to AIDS.

In the recent years, the interaction of Gp120 with chemokine receptors, enabled by the initial interaction with CD4, as been identified as the origin of many of the effects of Gp120 on the function of immune system cells and other tissues. By reviewing the recent literature, a general picture emerges that indicate that properties of Gp120 strongly associate with its chemokine receptor specificity and the activation status of the target cells. R5 Gp120 is able to activate resting cells, where X4 Gp120 seems to induce mainly anergy and apoptosis. On the other hand, X4 Gp120 is able to enhance the activation phenotype in cells that have been previously stimulated. A proper understanding of the influence of cell status on the effect of particular forms of Gp120 on cell viability and function is necessary to get an integrated view of the significance of free Gp120 for the HIV disease.

Several studies indicate that CD4 is not required for apoptosis of tumor cells induced by Gp120, since it can be mediated by CXCR4. In particular, the importance of CXCR4 expression for development and metastasis of breast cancer cells has been demonstrated, as well as for the Gp120-mediated apoptosis of these cells. Interestingly, HIV infected individuals do not present an increased incidence of this type of tumor, whereas they develop others (Amir et al., 2000; Herida et al., 2003; Pantanowitz & Dezube, 2001). Although other factors may determine this effect, participation of Gp120 can not be discarded. The complex structure and variability of Gp120 provides a substrate for the search of active molecules targeting chemokine receptor-expressing tumor cells.

#### **7. References**

82 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

The actual amount of Gp120 in tissues and fluids of the HIV infected individual is another important consideration regarding the role of free Gp120 in AIDS pathogenesis. The Gp120 Env subunit can shed from viral particles and infected cells in vitro to adopt a water-soluble form (McKeating et al., 1991; Smith-Franklin et al., 2002; Layne et al., 1992; Schneider et al., 1986). As described in the previous section of this review, a myriad of biological activities has been described for soluble Gp120, and thus the potential of this molecule to account for a significant portion of the physiological dysfunction observed during the HIV-1 infection is considerable. However, few studies have estimated the extension of the presence of Gp120 in the organism of HIV-infected subjects. Gp120 has been detected in the circulation of about on third of HIV-infected subjects at concentrations of 4-130 pM (Rychert et al., 2010) and 2- 20 pM (Gilbert et al., 1991) in early and chronic HIV-infected subjects, respectively. A different study by Oh and cols. (1992) reported a higher concentration in plasma, although the methodology used has been questioned (Klasse & Moore, 2004). The amount of Gp120 bound to tissues can be relevant to the understanding of the dynamics of this molecule in the body. A recent study by Santosuosso and cols. (2009) showed that concentration of Gp120 in secondary lymphoid tissues obtained from autopsies of HIV-infected subjects can be high (up to 9007 pg/ml, or 75 pM), even when Gp120 is not detected in plasma. Although a distinction among the amount of soluble Gp120 and virus or cell-associated Gp120 was not clear in this study, it was shown that Gp120 can accumulate in lymphoid tissues early in the HIV infection, and that levels of viral protein in these tissues can exceed significantly those

The presence of physiologically significant amounts of soluble Gp120 in vivo is still a matter of debate. Klasse and More (2004) have discussed several factors that may limit the effective concentration of Gp120 in fluids and tissues, like the capture of Gp120 by antibodies and serum lectins (Daniel et al., 1998), and the absorption of Gp120 by proteoglycans on cell surfaces (Mbemba et al., 1999;). The soluble mannose binding lectin (MBL), a innate immunity molecule present in the human serum, is able to capture HIV particles probably through the high-mannose glycosylation sites of the Gp120/Gp41 complex (Saifuddin et al., 2000). It has been proposed that MBL can participate in the clearance of HIV, since it activates complement and opzonise particles for binding to phagocytic cells (Mass et al., 1998; Pastinen et al., 1998). Soluble Gp120 could be also cleared or inactivated by MBL.

Gp120 is a molecule with remarkable properties, some of which are related to a probable evolutionary relationship with animal toxins, and others to its interaction and adaptation to the human immunological and physiological environment. The Gp120 primary role in viral entry using CD4 and chemokine receptors, allow it to induce signaling events which final outcome depends on the particular cell physiological status, thus leading to activation, altered function or death. The high mutation rate of the *env* gene, combined with a rapid replication and viral turnover rates and the pressure to change imposed for the immune response, allow HIV (and the secreted Gp120 molecule) to extent its range of functional capabilities and cellular tropism. Along with other viral proteins such as Nef and Tat, which also have a spectrum of biological effects as soluble proteins, Gp120 may be an important mediators of the bystander CD4+-T-cell death and chronic inflammation that are hallmark of

found in plasma.

**6. Conclusion** 

the disease leading to AIDS.


HIV Toxins: Gp120 as an Independent Modulator of Cell Function 85

Bodner, A., Maroney, A., Finn, J., Ghadge, G., Roos, R., & Miller, R. (2002).Mixed lineage

Bracci, L., & Neri, P.(1995). Molecular mimicry between the rabies virus and human

Bracci, L., Ballas, S., Spreafico, A., & Neri, P. (1997). Molecular mimicry between the rabies

Bren, G., Trushin, S., Whitman, J., Shepard, B., & Badley, A. (2009). HIV gp120 Induces, NF-

Busillo, J., & Benovic, J. (2007). Regulation of CXCR4 signaling. *Biochimica et Biophysica Acta* ,

Butzke, D., & Luch, A. (2010). High-molecular weight protein toxins of marine invertebrates

Canducci, F., Marinozzi, M., Sampaolo, M., Berrè, S., Bagnarelli, P., Degano, M., Gallotta, G.,

Cantin, R., Fortin, J., Lamontagne, G., & Tremblay, M. (1997). The acquisition of host-

Cheung, R., Ravyn, V., Wang, L., Ptasznik, A., & Collman, R. (2008). Signaling Mechanism of

Choudhary, S., Vrisekoop, N., Jansen, C., Otto, S., Schuitemaker, H., Miedema, F., &

Cicala, C., Arthos, J., Rubbert, A., Selig, S., Wildt, K., Cohen, O., & Fauci, A. (2000). HIV-1

Cicala, C., Arthos, J., Selig, S., Dennis, G. Jr., Hosack, D., Van Ryk, D., Spangler, M.,

Vol.82, No.6, (2002), pp.1424–1434, ISSN 0022-3042

Vol. 1768, No. 4, (Apr 2007), pp. 952-963, ISSN 0005-2736

*Retrovirology*, Vol.6, No.1, (Jan 2009), p. 4, ISSN. 1742-4690

Vol.60, No.2, (Jun 1996), pp. 386-406, ISSN 0146-0749

97, No.3, (Feb 2000), pp. 1178-1183, ISSN 0027-8424

Vol.99, No.14, (2002), pp.9380-9385, ISSN 0027-8424

(May 1995), pp. 391-393, ISSN 0003-9985

3623-3628, ISSN: 0006-4971

294X

4971

538X

(2009), p.e4875, ISSN 1932-6203

kinase 3 mediates gp120IIIB-induced neurotoxicity. *Journal of Neurochemistry*,

immunodeficiency virus. *Archives of Pathology & Laboratory Medicine*, Vol.119, No. 5,

virus glycoprotein and human immunodeficiency virus-1 GP120: cross-reacting antibodies induced by rabies vaccination. *Blood*, Vol. 90 , No. 9, (Nov 1997), pp.

kB Dependent, HIV Replication that Requires Procaspase 8. *PLoS One*, Vol.4, No.3,

and their elaborate modes of action. *EXS*, Vol.100, (2010), pp.213-232, ISSN 1023-

Mazzi, B., Lemey, P., Burioni, R., & Clementi, M. (2009). Dynamic features of the selective pressure on the human immunodeficiency virus type 1 (HIV-1) gp120 CD4-binding site in a group of long term non progressor (LTNP) subjects.

derived major histocompatibility complex class II glycoproteins by human immunodeficiency virus type 1 accelerates the process of virus entry and infection in human T-lymphoid cells. *Blood,* Vol.90, No. 3, (1997), pp. 1091-1100, ISSN. 0006-

HIV-1 gp120 and Virion-Induced IL-1β Release in Primary Human Macrophages. *The Journal of Immunology*, Vol.180, No.10, (2008), pp. 6675–6684, ISSN 0022-1767 Chirmule, N., & Pahwa, S. (1996). Envelope glycoproteins of human immunodeficiency

virus type 1: profound influences on immune functions. *Microbiological Reviews*,

Camerini, D. (2007). Low Immune Activation despite High Levels of Pathogenic Human Immunodeficiency Virus Type 1 Results in Long-Term Asymptomatic Disease. *The Journal of Virology*, Vol.81, No.16, (Aug 2007), pp. 8838-8842, ISSN 0022-

envelope induces activation of caspase-3 and cleavage of focal adhesion kinase in primary human CD4(+) T cells. *Proceedings of the National Academy of Sciences*, Vol.

Steenbeke, T., Khazanie, P., Gupta, N., Yang, J., Daucher, M., Lempicki, R., & Fauci, A. (2002). HIV envelope induces a cascade of cell signals in non-proliferating target cells that favor virus replication. *Proceedings of the National Academy of Sciences*,


Astoul, C., Peumans, W., Van Damme, E., & Rougé, P. (2000). Accessibility of the High-

Bachis, A., & Mocchetti, I. (2004). The Chemokine Receptor CXCR4 and Not the N-Methyl-

Badr, G., Borhis, G., Treton, D., Moog, C., Garraud, O., & Richard, Y. (2005). HIV type 1

Bahraoui, E., Benjouad, A., Guetard, D., Kolbe, H., Gluckman, J., & Montagnier, L. (1992).

Balabanian, K., Harriague, J., Décrion, C., Lagane, B., Shorte, S., Baleux, F., Virelizier, J.,

Bandres, J., Wang, Q., O'Leary, J., Baleaux, F., Amara, A., Hoxie, J., Zolla-Pazner, S., &

Banks, W., Freed, E., Wolf, K., Robinson, S., Franko, M., & Kumar V. (2001). Transport of

Bastiani, L., Laal, S., Kim, M., & Zolla-Pazner, S. (1997). Host cell-dependent alterations in

Binley, J., Ban, Y., Crooks, E., Eggink, D., Osawa, K., Schief, W., & Sanders, R., (2010). Role of

Blanpain, C., Libert, F., Vassart, G., & Parmentier, M. (2002). CCR5 and HIV infection.

*Receptors Channels,* Vol. 8, No. 1, (2002), pp.19–31, ISSN 1060-6823

*of Virology*, Vol. 71, No. 5, (May 1997), pp. 3444-3450, ISSN 0022-538X Berger, O., Gan, X., Gujuluva, C., Burns, A., Sulur, G., Stins, M., Way, D., Witte, M.,

Vol. 75, No.10, (May 2001), pp. 4681–4691, ISSN 0022-538X

5, No. 12, (Dec 1999), pp. 795-805, ISSN 1076-1551

2010), pp. 5637-5655, ISSN 0022-538X

Vol. 175, No. 1, (Jul 2005), pp. 302-310, ISSN 0022-1767

(May 1992), pp.565-573, ISSN 0889-2229

(Mar 1998), pp. 2500-2504, ISSN 0022-538X

ISSN 0006-291X

0360-4012

ISSN 0022-1767

Mannose Glycans of Glycoprotein gp120 from Human Immunodeficiency Virus Type 1 Probed by in Vitro Interaction with Mannose-Binding Lectins. *Biochemical and Biophysical Research Communications*, Vol. 274, No. 2, (August 2000), pp. 455-460,

D-Aspartate Receptor Mediates gp120 Neurotoxicity in Cerebellar Granule Cells. *Journal of Neuroscience Research,* Vol.75, No.1, (2004), pp.75–82, ISSN 0360-4012 Bachis, A., & Mocchetti, I.(2006). Semisynthetic sphingoglycolipid LIGA20 is

neuroprotective against human immunodeficiency virus-gp120-mediated apoptosis. *Journal of Neuroscience Research*, Vol.83, No.5, (2006), pp.890–896, ISSN

glycoprotein 120 inhibits human B cell chemotaxis to CXC chemokine ligand (CXCL) 12, CC chemokine ligand (CCL) 20, and CCL21. *The Journal of Immunology*,

Study of the interaction of HIV-1 and HIV-2 envelope glycoproteins with the CD4 receptor and role of N-glycans. *AIDS Research and Human Retroviruses*, Vol. 8, No. 5,

Arenzana-Seisdedos, F., & Chakrabarti, L. (2004). CXCR4-Tropic HIV-1 Envelope Glycoprotein Functions as a Viral Chemokine in Unstimulated Primary CD4+ T Lymphocytes. *The Journal of Immunology*, Vol.173, No.12, (2004), pp. 7150–7160,

Gorny, M. (1998). Human immunodeficiency virus (HIV) envelope binds to CXCR4 independently of CD4, and binding can be enhanced by interaction with soluble CD4 or by HIV envelope deglycosylation. *The Journal of Virology*, Vol. 72, No.3,

human immunodeficiency virus type 1 pseudoviruses across the blood-brain barrier: role of envelope proteins on adsorptive endocytosis. *The Journal of Virology*,

envelope components of human immunodeficiency virus type 1 virions. *The Journal* 

Weinand, M., Said, J., Kim, K., Taub, D., Graves, M., & Fiala, M. (1999). CXC and CC chemokine receptors on coronary and brain endothelia. *Molecular Medicine,* Vol.

complex carbohydrates in human immunodeficiency virus type 1 infection and resistance to antibody neutralization. *The Journal of Virology*, Vol. 84, No.11, (Jun


HIV Toxins: Gp120 as an Independent Modulator of Cell Function 87

*Academy of Sciences,* Vol.94, No.26, (1997), pp.14742-14747, ISSN 0027-8424 Edwards, L., & Hessinger, D. (2000). Portuguese man-of-war (Physalia physalis) venom

Edwards, L., & Evavold, B. (2011). T cell recognition of weak ligands: roles of signaling,

Eggink, D., Melchers, M., Wuhrer, M., Van Montfort, T., Dey, A., Naaijkens, B., David, K., Le

Endo, M., Inatsu, A., Hashimoto, K., Takamune, N., Shoji, S., & Misumi, S. (2008). Human

Endo, M., Gejima, S., Endo, A., Takamune, N., Shoji, S., & Misumi, S. (2010). Treatment of

Fantuzzi, L., Canini, I., Belardelli, F., & Gessani, S. (2001). HIV-1 gp120 Stimulates the

Fenouillet, E., Clerget-Raslain, B., Gluckman, J., Guétard, D., Montagnier, L., & Bahraoui, E.

Fernandis, A., Prasad, A., Band, H., Klösel, R., & Ganju, R. (2004). Regulation of CXCR4-

Fiala, M., Looney, D., Stins, M., Way, D., Zhang, L., Gan, X., Chiappelli, F., Schweitzer, E.,

Fox, J., Letellier, E., Oliphant, C., & Signoret, N. (2011). TLR2-dependent pathway of

Gerencer, M., Burek, V., Crowe, B., Barrett, N., & Dorner, F. (1998). The role of complement

*Molecular Medicine ,* Vol.3, No.8, (1997), pp. 553–564, ISSN 1076-1551 Fischer-Smith, T., Croul, S., Sverstiuk, A., Capini, C., L'Heureux, D., Regulier, E.,

*HIV Research,* Vol.6, No.1, (Jan 2008), pp. 34-42, ISSN 1570-162X

Vol.38, No.8, (2000), pp. 1015–1028, ISSN 0041-0101

39-48, ISSN 1559-0755

6158

2010), pp. 236-247, ISSN 0042-6822

5381–5387, ISSN 0022-1767

(1989), pp. 807-822, ISSN 0022-1007

No.1, (Jan 2004), pp.157-167, ISSN 0950-9232

Vol.7, No.6, (2001), pp. 528–541, ISSN 1355-0284

neurovirulent simian immunodeficiency virus strain. *Proceedings of the National* 

induces calcium influx into cells by permeabilizing plasma membranes. *Toxicon*,

receptor number, and affinity. *Immunologic Research*, Vol.50, No.1, (May 2011), pp.

Douce, V., Deelder, A., Kang, K., Olson, W., Berkhout, B., Hokke, C., Moore, J., & Sanders, R. (2010). Lack of complex N-glycans on HIV-1 envelope glycoproteins preserves protein conformation and entry function. *Virology*, Vol.401, No.2, (Jun

immunodeficiency virus-induced apoptosis of human breast cancer cells via CXCR4 is mediated by the viral envelope protein but does not require CD4. *Current* 

breast cancer cells with proteasome inhibitor lactacystin increases the level of sensitivity to cell death induced by human immunodeficiency virus type 1. *Biological & Pharmaceutical Bulletin*, Vol.33, No.11, (2010), pp. 1903-1906, ISSN 0918-

Production of β-Chemokines in Human Peripheral Blood Monocytes Through a CD4-Independent Mechanism. *The Journal of Immunology*, Vol.166, No.9, (2001), pp.

(1989). Role of N-linked glycans in the interaction between the envelope glycoprotein of human immunodeficiency virus and its CD4 cellular receptor. Structural enzymatic analysis. *The Journal of Experimental Medicine*, Vol.169, No.3,

mediated chemotaxis and chemoinvasion of breast cancer cells. *Oncogene*, Vol.23,

Shapshak, P., Weinand, M., Graves, M., Witte, M., & Kim, K. (1997). TNF-alpha opens a paracellular route for HIV-1 invasion across the bloodbrain barrier.

Richardson, M., Amini, S., Morgello, S., Khalili, K., & Rappaport, J. (2001). CNS invasion by CD14+/CD16+ peripheral blood-derived monocytes in HIV dementia: perivascular accumulation and reservoir of HIV infection. *Journal of Neurovirology*,

heterologous down-modulation for the CC chemokine receptors 1, 2, and 5 in human blood monocytes. *Blood*, Vol.117, No.6, (2011), pp.1851-1860, ISSN 0006-4971

and gp120-specific antibodies in virus lysis and CD4+ T cell depletion in HIV-1-


Cicala, C., Arthos, J., Martinelli, E., Censoplano, N., Cruz, C., Chung, E., Selig, S., Van Ryk,

Cicala, C., Arthos, J., Censoplano, N., Cruz, C., Chung, E., Martinelli, E., Lempicki, R.,

Cron, R., Bartz, S., Clausell, A., Bort, S., Klebanoff, S., & Lewis, D. (2000). NFAT1 enhances

Daniel, V., Süsal, C., Weimer, R., Zipperle, S., Kröpelin, M., Melk, A., Zimmermann, R.,

Daniel, V., Süsal, C., Weimer, R., Zimmermann, R., Huth-Kühne, A., & Opelz, G. (2001).

D'Aversa, T., Eugenin, E., & Berman, J. (2005). NeuroAIDS: contributions of the human

Davis, C., Dikic, I., Unutmaz, D., Hill, C., Arthos, J., Siani, M., Thompson, D., Schlessinger, J.,

Del Corno, M., Liu, Q., Schols, D., Clercq, E., Gessani, S., Freedman, B., & Collman, R. (2001).

DePaolo, R., Lathan, R., & Karpus, W. (2004). CCR5 Regulates High Dose Oral Tolerance by

Dobaczewski, M., Xia, Y., Bujak, M., Gonzalez-Quesada, C., & Frangogiannis, N. (2010).

*Journal of Virology*, Vol.65, No.4, (Apr 1991), pp. 2047-2055, ISSN 0022-538X Edinger, A., Mankowski, J., Doranz, B., Margulies, B., Lee, B., Rucker, J., Sharron, M.,

*of Pathology,* Vol.176, No.5, (May 2010), pp. 2177–2187, ISSN 0002-9440 Earl, P., Moss, B., & Doms, R. (1991). Folding, interaction with GRP78-BiP, assembly, and

*Immunology*, Vol.173, No.1, (2004), pp. 314-320, ISSN 0022-1767

Vol.103, No.10, (Mar 2006), pp. 3746-3751, ISSN 0027-8424

*Letters*, Vol.76, No.2, (Mar 2001), pp. 69-78, ISSN 0165-2478

No.10, (Nov 1997), pp.1793-1798, ISSN 0022-1007

(2006), pp. 105-114, ISSN 0042-6822.

1998), pp. 179-187, ISSN 0165-2478

ISSN 0360-4012

2916, ISSN 0006-4971

No.3, (Mar 2000), pp. 179-191, ISSN 1521-6616

D., Yang, J., Jagannatha, S., Chun, T., Ren, P., Lempicki, R., & Fauci, A. (2006a). R5 and X4 HIV envelopes induce distinct gene expression profiles in primary peripheral blood mononuclear cells. *Proceedings of the National Academy of Sciences*,

Natarajan, V., VanRyk, D., Daucher, M., & Fauci, A. (2006b). HIV-1 gp120 induces NFAT nuclear translocation in resting CD4+ T-cells. *Virology*, Vol. 345, No. 1,

HIV-1 gene expression in primary human CD4 T cells.*Clinical Immunology,* Vol.94,

Huth-Kühne, A., & Opelz, G. (1998). Association of viral load in plasma samples of HIV-infected hemophilia patients with autoantibodies and gp120-containing immune complexes on CD4+ lymphocytes. *Immunology Letters*, Vol.60, No.2-3, (Feb

Association of immune complexes and plasma viral load with CD4+ cell depletion, CD8+ DR+ and CD16+ cell counts in HIV+ hemophilia patients. Implications for the immunopathogenesis of HIV-induced CD4+ lymphocyte depletion. *Immunology* 

immunodeficiency virus-1 proteins Tat and gp120 as well as CD40 to microglial activation. *Journal of Neuroscience Research,* Vol.81, No.3, (Aug 2005), pp. 436-446,

& Littman, D. (1997). Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5. *The Journal of Experimental Medicine,* Vol.186,

HIV-1 gp120 and chemokine activation of Pyk2 and mitogen-activated protein kinases in primary macrophages mediated by calcium-dependent, pertussis toxininsensitive chemokine receptor signaling. *Blood*, Vol.98, No.10, (2001), pp. 2909-

Modulating CC Chemokine Ligand 2 Levels in the GALT. *The Journal of* 

CCR5 Signaling Suppresses Inflammation and Reduces Adverse Remodeling of the Infarcted Heart, Mediating Recruitment of Regulatory T Cells. *The American Journal* 

transport of the human immunodeficiency virus type 1 envelope protein. *The* 

Hoffman, T., Berson, J., Zink, M., Hirsch, V., Clements, J., & Doms, R. (1997). CD4 independent, CCR5-dependent infection of brain capillary endothelial cells by a neurovirulent simian immunodeficiency virus strain. *Proceedings of the National Academy of Sciences,* Vol.94, No.26, (1997), pp.14742-14747, ISSN 0027-8424


HIV Toxins: Gp120 as an Independent Modulator of Cell Function 89

Iyengar, S., Schwartz, D., & Hildreth, J. (1999). T Cell-Tropic HIV gp120 Mediates CD4 and

Janowska-Wieczorek, A., Marquez, L., Dobrowsky, A., Ratajczak, M., & Cabuhat, M. (2000).

Jones, P., Korte, T., & Blumenthal, R. (1998). Conformational changes in cell surface HIV-1

Kang, Y., Siegel, P., Shu, W., Drobnjak, M., Kakonen, S., Cordón-Cardo, C., Guise, T.,

Kanmogne, G., Primeaux, C., & Grammas, P. (2005). HIV-1 gp120 proteins alter tight

Kaufmann, A., Salentin, R., Gemsa, D., & Sprenger, H. (2001). Increase of CCR1 and CCR5

Khan, N., Di Cello, F., Stins, M., & Kim, K. (2007). Gp120-mediated cytotoxicity of human

Kim, M., Koh, Y., Kim, K., Koh, B., Nam, D., Alitalo, K., Kim, I., & Koh, G. (2010). CXCR4

Kinet, S., Bernard, F., Mongellaz, C., Perreau, M., Goldman, F., & Taylor, N. (2002). Gp120-

Kinoshita, S., Chen, B., Kaneshima, H., & Nolan, G. (1998). Host control of HIV-1 parasitism

Klasse, P., & Moore, J. (2004). Is there enough gp120 in the body fluids of HIV-1-infected

bone. *Cancer Cell*, Vol. 3, No. 6, (2003), pp. 537-549, ISSN 1535-6108

(Nov 2000), pp.1274-1285, ISSN 0301-472X

0022-1767

ISSN 0021-9258

ISSN 0271-678X

pp. 248-252, ISSN 0741-5400

pp. 595-604, ISSN 0092-8674

2004), pp.1-8, ISSN 0042-6822

251, ISSN 1355-0284

5472

4971

CD8 Cell Chemotaxis through CXCR4 Independent of CD4: Implications for HIV Pathogenesis. *The Journal of Immunology*, Vol.162, No.10, (1999), pp. 6263-6267, ISSN

Differential MMP and TIMP production by human marrow and peripheral blood CD34(+) cells in response to chemokines*. Experimental Hematology,* Vol.28, No.11,

envelope glycoproteins are triggered by cooperation between cell surface CD4 and co-receptors. *The Journal of Biological Chemistry,* Vol.273, No.1, (1998), pp. 404–409,

Massagué, J. (2003). A multigenic program mediating breast cancer metastasis to

junction protein expression and brain endothelial cell permeability: implications for the pathogenesis of HIV-associated dementia. *Journal of Neuropathology & Experimental Neurology*, Vol.64, No.6, (Jun 2005), pp. 498-505, ISSN 0022-3069 Kanmogne, G., Schall, K., Leibhart, J., Knipe, B., Gendelman, H., & Persidsky, Y. (2007).

HIV-1 gp120 compromises blood-brain barrier integrity and enhances monocyte migration across blood-brain barrier: implication for viral neuropathogenesis*. Journal of Cerebral Blood Flow & Metabolism*, Vol.27, No.1, (Jan 2007), pp. 123-134,

expression and enhanced functional response to MIP-1α during differentiation of human monocytes to macrophages. *Journal of Leukocyte Biology,* Vol.69, No.2, (2001),

brain microvascular endothelial cells is dependent on p38 mitogen-activated protein kinase activation. *Journal of Neurovirology*, Vol.13, No.3, (Jun 2007), pp. 242-

signaling regulates metastasis of chemoresistant melanoma cells by a lymphatic metastatic niche. *Cancer Research*, Vol.70, No.24, (2010), pp. 10411-10421, ISSN 0008-

mediated induction of the MAPK cascade is dependent on the activation state of CD4(+) lymphocytes. *Blood*, Vol.100, No.7, (Oct 2002), pp. 2546-2553, ISSN 0006-

in T cells by the nuclear factor of activated T cells. *Cell*, Vol.95, No.5, (Nov 1998),

individuals to have biologically significant effects?. *Virology*, Vol.323, No.1, (May

infected patients. *Microbial Pathogenesis* , Vol.25, No.5, (1998), pp.253-266, ISSN 0882-4010


Gilbert, M., Kirihara, J., & Mills, J. (1991). Enzyme-linked immunoassay for human

Goldman, F., Crabtree, J., Hollenback, C., & Koretzky, G. (1997). Sequestration of p56(lck) by

Gras, G., & Kaul, M. (2010). Molecular mechanisms of neuroinvasion by monocytes-

Green, D., Center, D., & Cruikshank, W. (2009). Human Immunodeficiency Virus Type 1

Hashimoto, F., Oyaizu, N., Kalyanaraman, V., & Pahwa, S. (1997). Modulation of Bcl-2

interleukin-2. *Blood*, Vol.90, No.2, (Jul 1997), pp. 745-753, ISSN 0006-4971 Hecht, I., Cahalon, L., Hershkoviz, R., Lahat, A., Franitza, S., & Lider, O. (2003).

Herbein, G., Mahlknecht, U., Batliwalla, F., Gregersen, P., Pappas, T., Butler, J., O'Brien, W.,

Herida, M., Mary-Krause, M., Kaphan, R., Cadranel, J., Poizot-Martin, I., Rabaud, C.,

*of Clinical Oncology*, Vol.21, No.18, (Sep 2003), pp. 3447-3453, ISSN 1527-7755 Hezareh, M., Moukil, M., Szanto, I., Pondarzewski, M., Mouche, S., Cherix, N., Brown, S.,

Hodgson, D., Yirmiya, R., Chiappelli, F., & Taylor, A. (1998). Intracerebral HIV glycoprotein

Holm, G., Zhang, C., Gorry, P., Peden, K., Schols, D., De Clercq, E., Gabuzda, D. (2004).

*Research*, Vol.781, No.1-2, (1998), pp. 244–251, ISSN 0006-8993.

*Microbiology*, Vol.29, No.1, (Jan 1991), pp.142-147, ISSN 0095-1137

(1997), pp. 2017-2024, ISSN 0022-1767

Vol. 15, No.1, (2003), pp. 29-38, ISSN 0953-8178

No.6698, (Sep 1998), pp.189-194, ISSN 0028-0836

0882-4010

4690

ISSN 0022-538X

ISSN 0956-3202

ISSN 0005-2736

infected patients. *Microbial Pathogenesis* , Vol.25, No.5, (1998), pp.253-266, ISSN

immunodeficiency virus type 1 envelope glycoprotein 120. *Journal of Clinical* 

gp120, a model for TCR desensitization. *The Journal of Immunology*, Vol. 158, No. 5,

macrophages in HIV-1 infection. *Retrovirology*, Vol.7, (Apr 2010), p. 30, ISSN 1742-

gp120 Reprogramming of CD4+ T-Cell Migration Provides a Mechanism for Lymphadenopathy. *Journal of Virology*, Vol.83, No.11, (Jun 2009), pp. 5765-5772,

protein by CD4 cross-linking: a possible mechanism for lymphocyte apoptosis in human immunodeficiency virus infection and for rescue of apoptosis by

Heterologous desensitization of T cell functions by CCR5 and CXCR4 ligands: inhibition of cellular signaling, adhesion and chemotaxis. *International Immunology*,

& Verdin, E. (1998). Apoptosis of CD8+ T cells is mediated by macrophages through interaction of HIV gp120 with chemokine receptor CXCR4. *Nature*, Vol.395,

Plaisance, N., Tissot-Dupont, H., Boue, F., Lang, J., Costagliola, D. (2003). Incidence of non-AIDS-defining cancers before and during the highly active antiretroviral therapy era in a cohort of human immunodeficiency virus-infected patients. *Journal* 

Carpentier, J., & Foti, M. (2004). Mechanisms of HIV receptor and co-receptor down-regulation by prostratin: role of conventional and novel PKC isoforms.*Antiviral Chemistry & Chemotherapy* , Vol.15, No.4, (Jul 2004), pp.207-222,

gp120. enhances tumor metastasis via centrally released interleukin-1. *Brain* 

Apoptosis of bystander T cells induced by human immunodeficiency virus type 1 with increased envelope/receptor affinity and coreceptor binding site exposure. *The Journal of Virology*, Vol.78, No.9, (May 2004), pp. 4541-4551, ISSN 0022-538X Iacovache, I., Van der Goot, F., & Pernot, L. (2008). Pore formation: An ancient yet complex

form of attack. *Biochimica et Biophysica Acta*, Vol.1778, No.7-8, (2008), pp.1611–1623,


HIV Toxins: Gp120 as an Independent Modulator of Cell Function 91

Masci, A., Galgani, M., Cassano, S., De Simone, S., Gallo, A., De Rosa, V., Zappacosta, S., &

Mbemba, E., Benjouad, A., Saffar, L., & Gattegno, L.(1999). Glycans and proteoglycans are

McKeating, J., McKnight, A., & Moore, J. (1991). Differential loss of envelope glycoprotein

Means, R., & Desrosiers, R. (2000). Resistance of native, oligomeric envelope on simian

Medders, K., Sejbuk, N., Maung, R., Desai, M., & Kaul, M. (2010). Activation of p38 MAPK is

Medina, I., Ghose, S., & Ben-Ari, Y. (1999). Mobilization of intracellular calcium stores

Melar, M., Ott, D., & Hope, T. (2007). Physiological levels of virion-associated human

Melli, G., Keswani, S., Fischer, A., Chen, W., & Hoke, A. (2006). Spatially distinct and

Meucci, O., & Miller, R. (1996). gp120-induced neurotoxicity in hippocampal pyramidal

Missé, D., Cerutti, M., Noraz, N., Jourdan, P., Favero, J., Devauchelle, G., Yssel, H., Taylor,

*Virology*, Vol. 265, No.2, (Dec 1999), pp. 354-364, ISSN 0042-6822

Vol.74, No.6, (2003), pp. 1117–1124, ISSN 0741-5400

No.23, (Dec 2000), pp. 11181-11190, ISSN 0022-538X

156-166, ISSN 1021-7770

852-860, ISSN 0022-538X

(2000), pp.413–423, ISSN 0021-9525

No.13, (1996), pp. 4080–4088, ISSN 0270-6474

No.9, (Jun 2005), pp. 897-905, ISSN 0269-9370

ISSN 0022-1767

0953-816X

0006-8950

galactosylceramide in the cytopathic effects induced by HIV-1 gp120 in the HT-29- D4 intestinal cell line. *Journal of Biomedical Science*, Vol.10, No.1, (Jan-Feb 2003), pp.

Racioppi, L.(2003). HIV-1 gp120 induces anergy in naive T lymphocytes through. CD4-independent protein kinase-A-mediated signaling. *Journal of Leukocyte Biology,*

involved in the interactions of human immunodeficiency virus type 1 envelope glycoprotein and of SDF-1alpha with membrane ligands of CD4(+) CXCR4(+) cells.

gp120 from virions of human immunodeficiency virus type 1 isolates: effects on infectivity and neutralization. *The Journal of Virology,*Vol.65, No.2, (Feb 1991), pp.

immunodeficiency virus to digestion by glycosidases. *The Journal of Virology,* Vol.74,

required in monocytic and neuronal cells for HIV glycoprotein 120-induced neurotoxicity. *The Journal of Immunology*, Vol.185, No.8, (Oct 2010), pp. 4883-4895,

participates in the rise of [Ca21]i and the toxic actions of the HIV coat protein gp120. *European Journal of Neuroscience*, Vol. 11, No. 4, (1999), pp. 1167–1178, ISSN

immunodeficiency virus type 1 envelope induce coreceptor-dependent calcium flux. *The Journal of Virology,* Vol. 81, No.4, (2007), pp.1773-1785, ISSN 0022-538X Melikyan, G., Markosyan, R., Hemmati, H., Delmedico, M., Lambert, D., & Cohen, F.(2000).

Evidence that the transition of HIV-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion. *The Journal of Cell Biology*, Vol. 151, No.2,

functionally independent mechanisms of axonal degeneration in a model of HIVassociated sensory neuropathy. *Brain*, Vol.129, No.5, (2006), pp.1330–1338, ISSN

neuron cultures: protective action of TGF-beta1. *The Journal of Neuroscience,* Vol.16,

N., & Veas, F. (1999). A CD4-Independent Interaction of Human Immunodeficiency Virus-1 gp120 With CXCR4 Induces Their Cointernalization, Cell Signaling, and T-Cell Chemotaxis. *Blood*, Vol. 93, No. 8, (1999), pp. 2454-2462, ISSN 0006-4971 Missé, D., Gajardo, J., Oblet, C., Religa, A., Riquet, N., Mathieu, D., Yssel, H., & Veas, F.

(2005). Soluble HIV-1 gp120 enhances HIV-1 replication in non-dividing CD4+ T cells, mediated via cell signaling and Tat cofactor overexpression. *AIDS*, Vol.19,


Korber, B., Gaschen, B., Yusim, K., Thakallapally, R., Kesmir, C., & Detours, V. (2001).

Kristan, K., Viero, G., Dalla Serra, M., Macek, P., & Anderluh, G. (2009). Molecular

Kryworuchko, M., Pasquier, V., & Thèze, J. (2003). Human immunodeficiency virus-1

Kucia, M., Reca, R., Miekus, K., Wanzeck, J., Wojakowski, W., Janowska-Wieczorek, A.,

Land, A., & Braakman, I. (2001). Folding of the human immunodeficiency virus type 1

Layne, S., Merges, M., Dembo, M., Spouge, J., Conley, S., Moore, J., Raina, J., Renz, H.,

Lévesque, J., Hendy, J., Takamatsu, Y., Simmons, P., & Bendall, L. (2003). Disruption of the

Li, Y., Luo, L., Rasool, N., & Kang, C. (1993). Glycosylation is necessary for the correct

Liang, Z., Yoon, Y., Votaw, J., Goodman, M., Williams, L., & Shim, H. (2005). Silencing of

Louboutin, J., Reyes, B., Agrawal, L., Maxwell, C., Van Bockstaele, E., & Strayer, D. (2010).

Maas, J., De Roda Husman, A., Brouwer, M., Krol, A., Coutinho, R., Keet, I., Van Leeuwen,

Maccarrone, M., Navarra, M., Catani, V., Corasaniti, T., Bagetta, G., & Finazzi-Agro, A.

Marchese, A., Paing, M., Temple, B., &Trejo, J.(2008). G protein-coupled receptor sorting to

Maresca, M., Mahfoud, R., Garmy, N., Kotler, D., Fantini, J., & Clayton, F. (2003). The

*Investigation*, Vol.111, No.2, (Jan 2003), pp.187–196, ISSN 0021-9738

*Virology*, Vol.67, No.1, (Jan 1993), pp. 584-588, ISSN 0022-538X

*AIDS.,* Vol.12, No.17, (1998), pp. 2275-2280, ISSN 0269-9370

*British Medical Bulletin,* Vol.58, (2001), pp.19–42, ISSN 0007-1420

*Immunology,* Vol. 131, No.3, (2003), pp. 422–427, ISSN 0009-9104

axis. *Stem Cells*, Vol.23, No.7, (2005), pp.879-894, ISSN 1066-5099

1125-1134, ISSN 0041-0101

2001), pp. 783-790, ISSN 0300-9084

pp. 967-971, ISSN 0008-5472

(2010), pp. 313–325, ISSN 0969-9961

(2002), pp. 1444–1452, ISSN 0022-3042

(2008), pp. 601–629, ISSN 0362-1642

Vol.189, No.2, (1992), pp. 695-714, ISSN 0042-6822

Evolutionary and immunological implications of contemporary HIV-1 variation*.* 

mechanism of pore formation by actinoporins. *Toxicon*, Vol.54, No.8, (Dec 2009), pp.

envelope glycoproteins and anti-CD4 antibodies inhibit interleukin-2-induced Jak/STAT signaling in human CD4 T lymphocytes*. Clinical & Experimental* 

Ratajczak, J., & Ratajczak, M. (2005).Trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms: pivotal role of the SDF-1-CXCR4

envelope glycoprotein in the endoplasmic reticulum. *Biochimie*, Vol.83, No.8, (Aug

Gelderblom, H., & Nara, P. (1992). Factors underlying spontaneous inactivation and susceptibility to neutralization of human immunodeficiency virus. *Virology,* 

CXCR4/CXCL12 chemotactic interaction during hematopoietic stem cell mobilization induced by GCSF or cyclophosphamide. *The Journal of Clinical* 

folding of human immunodeficiency virus gp120 in CD4 binding. *The Journal of* 

CXCR4 blocks breast cancer metastasis. *Cancer Research*, Vol.65, No.3, (Feb 2005),

Blood–brain barrier abnormalities caused by exposure to HIV-1 gp120 -Protection by gene delivery of antioxidant enzymes. *Neurobiology of Disease*, Vol.38, No.2,

R., & Schuitemaker, H. (1998). Presence of the variant mannose binding lectin alleles associated with slower progression to AIDS. Amsterdam Cohort Study.

(2002). Cholesterol-dependent modulation of the toxicity of HIV-1 coat protein gp120 in human neuroblastoma cells. *Journal of Neurochemistry*, Vol. 82, No.6,

endosomes and lysosomes. *Annual Review of Pharmacology and Toxicology* , Vol.48,

virotoxin model of HIV-1 enteropathy: involvement of GPR15/Bob and

galactosylceramide in the cytopathic effects induced by HIV-1 gp120 in the HT-29- D4 intestinal cell line. *Journal of Biomedical Science*, Vol.10, No.1, (Jan-Feb 2003), pp. 156-166, ISSN 1021-7770


HIV Toxins: Gp120 as an Independent Modulator of Cell Function 93

Otteken, A., Earl, P., & Moss, B. (1996). Folding, assembly, and intracellular trafficking of the

Pal, R., Di Marzo Veronese, F., Nair, B., Rittenhouse, S., Hoke, G., Mumbauer, S., &

Pantanowitz, L., Dezube, B. (2001). Breast cancer in women with HIV/AIDS. *The Journal of* 

Pastinen, T., Liitsola, K., Niini, P., Salminen, M., & Syvanen, A. (1998). Contribution of the

Pawlak, J., Mackessy, S., Fry, B., Bhatia, M., Mourier, G., Fruchart-Gaillard, C., Servent, D.,

Peng, F., Dhillon, N., Callen, S., Yao, H., Bokhari, S., Zhu, X., Baydoun, H., & Buch, S. (2008).

Price, O., Ercal, N., Nakaoke, R., & Banks, W. (2005). HIV-1 viral proteins gp120 and Tat

Rahangdale, S., Morgan, R., Heijens, C., Ryan, T., Yamasaki, H., Bentley, E., Sullivan, E.,

Roggero, R., Robert-Hebmann, V., Harrington, S., Roland, J., Vergne, L., Jaleco, S., Devaux,

Rychert, J., Strick, D., Bazner, S., Robinson, J., & Rosenberg, E. (2010). Detection of HIV

*Virology,* Vol.75, No.16, (2001), pp.7637-7650, ISSN 0022-538X

*Virology*, Vol.70, No.6, (Jun 1996), pp.3407-3415, ISSN 0022-538X

Vol.196, No.3, (Nov 1993), pp.1335-1342, ISSN 0006-291X

7484

ISSN 0889-2229

0042-6822

0021-9258

29041, ISSN 0021-9258

(2005), pp. 57–63, ISSN 0006-8993

human immunodeficiency virus type 1 envelope glycoprotein analyzed with monoclonal antibodies recognizing maturational intermediates. *The Journal of* 

Sarngadharan, M.(1993). Glycoprotein of human immunodeficiency virus type 1 synthesized in chronically infected Molt3 cells acquires heterogeneous oligosaccharide structures. *Biochemical and Biophysical Research Communications,*

*the American Medical Association,*Vol.285, No.24, (2001), pp. 3090-3091, ISSN 0098-

CCR5 and MBL genes to susceptibility to HIV type 1 infection in the Finnish population. *AIDS Research and Human Retroviruses*, Vol.14, No.8, (1998), pp. 695-698,

Ménez, R., Stura, E., Ménez, A., & Kini, R. (2006). Denmotoxin, a three-finger toxin from the colubrid snake Boiga dendrophila (Mangrove Catsnake) with bird-specific activity. *The Journal of Biological Chemistry,* Vol.281, No.39, (Sep 2006), pp. 29030-

Platelet-Derived Growth Factor Protects Neurons against Gp120- Mediated Toxicity. *Journal of Neurovirology,* Vol.14, No.1, (2008), pp. 62–72, ISSN 1355-0284 Polzer, S., Dittmar, M., Schmitz, H., & Schreiber, M. (2002). The N-linked glycan g15 within

the V3 loop of the HIV-1 external glycoprotein gp120 affects coreceptor usage, cellular tropism, and neutralization. *Virology*, Vol. 304, No.1, (2002), pp. 70-80, ISSN

induce oxidative stress in brain endothelial cells. *Brain Research*, Vol.1045, No.1-2,

Center, D., & Cruikshank. W. ( 2006). Chemokine receptor CXCR3 desensitization by IL-16/CD4 signaling is dependent on CCR5 and intact membrane cholesterol. *The Journal of Immunology,* Vol.176, No.4, (2006), pp. 2337-2345, ISSN 0022-1767 Raska, M., Takahashi, K., Czernekova, L., Zachova, K., Hall, S., Moldoveanu, Z., Elliott, M.,

Wilson, L., Brown, R., Jancova, D., Barnes, S., Vrbkova, J., Tomana, M., Smith, P., Mestecky, J., Renfrow, M., & Novak, J.(2010). Glycosylation patterns of HIV-1 gp120 depend on the type of expressing cells and affect antibody recognition. *The Journal of Biological Chemistry,* Vol.285, No.27, (Jul 2010), pp. 20860-20869. ISSN

C., & Biard-Piechaczyk, M. (2001). Binding of human immunodeficiency virus type 1 gp120 to CXCR4 induces mitochondrial transmembrane depolarization and cytochrome *c*-mediated apoptosis independently of Fas signaling. *The Journal of* 

gp120 in plasma during early HIV infection is associated with increased


Mizuochi, T., Matthews, T., Kato, M., Hamako, J., Titani, K., Solomon, J., & Feizi, T.(1990).

Moir, S., Chun, T., & Fauci, A. (2011). Pathogenic Mechanisms of HIV Disease. *Annual* 

Muller, A., Homey, B., Soto, H., Ge, N., Catron, D., Buchanan, M., McClanahan, T., Murphy,

Mulroney, S., McDonnell, K., Pert, C., Ruff, M., Resch, Z., Samson, W., & Lumpkin, M.

Murdoch, C., Monk, P., & Finn, A. (1999). CXC chemokine receptor expression on human endothelial cells. *Cytokine*, Vol.11, No.9, (1999), pp. 704–712, ISSN 1043-4666 Muthumani, K., Wadsworth, S., Dayes, N., Hwang, D., Choo, A., Abeysinghe, H., Siekierka,

Neri, P., Bracci, L., Rustici, M., & Santucci, A.(1990). Sequence homology between HIV

Nimmagadda, S., Pullambhatla, M., Stone, K., Green, G., Bhujwalla, Z., & Pomper, M.

Ogert, R., Lee, M., Ross, W., Buckler-White, A., Martin, M., & Cho, M. (2001). N-linked

Oh, S., Cruikshank, W., Raina, J., Blanchard, G., Adler, W., Walker, J., & Kornfeld, H.(1992).

Okamoto, S., Kang, Y., Brechtel, C., Siviglia, E., Russo, R., Clemente, A., Harrop, A.,

Oppermann, M. (2004). Chemokine receptor CCR5: insights into structure, function, and

Vol.70, No.10, (May 2010), pp. 3935-3944, ISSN 0008-5472

*Signal*, Vol.12, No.1, (2000), pp. 1-13, ISSN 0898-6568

*Review Of Pathology* ,Vol.6, (2011), pp. 223-248, ISSN 1553-4014

No.6824, (2001), pp.50-56, ISSN 0028-0836

pp.739-748, ISSN 0269-9370

ISSN 0022-538X

256, ISSN 0894-9255

0898-6568

(1990), pp. 265-269, ISSN 0304-8608

9258

Diversity of oligosaccharide structures on the envelope glycoprotein gp 120 of human immunodeficiency virus 1 from the lymphoblastoid cell line H9. Presence of complex-type oligosaccharides with bisecting N-acetylglucosamine residues. *The Journal of Biological Chemistry,* Vol.265, No.15, (May 1990), pp. 8519-8524, ISSN 0021-

E., Yuan, W., Wagner, S., Barrera, J., Mohar, A., Verastegui, E., & Zlotnik, A. (2001). Involvement of chemokine receptors in breast cancer metastasis. *Nature,* Vol.410,

(1998). HIV gp120 inhibits the somatotropic axis: a possible GH-releasing hormone receptor mechanismfor the pathogenesis of AIDS wasting. *Proceedings of the National Academy of Sciences*, Vol.95, No.4, (Feb 1998), pp.1927-1932, ISSN 0027-8424

J., & Weiner, D. (2004). Suppression of HIV-1 viral replication and cellular pathogenesis by a novel p38/JNK kinase inhibitor. *AIDS*., Vol.18, No.5, (Mar 2004),

gp120, rabies virus glycoprotein, and snake venom neurotoxins. Is the nicotinic acetylcholine receptor an HIV receptor?. *Archives of Virology*, Vol.114, No.3-4,

(2010). Molecular imaging of CXCR4 receptor expression in human cancer xenografts with [64Cu]AMD3100 positron emission tomography. *Cancer Research,*

glycosylation sites adjacent to and within the V1/V2 and the V3 loops of dualtropic human immunodeficiency virus type 1 isolate DH12 gp120 affect coreceptor usage and cellular tropism. *The Journal of Virology,* Vol.75, No.13, (Jul 2001), pp. 5998-6006,

Identification of HIV-1 envelope glycoprotein in the serum of AIDS and ARC patients. *Journal of Acquired Immune Deficiency Syndromes,*Vol.5, No.3, (1992), pp.251-

Mckercher, S., Kaul, M., & Lipton, R. (2007). HIV/gp120 decreases adult neural progenitor cell proliferation via checkpoint kinase-mediated cell cycle withdrawal and G1 Arrest. *Cell Stem Cell,* Vol.1, No.2, (2007), pp. 230-236, ISSN 1934-5909 Ono, K., & Han, J. (2000). The p38 signal transduction pathway: activation and function. *Cell* 

regulation. *Cellular Signalling* ,Vol.16, No. 11, (November 2004), pp. 1201-1210, ISSN


HIV Toxins: Gp120 as an Independent Modulator of Cell Function 95

Sullivan, N., Sun, Y., Sattentau, Q., Thali, M., Wu, D., Denisova, G., Gershoni, J., Robinson,

Sunila, I., Vaccarezza, M., Pantaleo, G., Fauci, A., & Orenstein, J. (1997). gp120 is present on

Suput, D.(2009). In vivo effects of cnidarian toxins and venoms. *Toxicon*, Vol.54, No.8, (2009),

Thieblemont, N., Weiss, L., Sadeghi, H., Estcourt, C., & Haeffner-Cavaillon, N. (1995).

Toborek, M., Lee, Y., Flora, G., Pu, H., András, I., Wylegala, E., Hennig, B., & Nath, A.(2005).

*Molecular Neurobiology,* Vol.25, No.1, (Feb 2005), pp.181-199, ISSN 0272-4340 Trkola, A., Dragic, T., Arthos, J., Binley, J., Olson, W., Allaway, G., Cheng-Mayer, C.,

Trushin, S., Algeciras-Schimnich, A., Vlahakis, S., Bren, G., Warren, S., Schnepple, D., &

*The Open Virology Journal,* Vol. 4, (Jun 2010), pp.157-62, ISSN 1874-3579 Van Drenth, C., Jenkins, A., Ledwich, L., Ryan, T., Mashikian, M., Brazer, W., Center, D., &

Visalli, V., Muscoli, C., Sacco, I., Sculco, F., Palma, E., Costa, N., Colica, C., Rotiroti, D., &

Vlahakis, S., Villasis-Keever, A., Gomez, T., Bren, G., & Paya, C. (2003). Human

Wang, Q., Diao, X., Sun, J., & Chen, Z. (2011). Regulation of VEGF, MMP-9, and metastasis

Vol.165, No.11, (2000), pp. 6356-6363, ISSN 0022-1767

*Neuroscience*, Vol.8, No.1, (2007), p.106, ISSN 1471-2202

4703, ISSN 0022-538X

(Jan 1997), pp. 27-32, ISSN 0269-9370

(1996), pp.184–187, ISSN 0028-0836

pp. 6050-6062, ISSN 0008-5472

2011), pp. 897-904, ISSN 1095-8355

No.12, (1995), pp.3418–3424, ISSN 0014-2980

pp. 1190-1200, ISSN 0041-0101

J., Moore, J., & Sodroski, J.(1998). CD4-induced conformational changes in the human immunodeficiency virus type 1 gp120 glycoprotein: consequences for virus entry and neutralization. *The Journal of Virology*, Vol. 72, No. 6, (1998), pp. 4694–

the plasma membrane of apoptotic CD4 cells prepared from lymph nodes of HIV-1 infected individuals: an immunoelectron microscopic study. *AIDS*., Vol.11, No.1,

CD14lowCD16high: a cytokine-producing monocyte subset which expands during human immunodeficiency virus infection. *European Journal of Immunology,* Vol.25,

Mechanisms of the blood-brain barrier disruption in HIV-1 infection. *Cellular and* 

Robinson, J., Maddon, P., & Moore, J.(1996). CD4-dependent, antibody-sensitive interactions between HIV-1 and its co-receptor CCR-5. *Nature*, Vol.384, No.6605,

Badley, A. (2007). Glycoprotein 120 binding to CXCR4 causes p38-dependent primary T cell death that is facilitated by, but does not require cell-associated CD4. *The Journal of Immunology,* Vol.178, No.8, (2007), pp. 4846-4853, ISSN 0022-1767 Trushin, S., Bren, G., & Badley, A. (2010). CXCR4 Tropic HIV-1 gp120 Inhibition of SDF-1α-

Induced Chemotaxis Requires Lck and is Associated with Cofilin Phosphorylation*.* 

Cruikshank, W. (2000). Desensitization of CXC chemokine receptor 4, mediated by IL-16/CD4, is independent of p56lck enzymatic activity. *The Journal of Immunology,* 

Mollace, V. (2007). N-acetylcysteine prevents HIV gp 120-related damage of human cultured astrocytes: correlation with glutamine synthase dysfunction.*BMC* 

immunodeficiency virus-induced apoptosis of human hepatocytes via CXCR4. *Journal of Infectious Diseases,* Vol.188, No.10, (2003), pp. 1455-1460, ISSN 1537-6613 Wang, Q., Diskin, S., Rappaport, E., Attiyeh, E., Mosse, Y., Shue, D., Seiser, E., Jagannathan,

J., Shusterman, S., & Bansal, M. (2006). Integrative genomics identifies distinct molecular classes of neuroblastoma and shows that multiple genes are targeted by regional alterations in DNA copy number. *Cancer Research,* Vol.66, No.12, (2006),

by CXCR4 in a prostate cancer cell line. *Cell Biology International,* Vol.35, No.9, (Feb

proinflammatory and immunoregulatory cytokines*. AIDS Research and Human Retroviruses*, Vol.26, No.10, (Oct 2010), pp.1139-1145, ISSN 1931-8405


*Retroviruses*, Vol.26, No.10, (Oct 2010), pp.1139-1145, ISSN 1931-8405 Saifuddin, M., Hart, M., Gewurz, H., Zhang, Y., & Spear, G.(2000). Interaction of mannose-

Vol.200, No.7, (Oct 2009), pp. 1050-1053, ISSN 0022-1899

pp.2533-2538, ISSN 0022-1317

(Jan 2009), pp. 178-184, ISSN 1535-7163

2002), pp. 2408-2414, ISSN 0022-1767

2004), pp. 2900-2907, ISSN 0006-4971

208, ISSN 1551-4056

pp. 275-284, ISSN 1062-3329

e1000766, ISSN 1553-7366

pp. 96-115, ISSN 1872-6240

538X

ISSN 0732-0582

proinflammatory and immunoregulatory cytokines*. AIDS Research and Human* 

binding lectin with primary isolates of human immunodeficiency virus type 1. *Journal of General Virology*, Vol.81, No.4, (Apr 2000), pp. 949-955, ISSN 0022-1317 Santosuosso, M., Righi, E., Lindstrom, V., Leblanc, P., & Poznansky, M.(2009). HIV-1

envelope protein gp120 is present at high concentrations in secondary lymphoid organs of individuals with chronic HIV-1 infection. *Journal of Infectious Diseases,* 

immunodeficiency virus envelope glycoprotein by soluble CD4 binding, *The Journal* 

interspecies type sero-reactivity of the envelope glycopolypeptide gp120 of the human immunodeficiency virus. *Journal of General Virology,* Vol.67, No.11, (1986),

anergy in human peripheral blood lymphocytes by inducing interleukin-10 production. *The Journal of Virology,* Vol.70, No.8, (1996), pp. 4953-4960, ISSN 0022-

CXCR4-gp120-IIIB interactions induce caspase-mediated apoptosis of prostate cancer cells and inhibit tumor growth. *Molecular Cancer Therapeutics,* Vol.8, No.1,

G.(2002). Follicular dendritic cells and the persistence of HIV infectivity: the role of antibodies and Fc gamma receptors. *The Journal of Immunology,* Vol.168, No.5, (Mar

Lapidot, T.(2004) Unique SDF-1-induced activation of human precursor-B ALL cells as a result of altered CXCR4 expression and signaling. *Blood*, Vol.103, No.8, (Apr

Virus Glycoprotein. *Yale Journal of biology and medicine*, Vol. 83, No.4, (2010), pp.201-

receptors on human brain microvascular endothelial cells, implications for human immunodeficiency virus type 1 pathogenesis. *Endothelium*, Vol.11, No.5-6, (2004),

to expanded HIV tropism in vivo. *PLoS Pathogens*, Vol.6, No.2, (Feb 2010), pp.

The effects of HIV-1 on the blood-brain barrier. *Brain Research* , Vol.1399, (Jul 2011),

Sattentau, Q., & Moore, J. (1991). Conformational changes induced in the human

*of Experimental Medicine*, Vol.174, No.2, (1991), pp. 407–415, ISSN 0022-1007 Schneider, J., Kaaden, O., Copeland, T., Oroszlan, S., & Hunsmann, G. (1986). Shedding and

Schols, D., De Clercq, E.(1996). Human immunodeficiency virus type 1 gp120 induces

Schwartz, R.(2003). T cell anergy. *Annual Review of Immunology,* Vol.21, (2003), pp. 305–334,

Singh, S., Bond, V., Powell, M., Singh, U., Bumpers, H., Grizzle, W., & Lillard, J.(2009).

Smith-Franklin, B., Keele, B., Tew, J., Gartner, S., Szakal, A., Estes, J., Thacker, T., & Burton,

Spiegel, A., Kollet, O., Peled, A., Abel, L., Nagler, A., Bielorai, B., Rechavi, G., Vormoor, J., &

Stansell, E., & Desrosiers, R. (2010). Functional Contributions of Carbohydrate on AIDS

Stins, M., Pearce, D., Choi, H., Di Cello, F., Pardo, C., & Kim, K. (2004). CD4 and chemokine

Stoddart, C., Keir, M., & McCune, J. (2010). IFN-alpha-induced upregulation of CCR5 leads

Strazza, M., Pirrone, V., Wigdahl, B., & Nonnemacher, M. (2011). Breaking down the barrier:


**4** 

*Australia* 

*The University of Sydney, Sydney* 

**HIV Recombination and Pathogenesis –** 

*2Perinatal Medicine Group, Kolling Institute of Medical Research,* 

*Royal North Shore Hospital, St Leonards, NSW, Sydney* 

**Biological and Epidemiological Implications** 

Nitin K. Saksena1, Katherine A. Lau2, Dominic E. Dwyer3 and Bin Wang1 *1Retroviral Genetics Division, Centre for Virus Research, Westmead Millennium Institute,* 

*3Department of Virology, Institute for Clinical Pathology and Medical Research, Sydney* 

**1.1 The origin, evolution and distribution of HIV: Introduction of HIV into the human** 

There are several lines of evidence, which suggest that HIV-1 and HIV-2 originated from primates and were introduced into the human population via cross-species transmission events (Sharp et al., 1995). The viral genome structure of the simian form of the virus, simian immunodeficiency virus (SIV) is very similar to that of HIV (Huet et al., 1990), and phylogenetic relatedness has been established (Gao et al., 1999; Hirsch et al., 1989). SIV has been shown to infect primates that live in geographical areas where HIV is endemic (Gao et al., 1999; Hirsch et al., 1989), and possible routes of transmission, such as the butchering of primates for food and keeping monkeys as pets, have been proposed (Gao

A natural primate host for HIV-1 has been proposed however there still remains some controversy. Strains of SIV (SIVcpz) from the chimpanzee Pan troglodytes troglodytes, phylogenetically cluster closer to HIV-1 strains than many other characterized SIV strains (Gao et al., 1999). However there is still a moderate amount of diversity between SIVcpz and HIV-1, and the prevalence of SIV infections in wild chimpanzees is low. There are three major groups within HIV-1, M (main), O (outlier) and N (new, or 'non-O non-M'). Each of the three groups of HIV-1 share a common branch with SIVcpz strains, but do not diverge from a common stem with SIVcpz, suggesting that they each arose from different crossspecies transmissions (Gao et al., 1999; Thomson et al., 2002b). To support the idea that P.t. troglodytes is the natural reservoir of HIV, the HIV-1 N group was shown to be a recombinant between diverse viral strains within the HIV/SIVcpz group, suggesting an ancestral recombination in this sub-species of chimpanzee (Gao et al., 1999; Garcia et al.,

It is thought that the HIV-1 M group originated in the Democratic Republic of Congo, as an extremely high genetic diversity is seen within the region (Vidal et al., 2000) and the earliest confirmed HIV-1 infection was found there in a stored serum sample from 1959 (Zhu et al.,

**1. Introduction** 

**population** 

et al., 1999).

1999).


## **HIV Recombination and Pathogenesis – Biological and Epidemiological Implications**

Nitin K. Saksena1, Katherine A. Lau2, Dominic E. Dwyer3 and Bin Wang1 *1Retroviral Genetics Division, Centre for Virus Research, Westmead Millennium Institute,* 

*3Department of Virology, Institute for Clinical Pathology and Medical Research, Sydney Australia* 

## **1. Introduction**

96 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Wei, X., Decker, J., Wang, S., Hui, H., Kappes, J., Wu, X., Salazar-Gonzalez, J., Salazar, M.,

Weiss, J., Nath, A., Major, E., & Berman, J.(1999). HIV-1 Tat induces monocyte

Weissman, D., Rabin, R., Arthos, J., Rubbert, A., Dybul, M., Swofford, R., Venkatesan, S.,

Williams, S., & Greene, W.(2007). Regulation of HIV-1 latency by T-cell activation. *Cytokine*,

Wu, L., Gerard, N., Wyatt, R., Choe, H., Parolin, C., Ruffing, N., Borsetti, A., Cardoso, A.,

CCR-5, *Nature,* Vol.384, No.6605, (1996), pp. 179–183, ISSN 0028-0836 Wu, Y., & Yoder, A. (2009). Chemokine coreceptor signaling in HIV-1 infection and pathogenesis. *PLoS Pathogens*, Vol.5, No.12, (Dec 2009), p., ISSN 1553-7366 Wyatt, R., & Sodroski, J.(1998). The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens, *Science*, Vol.280, No.5371, (1998), pp.1884–1888, ISSN 0036-8075 Wysoczynski, M., Reca, R., Ratajczak, J., Kucia, M., Shirvaikar, N., Honczarenko, M., Mills,

(2003), pp. 307-312, ISSN 0028-0836

(1997), pp. 981-985, ISSN 0028-0836

2005), pp.40-48, ISSN 0006-4971

1007, ISSN 1098-1136

Vol.39, No.1, (Jul 2007) , pp. 63-74, ISSN 1043-4666

0022-1767

Kilby, J., Saag, M., Komarova, N., Nowak, M., Hahn, B., Kwong, P., & Shaw, G. (2003). Antibody neutralization and escape by HIV-1. *Nature,* Vol.422, No.6929,

chemoattractant protein-1-mediated monocyte transmigration across a model of the human blood-brain barrier and up-regulates CCR5 expression on human monocytes. *The Journal of Immunology,* Vol.163, No.5, (1999), pp. 2953-2959, ISSN

Farber, J., & Fauci, A. (1997). Macrophage-tropic HIV and SIV envelope proteins induce a signal through the CCR5 chemokine receptor. *Nature*, Vol.389, No.6654,

Desjardin, E., Newman, W., Gerard, C., & Sodroski, J. (1996). CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor

M., Wanzeck, J., Janowska-Wieczorek, A., & Ratajczak, M. (2005). Incorporation of CXCR4 into membrane lipid rafts primes homing-related responses of hematopoietic stem/progenitor cells to an SDF-1 gradient. *Blood*, Vol.105, No.1, (Jan

enhances outward potassium current via CXCR4 and cAMP-dependent protein kinase a signaling in cultured rat microglia. *Glia*, Vol.59, No.6, (Mar 2011), pp.997-

Stephany, D., Cooper, J., Marsh, J., & Wu, Y.(2008). HIV Envelope-CXCR4 Signaling Activates Cofilin to Overcome Cortical Actin Restriction in Resting CD4 T Cells.

Tracking global patterns of *N*-linked glycosylation site variation in highly variable viral glycoproteins: HIV, SIV, and HCV envelopes and influenza hemagglutinin.

Dewhurst, S., & Maggirwar, S. (2006). Inhibition of Mixed Lineage Kinase 3 Prevents HIV-1 Tat-Mediated Neurotoxicity and Monocyte Activation. *The Journal* 

Xu, C., Liu, J., Chen, L., Liang, S., Fujii, N., Tamamura, H., & Xiong, H. (2011). HIV-1 gp120

Yoder, A., Yu, D., Dong, L., Iyer, S., Xu, X., Kelly, J., Liu, J., Wang, W., Vorster, P., Agulto, L.,

Zhang, M., Gaschen, B., Blay, W., Foley, B., Haigwood, N., Kuiken, C., & Korber, B.(2004).

Ziye, S., Fan, S., Sniderhan, L., Reisinger, E., Litzburg, A., Schifitto, G., Gelbard, H.,

*Glycobiology*, Vol.14, No.12, (2004), pp. 1229-1246, ISSN 0959-6658

*of Immunology,* Vol.177, No.1, (2006), pp.702-711, ISSN 0022-1767

*Cell*, Vol.134, No.5, (2008), pp. 782–792, ISSN 0092-8674

#### **1.1 The origin, evolution and distribution of HIV: Introduction of HIV into the human population**

There are several lines of evidence, which suggest that HIV-1 and HIV-2 originated from primates and were introduced into the human population via cross-species transmission events (Sharp et al., 1995). The viral genome structure of the simian form of the virus, simian immunodeficiency virus (SIV) is very similar to that of HIV (Huet et al., 1990), and phylogenetic relatedness has been established (Gao et al., 1999; Hirsch et al., 1989). SIV has been shown to infect primates that live in geographical areas where HIV is endemic (Gao et al., 1999; Hirsch et al., 1989), and possible routes of transmission, such as the butchering of primates for food and keeping monkeys as pets, have been proposed (Gao et al., 1999).

A natural primate host for HIV-1 has been proposed however there still remains some controversy. Strains of SIV (SIVcpz) from the chimpanzee Pan troglodytes troglodytes, phylogenetically cluster closer to HIV-1 strains than many other characterized SIV strains (Gao et al., 1999). However there is still a moderate amount of diversity between SIVcpz and HIV-1, and the prevalence of SIV infections in wild chimpanzees is low. There are three major groups within HIV-1, M (main), O (outlier) and N (new, or 'non-O non-M'). Each of the three groups of HIV-1 share a common branch with SIVcpz strains, but do not diverge from a common stem with SIVcpz, suggesting that they each arose from different crossspecies transmissions (Gao et al., 1999; Thomson et al., 2002b). To support the idea that P.t. troglodytes is the natural reservoir of HIV, the HIV-1 N group was shown to be a recombinant between diverse viral strains within the HIV/SIVcpz group, suggesting an ancestral recombination in this sub-species of chimpanzee (Gao et al., 1999; Garcia et al., 1999).

It is thought that the HIV-1 M group originated in the Democratic Republic of Congo, as an extremely high genetic diversity is seen within the region (Vidal et al., 2000) and the earliest confirmed HIV-1 infection was found there in a stored serum sample from 1959 (Zhu et al.,

*The University of Sydney, Sydney* 

*<sup>2</sup>Perinatal Medicine Group, Kolling Institute of Medical Research,* 

*Royal North Shore Hospital, St Leonards, NSW, Sydney* 

HIV Recombination and Pathogenesis – Biological and Epidemiological Implications 99

Fig. 2. Map showing the global dispersal of diverse HIV subtypes (represented by single

One of the major hallmarks of HIV infection is high genetic diversity. This high genetic diversity in HIV-1 is attributed to the infidelity of its reverse transcriptase enzyme, which leads to high rates of mutation [Preston et al., 1988] and rapid viral variant turnover in patients-termed quasispecies [Ho et al., 1995 and Wei et al., 1995]. This quasispecies or the swarm of viral variants is a powerful asset of HIV in creating recombinants with superior ability to survive, evade and infect in vivo. In addition, the subtypic diversity within HIV-1 M group also provides the virus with a wider selection of strains to recombine with and disperse effectively by creating creating virulent forms. Currently, major subtypes are being replaced

by circulating recombinant forms (CRFs), the evidence of which can be seen in **figure 2**.

The HIV-1 M group can be divided into 9 subtypes (A-D, F-H, J and K), and some subtypes are further broken down into subsubtypes, according to their topologies within phylogenetic trees. There is possibly a 10th subtype, L, for which 2 full-length sequences have been identified (Mokili et al., 2002). All of these subtypes can be found in Africa, where they are thought to have originated (Thomson et al., 2002b). Other continents have a variety of subtypes circulating, resulting in an uneven representation of the subtypes. Based on the amino acid sequence of the env region, each subtype is separated by approximately 25-30% genetic distance (Robertson et al., 1999; Thomson et al., 2002b). Subtypes A and F can be further broken down into subsubtypes, A1-A4 and F1 and F2 (Vidal et al., 2006). Technically

letter codes) and their replacement by circulating recombinant forms (CRFs).

**2. Genetic diversity in HIV: Recombination – A unique trait for the** 

**continuation of viral progeny** 

1998). Based on sequence analysis of the HIV-1 M group, it was estimated that these strains arose from a common ancestor in about 1931 (Korber et al., 2000).

Unlike HIV-1, the origin of HIV-2 is more definitive. The discovery of a form of SIV in sooty mangabeys (SIVsm) which is nearly identical to HIV-2, and which is found in these primates from the area where HIV-2 circulates in humans, provides very strong evidence that HIV-2 came from these primates (Gao et al., 1992). At present, there are eight designated groups of HIV-2, A-H, which are analogous to the HIV-1 groups (M, N and O) although, groups C-H have only been identified in single individuals (Chen et al., 1997; Damond et al., 2004). All groups of HIV-2 are believed to have arisen from individual cross-species transmission events from sooty mangabeys (Chen et al., 1996). Analysis conducted with HIV-2 strains from subtypes A and B, dated a recent common ancestor to around 1940 and 1945 respectively (Lemey et al., 2003).

Currently, there are 33 million people living with HIV/AIDS globally with 16,000 new infections happening every day (UNAIDS 2010) (**Figure 1**). As the acquired immune deficiency syndrome (AIDS) pandemic enters its third decade, the number of people living with human immunodeficiency virus (HIV) infection continues to increase. Although the HIV/AIDS epidemic was recognized in Southeast Asia later than elsewhere, local risk behaviors have allowed the epidemic to expand rapidly. Today, injecting drug use (IDU) accounts for up to 70% of HIV-1 transmission in many Asian countries, including China, Indonesia, Malaysia, Myanmar, Eastern India and Vietnam (Saksena et al., 2005). Also, there is ample evidence that heterosexual transmission through commercial sex workers has increased over the last few years (Saksena et al., 2005).

Fig. 1. Estimation of global adult prevalence in 2007, with an approximately 33 million of people living with HIV. Diagram source: UNAIDS, 2008: Report on the global AIDS epidemic.

1998). Based on sequence analysis of the HIV-1 M group, it was estimated that these strains

Unlike HIV-1, the origin of HIV-2 is more definitive. The discovery of a form of SIV in sooty mangabeys (SIVsm) which is nearly identical to HIV-2, and which is found in these primates from the area where HIV-2 circulates in humans, provides very strong evidence that HIV-2 came from these primates (Gao et al., 1992). At present, there are eight designated groups of HIV-2, A-H, which are analogous to the HIV-1 groups (M, N and O) although, groups C-H have only been identified in single individuals (Chen et al., 1997; Damond et al., 2004). All groups of HIV-2 are believed to have arisen from individual cross-species transmission events from sooty mangabeys (Chen et al., 1996). Analysis conducted with HIV-2 strains from subtypes A and B, dated a recent common ancestor to around 1940 and 1945

Currently, there are 33 million people living with HIV/AIDS globally with 16,000 new infections happening every day (UNAIDS 2010) (**Figure 1**). As the acquired immune deficiency syndrome (AIDS) pandemic enters its third decade, the number of people living with human immunodeficiency virus (HIV) infection continues to increase. Although the HIV/AIDS epidemic was recognized in Southeast Asia later than elsewhere, local risk behaviors have allowed the epidemic to expand rapidly. Today, injecting drug use (IDU) accounts for up to 70% of HIV-1 transmission in many Asian countries, including China, Indonesia, Malaysia, Myanmar, Eastern India and Vietnam (Saksena et al., 2005). Also, there is ample evidence that heterosexual transmission through commercial sex workers has

Fig. 1. Estimation of global adult prevalence in 2007, with an approximately 33 million of people living with HIV. Diagram source: UNAIDS, 2008: Report on the global AIDS

arose from a common ancestor in about 1931 (Korber et al., 2000).

respectively (Lemey et al., 2003).

epidemic.

increased over the last few years (Saksena et al., 2005).

Fig. 2. Map showing the global dispersal of diverse HIV subtypes (represented by single letter codes) and their replacement by circulating recombinant forms (CRFs).

## **2. Genetic diversity in HIV: Recombination – A unique trait for the continuation of viral progeny**

One of the major hallmarks of HIV infection is high genetic diversity. This high genetic diversity in HIV-1 is attributed to the infidelity of its reverse transcriptase enzyme, which leads to high rates of mutation [Preston et al., 1988] and rapid viral variant turnover in patients-termed quasispecies [Ho et al., 1995 and Wei et al., 1995]. This quasispecies or the swarm of viral variants is a powerful asset of HIV in creating recombinants with superior ability to survive, evade and infect in vivo. In addition, the subtypic diversity within HIV-1 M group also provides the virus with a wider selection of strains to recombine with and disperse effectively by creating creating virulent forms. Currently, major subtypes are being replaced by circulating recombinant forms (CRFs), the evidence of which can be seen in **figure 2**.

The HIV-1 M group can be divided into 9 subtypes (A-D, F-H, J and K), and some subtypes are further broken down into subsubtypes, according to their topologies within phylogenetic trees. There is possibly a 10th subtype, L, for which 2 full-length sequences have been identified (Mokili et al., 2002). All of these subtypes can be found in Africa, where they are thought to have originated (Thomson et al., 2002b). Other continents have a variety of subtypes circulating, resulting in an uneven representation of the subtypes. Based on the amino acid sequence of the env region, each subtype is separated by approximately 25-30% genetic distance (Robertson et al., 1999; Thomson et al., 2002b). Subtypes A and F can be further broken down into subsubtypes, A1-A4 and F1 and F2 (Vidal et al., 2006). Technically

HIV Recombination and Pathogenesis – Biological and Epidemiological Implications 101

Multiple infection of a single cell with HIV can occur simultaneously or sequentially. There is some debate regarding the sequential infection, as once a cell is infected, HIV downregulates the CD4 and CCR5 receptor molecules (Michel et al., 2005), and therefore simultaneous infection may be the primary mechanism involved. Interestingly, a study by Dang et al. (2004) showed that dual infections of cells occurs at a much higher rate than predicted by chance, in both a T cell line, and primary T cells, regardless of how the virus was transmitted. In a follow up study it was also shown that coreceptor differences were not a barrier to recombination, as viruses using different co-receptors, CCR5 or CXCR4 also exhibited rates of double infection that were higher than predicted from a random distribution (Chen et al., 2005). Likewise, other studies have also shown co-infection of cells to be common, which implies that opportunities

Fig. 3. HIV dual infection of a cell, heterozygous virion formation and recombination.

There have been several studies addressing minus-strand recombination, and it has been shown to occur frequently, with an average of three crossovers occurring per replication

The first mechanism proposed to account for recombination was the forced copy-choice model (Coffin, 1979), and was based on evidence that suggested the genomic RNA of retroviruses is fragmented. A break in the RNA would halt reverse transcription and consequently force the reverse transcriptase to switch to the second copy of RNA in order to continue synthesis, that is, a strand transfer event would occur (Hu and Temin, 1990b). In order for this to take place, the RT enzyme must be transferred to a homologous region on the second RNA copy. This model assumes therefore that recombination occurs during

for recombination are favoured (Jung et al., 2002).

Modified from: Najera et al. (2002).

**3.1 Minus-strand recombination** 

cycle (Yu et al., 1998).

subtypes B and D are closely related and could be classified together as sub-subtypes, however they are generally still mentioned separately for consistency (Lal et al., 2005). In addition, within subtypes there are clusters that represent strains from different geographical locations, such as subtype B strains from Thailand, which are referred to as B' or Thai B (Kalish et al., 1995). These clusters most likely arose due to the initial introduction of only a small number of strains into a particular region, and their subsequent diversification and spread (known as a founder effect) (Thomson et al., 2002b). In addition to the nine subtypes of HIV-1, there exists circulating recombinant forms (CRFs), which are mosaic viruses of two or more HIV-1 subtypes. To be classified as a CRF, an inter-subtype recombinant virus must be isolated from at least two (preferably three) unlinked individuals and sequenced in full (Carr et al., 1998). Currently 34 CRFs have been recognized, with 26 described in detail (Los Alamos HIV Database) (**Figure 6**). These CRFs now include second generation CRFs such as CRF09\_cpx and CRF15\_01B, which is contain the primary CRFs CRF02\_AG and CRF01\_AE respectively. Regions within CRFs that cannot be classified as a known subtype are designated U for unknown, and a CRF comprised of more than two subtypes is denoted by cpx for complex (Peeters, 2000; Robertson et al., 1999). The first CRF identified was CRF01\_AE, and was originally classified as subtype E, before it was designated a recombinant (CRF01\_AE has subtype A gag and pol regions and a subtype E env) (Gao et al., 1996), and interestingly a fulllength subtype E virus has not been identified, possibly indicating that the gag and pol regions were lost through an ancestral recombination event (Gao et al., 1996).

Recombination is common in HIV and it uses recombination in order to acquire viral fitness, virulence and ability to evade the host immune system. Recombination occurs as a result of the low affinity binding of RT, which is necessary for the strand transfers of reverse transcription to occur. As HIV carries two copies of its viral RNA, during reverse transcription both of these can serve as templates to generate the provirus. If the two copies of RNA encapsulated with a virion are distinct, and are both used during reverse transcription, then the resulting provirus will be a recombinant. Thus the first requirement for recombination is a dually infected cell, which can give rise to a heterozygous virion, which carries two distinct copies of RNA. Once a heterozygous virion is formed recombination then occurs upon infection of a new cell (**Figure 3**). The theories behind how recombination occurs at the molecular level can be divided into two groups, recombination that occurs during the synthesis of the minus-strand DNA and that which occurs during the synthesis of the plus-strand DNA. Each theory has its own explanation and some supporting experimental evidence, implying that both mechanisms can occur (Hu and Temin, 1990b).

#### **3. Heterozygous virions and their formation**

A requirement for the generation of recombinant HIV genomes is a heterozygous virion, that is, a virion with two non-identical RNA strands (Hu and Temin, 1990a; Weiss et al., 1973). Heterozygous virions are generated in individual cells which are infected with two or more different viral variants, which integrate their proviral genome, and generate new full length viral RNA.

As packaging of the viral RNA is not selective for specific RNA copies (D'Souza and Summers, 2005), a heterozygous virus can be formed, by encapsidation of an RNA copy from each viral variant. Heterozygous virions can then infect other cells and recombination between the two co-packaged viral RNAs can occur during reverse transcription (Duesberg, 1968). (**Figure 3**). The different viral RNAs can come from variants within the viral quasispecies, or from other HIV strains or subtypes, if the patient has a dual infection.

subtypes B and D are closely related and could be classified together as sub-subtypes, however they are generally still mentioned separately for consistency (Lal et al., 2005). In addition, within subtypes there are clusters that represent strains from different geographical locations, such as subtype B strains from Thailand, which are referred to as B' or Thai B (Kalish et al., 1995). These clusters most likely arose due to the initial introduction of only a small number of strains into a particular region, and their subsequent diversification and spread (known as a founder effect) (Thomson et al., 2002b). In addition to the nine subtypes of HIV-1, there exists circulating recombinant forms (CRFs), which are mosaic viruses of two or more HIV-1 subtypes. To be classified as a CRF, an inter-subtype recombinant virus must be isolated from at least two (preferably three) unlinked individuals and sequenced in full (Carr et al., 1998). Currently 34 CRFs have been recognized, with 26 described in detail (Los Alamos HIV Database) (**Figure 6**). These CRFs now include second generation CRFs such as CRF09\_cpx and CRF15\_01B, which is contain the primary CRFs CRF02\_AG and CRF01\_AE respectively. Regions within CRFs that cannot be classified as a known subtype are designated U for unknown, and a CRF comprised of more than two subtypes is denoted by cpx for complex (Peeters, 2000; Robertson et al., 1999). The first CRF identified was CRF01\_AE, and was originally classified as subtype E, before it was designated a recombinant (CRF01\_AE has subtype A gag and pol regions and a subtype E env) (Gao et al., 1996), and interestingly a fulllength subtype E virus has not been identified, possibly indicating that the gag and pol regions

Recombination is common in HIV and it uses recombination in order to acquire viral fitness, virulence and ability to evade the host immune system. Recombination occurs as a result of the low affinity binding of RT, which is necessary for the strand transfers of reverse transcription to occur. As HIV carries two copies of its viral RNA, during reverse transcription both of these can serve as templates to generate the provirus. If the two copies of RNA encapsulated with a virion are distinct, and are both used during reverse transcription, then the resulting provirus will be a recombinant. Thus the first requirement for recombination is a dually infected cell, which can give rise to a heterozygous virion, which carries two distinct copies of RNA. Once a heterozygous virion is formed recombination then occurs upon infection of a new cell (**Figure 3**). The theories behind how recombination occurs at the molecular level can be divided into two groups, recombination that occurs during the synthesis of the minus-strand DNA and that which occurs during the synthesis of the plus-strand DNA. Each theory has its own explanation and some supporting experimental evidence, implying that both mechanisms can

A requirement for the generation of recombinant HIV genomes is a heterozygous virion, that is, a virion with two non-identical RNA strands (Hu and Temin, 1990a; Weiss et al., 1973). Heterozygous virions are generated in individual cells which are infected with two or more different viral variants, which integrate their proviral genome, and generate new full

As packaging of the viral RNA is not selective for specific RNA copies (D'Souza and Summers, 2005), a heterozygous virus can be formed, by encapsidation of an RNA copy from each viral variant. Heterozygous virions can then infect other cells and recombination between the two co-packaged viral RNAs can occur during reverse transcription (Duesberg, 1968). (**Figure 3**). The different viral RNAs can come from variants within the viral quasispecies, or from other HIV strains or subtypes, if the patient has a dual infection.

were lost through an ancestral recombination event (Gao et al., 1996).

occur (Hu and Temin, 1990b).

length viral RNA.

**3. Heterozygous virions and their formation** 

Multiple infection of a single cell with HIV can occur simultaneously or sequentially. There is some debate regarding the sequential infection, as once a cell is infected, HIV downregulates the CD4 and CCR5 receptor molecules (Michel et al., 2005), and therefore simultaneous infection may be the primary mechanism involved. Interestingly, a study by Dang et al. (2004) showed that dual infections of cells occurs at a much higher rate than predicted by chance, in both a T cell line, and primary T cells, regardless of how the virus was transmitted. In a follow up study it was also shown that coreceptor differences were not a barrier to recombination, as viruses using different co-receptors, CCR5 or CXCR4 also exhibited rates of double infection that were higher than predicted from a random distribution (Chen et al., 2005). Likewise, other studies have also shown co-infection of cells to be common, which implies that opportunities for recombination are favoured (Jung et al., 2002).

Fig. 3. HIV dual infection of a cell, heterozygous virion formation and recombination. Modified from: Najera et al. (2002).

#### **3.1 Minus-strand recombination**

There have been several studies addressing minus-strand recombination, and it has been shown to occur frequently, with an average of three crossovers occurring per replication cycle (Yu et al., 1998).

The first mechanism proposed to account for recombination was the forced copy-choice model (Coffin, 1979), and was based on evidence that suggested the genomic RNA of retroviruses is fragmented. A break in the RNA would halt reverse transcription and consequently force the reverse transcriptase to switch to the second copy of RNA in order to continue synthesis, that is, a strand transfer event would occur (Hu and Temin, 1990b). In order for this to take place, the RT enzyme must be transferred to a homologous region on the second RNA copy. This model assumes therefore that recombination occurs during

HIV Recombination and Pathogenesis – Biological and Epidemiological Implications 103

Across the globe there is an uneven representation of the HIV-1 M group subtypes (**Figure 2**), along with the presence of diverse subtypes and its CRFs in Asia (**Figure 5**). This uneven distribution is thought to have arisen partially due to a founder effect, where one subtype is introduced into a region by a single or a few individuals from which the epidemic radiates out (Korber et al., 2000). Subtype A is currently divided in to four sub-subtypes: A1-A4. Subsubtype A1 is one of the more common variants and is found throughout Western, Central and Eastern Africa and Eastern Europe, and two CRF forms containing subtype A1 (CRF02\_AG and CRF03\_AB) are also widely spread in these regions (Andersson et al., 1999; Bobkov et al., 2004; Dowling et al., 2002; Steain et al., 2005). Subtype A (sub-subtypes 1 and 2) strains are highly predominant in Kenya, with one study showing 93% of all strains found in the region being subtype A or a recombinant containing subtype A (Dowling et al., 2002). In addition, CRF01\_AE is one of the major strains found in Thailand and Southeast Asia (McCutchan et al., 1992; Tovanabutra et al., 2004). Subsubtype A2 was first identified from sequences originating in the Democratic Republic of Congo and Cyprus (Gao et al., 2001), and is now also found in Kenya (Visawapoka et al., 2006). In addition, there have been 2 recognised CRF forms, CRF16\_A2D and CRF21\_A2D, both of which were also identified in Kenya (Visawapoka et al., 2006). CRF16\_A2D has also achieved a global spread and has been identified in Korea and Argentina (Gomez-Carrillo et al., 2004). Subsubtype A3 has only been recently described (Meloni et al., 2004a), and has thus far only been identified in areas of West and Central Africa (Meloni et al., 2004a; Meloni et al., 2004b). Similarly subsubtype A4 is a relatively new strain, and has only

**4. The global distribution of HIV-1 subtypes and CRFs** 

been identified within the Democratic Republic of Congo (Vidal et al., 2006).

intersubtype recombinants (Soares et al., 2005; Tatt et al., 2004).

Subtype B is the most common subtype in Australia, as well as the USA, and Western and Central Europe (de Oliveira et al., 2000; Essex, 1999; Herring et al., 2003). It is also found in South America, where CFR012\_BF also circulates (Montano et al., 2005), Thailand (B' strains), with CRF15\_01B (Tovanabutra et al., 2001), China, with CRF07\_BC and CRF08\_BC (Saksena et al., 2005), Spain with CRF14\_BG (Delgado et al., 2002) and Eastern Europe with CRF03\_AB (Liitsola et al., 1998). This subtype was one of the first to spread globally, however generally seems to be on the decline, with the wider spread of other subtypes and

Subtype C is currently the most prevalent subtype. It is found circulating widely through South Africa, East Africa and India (Bessong et al., 2005) and to a lesser extent in South America, Eastern Europe and Chine (Saksena et al., 2005; Soares et al., 2005). It has been suggested that strains of subtype C possess some selective advantage due to its rapid

Subtype D is found across most of Africa, particularly East African countries, where in Uganda it has been reported as a predominating strain (Harris et al., 2002). It has also been reported in other continents, though usually as a minor variant (Tatt et al., 2004). In addition, subtype D is frequently a component of unique intersubtype recombinants, from Kenya and other East African countries (Steain et al., 2005), and it has been suggested that recombinants between subtypes A and D are selected for in dually infected patients (Songok et al., 2004). Subsubtype F1 is found in South America and Europe, whereas subtype F strains from Africa (eg Cameroon) more commonly belong to subsubtype F2 (Laukkanen et al., 2000). Subtype F also forms part of CRF12\_BF, which has become widespread across South America, and more recently as a part of CRF17\_BF, CRF28\_BF and CRF29\_BF, which have also been identified in South America (De Sa Filho et al., 2006; Hierholzer et al., 2002).

dispersal across the globe that has been seen in recent years (Walker et al., 2005).

minus-strand synthesis, and is comparable to the minus-strand strong stop strand transfer that occurs during reverse transcription (Negroni and Buc, 2001) (**Figure 4**). However, no studies have been able to establish a firm connection between strand switching and the frequency of RNA breaks. Further, experiments have shown that a strand break is not necessary for a template switch (Hu and Temin, 1990b). Consequently, the minus-strand exchange model was proposed which suggests that the low processivity (loose adherence to the RNA template) of RT causes strand transfers (Coffin, 1979; Yu et al., 1998). It has also been shown that obstacles to reverse transcription, causing the enzyme to pause, can trigger a strand transfer (Wu et al., 1995). In addition, studies have also suggested that secondary structures of the RNA templates could also increase template switching without a pause, by bringing the two templates into close proximity (Balakrishnan et al., 2001) (**Figure 4**).

#### **3.2 Plus-strand recombination**

Recombination that occurs during the synthesis of the positive strand of DNA is referred to as the strand displacement assimilation model (Hu and Temin, 1990b). This model suggests that both copies of viral RNA are transcribed to produce two minus-strand DNA copies. Synthesis of plus-strand DNA is initially discontinuous, and internally initiated fragments occur eg. at the cPPT (Hsu and Taylor, 1982). Therefore, it has been proposed that an internally initiated fragment can be displaced by an elongating upstream DNA fragment, and this can cause the internally initiated fragment to dissociate and re-anneal to the complementary region of the second minus-strand DNA. The resulting double stranded DNA will have a mismatched region and mismatch DNA repair will then correct the sequence differences (Hu and Temin, 1990b) (**Figure 4**). Two positive strands of RNA, which may contain breaks. During minus-strand synthesis, the RT can switch to the other template at a break.

Fig. 4. Model of retrovirus recombination: minus-strand recombination (forced-copy choice) and plus-strand recombination (strand displacement assimilation).

## **4. The global distribution of HIV-1 subtypes and CRFs**

102 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

minus-strand synthesis, and is comparable to the minus-strand strong stop strand transfer that occurs during reverse transcription (Negroni and Buc, 2001) (**Figure 4**). However, no studies have been able to establish a firm connection between strand switching and the frequency of RNA breaks. Further, experiments have shown that a strand break is not necessary for a template switch (Hu and Temin, 1990b). Consequently, the minus-strand exchange model was proposed which suggests that the low processivity (loose adherence to the RNA template) of RT causes strand transfers (Coffin, 1979; Yu et al., 1998). It has also been shown that obstacles to reverse transcription, causing the enzyme to pause, can trigger a strand transfer (Wu et al., 1995). In addition, studies have also suggested that secondary structures of the RNA templates could also increase template switching without a pause, by

bringing the two templates into close proximity (Balakrishnan et al., 2001) (**Figure 4**).

Recombination that occurs during the synthesis of the positive strand of DNA is referred to as the strand displacement assimilation model (Hu and Temin, 1990b). This model suggests that both copies of viral RNA are transcribed to produce two minus-strand DNA copies. Synthesis of plus-strand DNA is initially discontinuous, and internally initiated fragments occur eg. at the cPPT (Hsu and Taylor, 1982). Therefore, it has been proposed that an internally initiated fragment can be displaced by an elongating upstream DNA fragment, and this can cause the internally initiated fragment to dissociate and re-anneal to the complementary region of the second minus-strand DNA. The resulting double stranded DNA will have a mismatched region and mismatch DNA repair will then correct the sequence differences (Hu and Temin, 1990b) (**Figure 4**). Two positive strands of RNA, which may contain breaks. During minus-strand synthesis, the RT can switch to the other template

Fig. 4. Model of retrovirus recombination: minus-strand recombination (forced-copy choice)

and plus-strand recombination (strand displacement assimilation).

**3.2 Plus-strand recombination** 

at a break.

Across the globe there is an uneven representation of the HIV-1 M group subtypes (**Figure 2**), along with the presence of diverse subtypes and its CRFs in Asia (**Figure 5**). This uneven distribution is thought to have arisen partially due to a founder effect, where one subtype is introduced into a region by a single or a few individuals from which the epidemic radiates out (Korber et al., 2000). Subtype A is currently divided in to four sub-subtypes: A1-A4. Subsubtype A1 is one of the more common variants and is found throughout Western, Central and Eastern Africa and Eastern Europe, and two CRF forms containing subtype A1 (CRF02\_AG and CRF03\_AB) are also widely spread in these regions (Andersson et al., 1999; Bobkov et al., 2004; Dowling et al., 2002; Steain et al., 2005). Subtype A (sub-subtypes 1 and 2) strains are highly predominant in Kenya, with one study showing 93% of all strains found in the region being subtype A or a recombinant containing subtype A (Dowling et al., 2002). In addition, CRF01\_AE is one of the major strains found in Thailand and Southeast Asia (McCutchan et al., 1992; Tovanabutra et al., 2004). Subsubtype A2 was first identified from sequences originating in the Democratic Republic of Congo and Cyprus (Gao et al., 2001), and is now also found in Kenya (Visawapoka et al., 2006). In addition, there have been 2 recognised CRF forms, CRF16\_A2D and CRF21\_A2D, both of which were also identified in Kenya (Visawapoka et al., 2006). CRF16\_A2D has also achieved a global spread and has been identified in Korea and Argentina (Gomez-Carrillo et al., 2004). Subsubtype A3 has only been recently described (Meloni et al., 2004a), and has thus far only been identified in areas of West and Central Africa (Meloni et al., 2004a; Meloni et al., 2004b). Similarly subsubtype A4 is a relatively new strain, and has only been identified within the Democratic Republic of Congo (Vidal et al., 2006).

Subtype B is the most common subtype in Australia, as well as the USA, and Western and Central Europe (de Oliveira et al., 2000; Essex, 1999; Herring et al., 2003). It is also found in South America, where CFR012\_BF also circulates (Montano et al., 2005), Thailand (B' strains), with CRF15\_01B (Tovanabutra et al., 2001), China, with CRF07\_BC and CRF08\_BC (Saksena et al., 2005), Spain with CRF14\_BG (Delgado et al., 2002) and Eastern Europe with CRF03\_AB (Liitsola et al., 1998). This subtype was one of the first to spread globally, however generally seems to be on the decline, with the wider spread of other subtypes and intersubtype recombinants (Soares et al., 2005; Tatt et al., 2004).

Subtype C is currently the most prevalent subtype. It is found circulating widely through South Africa, East Africa and India (Bessong et al., 2005) and to a lesser extent in South America, Eastern Europe and Chine (Saksena et al., 2005; Soares et al., 2005). It has been suggested that strains of subtype C possess some selective advantage due to its rapid dispersal across the globe that has been seen in recent years (Walker et al., 2005).

Subtype D is found across most of Africa, particularly East African countries, where in Uganda it has been reported as a predominating strain (Harris et al., 2002). It has also been reported in other continents, though usually as a minor variant (Tatt et al., 2004). In addition, subtype D is frequently a component of unique intersubtype recombinants, from Kenya and other East African countries (Steain et al., 2005), and it has been suggested that recombinants between subtypes A and D are selected for in dually infected patients (Songok et al., 2004). Subsubtype F1 is found in South America and Europe, whereas subtype F strains from Africa (eg Cameroon) more commonly belong to subsubtype F2 (Laukkanen et al., 2000). Subtype F also forms part of CRF12\_BF, which has become widespread across South America, and more recently as a part of CRF17\_BF, CRF28\_BF and CRF29\_BF, which have also been identified in South America (De Sa Filho et al., 2006; Hierholzer et al., 2002).

HIV Recombination and Pathogenesis – Biological and Epidemiological Implications 105

recombinants are also found in Spain and neighbouring regions (Perez-Alvarez et al., 2003). Subtypes H, J and K have had only a minor impact on the HIV epidemic and are generally only found in small numbers across West and Central Africa (Mokili et al., 1999; Thomson et al., 2002b; Vidal et al., 2000; Yang et al., 2005a). The remaining CRFs are also found in varying proportions, usually each within a distinct geographical region. Whole spectrum of emerging

The magnitude of genetic diversity in a host relates to the size of the viral population, the extent of replication, the mutation and recombination rates and the selective pressures placed on the virus (Saksena et al., 2001). Overall the divergence between the circulating HIV strains within a single individual and the original infecting strain/s is thought to increase by around 1% per year in early infection (Shankarappa et al., 1999). Thus, within an individual a heterogeneous viral population exists which has been termed a 'quasispecies'. Within an individual this viral diversity can reach as 15% (Lukashov and Goudsmit, 1997). The reasons behind such viral diversity include a fast turnover of virions, approximately 109 new virions are produced each day (Ho et al., 1995), and the low fidelity of reverse transcriptase. Reverse transcriptase lacks a proof-reading function, meaning that any nucleotides that are mis-incorporated during DNA synthesis are not corrected (Battula and Loeb, 1976). Studies have shown the mutation rate of HIV RT to be 3.4 x 10-5 mutations per base pair per cycle, which is relatively high compared to other retroviruses (Mansky and Temin, 1995). However, as the rate of recombination, 3-9 crossovers per round of replication (Jetzt et al., 2000), exceeds the mutation rate, recombination is the largest contributing factor

Intra-strain recombination between variants within the viral quasispecies, as well as interstrain/intersubtype recombination, increases genetic diversity and thereby increases the chance of survival for the virus. Recombination can generate strains capable of evading the host's immune system, strains that are resistant to one or more antiretroviral drugs, or that can replicate faster and more efficiently (Steain et al., 2004). It can also result in the formation of novel genes (Sharp et al., 1996). Recombination can also be a repair mechanism for HIV, allowing viral replication to continue in the presence of a break in a strand of RNA. However, recombination can also result in the emergence of less fit viral strains, as it can break-up favourable combinations of genes and therefore is not always advantageous for

Studies have shown that multidrug resistant viruses emerge rapidly in the presence of two drugs, due to recombination between strains that were each resistant to a single drug (Moutouh et al., 1996). In vivo, this could allow the emergence of strains that are resistant to many different classes of drugs, and recently intrapatient recombination leading to a multidrug resistant strain has been reported (Weiser et al., 2005). Further, selective pressure placed on the pol region in the presence of anti-retroviral drugs, does not need to be carried across the entire genome as recombination could occur between strains with diverse gag and env regions with an escape mutant in the pol region (Charpentier et al., 2006). Similarly, a study of two patients by van Rij et al. (2003) observed that after the emergence of X4 utilising strains, the R5 and X4 gp120 envelope sequences diverged from each other, whereas the respective gag p17 regions did not. Thus it was proposed that recombination

and well-established CRFs have been identified, which are listed in **Figure 6**.

**5. Dual infections and recombination of HIV** 

in viral evolution (Bocharov et al., 2005).

the virus (Bretscher et al., 2004).

was occurring between the two strains in vivo.

CRF05\_DF has also been reported in Europe, although is thought to have arisen in Africa (Casado et al., 2003; Laukkanen et al., 2000).

Fig. 5. The complex HIV-1 genetic diversity and the spread of the predominant HIV-1 strains in Asia. Distribution of different HIV-1 subtypes and CRFs (demonstrated in descending order; from the most to the least prevalent strain) in major Asian countries, including subtypes A; B; C; D; G, CRFs such as 01\_AE, CRF01\_AE; 02\_AG, CRF02\_AG; 07\_BC, CRF07\_BC; 08\_BC, CRF08\_BC; 33\_01B, CRF33\_01B and other HIV-1 recombinant forms: BC, B/C inter-subtype recombinant; 01B, CRF01\_AE/B; 01C, CRF01\_AE/C and 01BC, CRF01\_AE/B/C. The three different routes of spread for subtype C, CRF01\_AE as well as subtype B, C and B/C recombinants are highlighted in green, red and blue, respectively. Diagram source: Lau *et al.* (2007).

Subtype G has been reported across Africa, and parts of Europe (Esteves et al., 2003; Gutierrez et al., 2004; Parreira et al., 2005; Yang et al., 2005a), however it is its CRF02\_AG that has had the greater impact. This strain is currently the most prevalent CRF and is found predominantly across West and Central Africa (Mamadou et al., 2003). CRF14\_BG, as well as other G recombinants are also found in Spain and neighbouring regions (Perez-Alvarez et al., 2003). Subtypes H, J and K have had only a minor impact on the HIV epidemic and are generally only found in small numbers across West and Central Africa (Mokili et al., 1999; Thomson et al., 2002b; Vidal et al., 2000; Yang et al., 2005a). The remaining CRFs are also found in varying proportions, usually each within a distinct geographical region. Whole spectrum of emerging and well-established CRFs have been identified, which are listed in **Figure 6**.

## **5. Dual infections and recombination of HIV**

104 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

CRF05\_DF has also been reported in Europe, although is thought to have arisen in Africa

Fig. 5. The complex HIV-1 genetic diversity and the spread of the predominant HIV-1 strains in Asia. Distribution of different HIV-1 subtypes and CRFs (demonstrated in descending order; from the most to the least prevalent strain) in major Asian countries, including subtypes A; B; C; D; G, CRFs such as 01\_AE, CRF01\_AE; 02\_AG, CRF02\_AG; 07\_BC, CRF07\_BC; 08\_BC, CRF08\_BC; 33\_01B, CRF33\_01B and other HIV-1 recombinant forms: BC, B/C inter-subtype recombinant; 01B, CRF01\_AE/B; 01C, CRF01\_AE/C and 01BC, CRF01\_AE/B/C. The three different routes of spread for subtype C, CRF01\_AE as well as subtype B, C and B/C recombinants are highlighted in green, red and blue,

Subtype G has been reported across Africa, and parts of Europe (Esteves et al., 2003; Gutierrez et al., 2004; Parreira et al., 2005; Yang et al., 2005a), however it is its CRF02\_AG that has had the greater impact. This strain is currently the most prevalent CRF and is found predominantly across West and Central Africa (Mamadou et al., 2003). CRF14\_BG, as well as other G

(Casado et al., 2003; Laukkanen et al., 2000).

respectively. Diagram source: Lau *et al.* (2007).

The magnitude of genetic diversity in a host relates to the size of the viral population, the extent of replication, the mutation and recombination rates and the selective pressures placed on the virus (Saksena et al., 2001). Overall the divergence between the circulating HIV strains within a single individual and the original infecting strain/s is thought to increase by around 1% per year in early infection (Shankarappa et al., 1999). Thus, within an individual a heterogeneous viral population exists which has been termed a 'quasispecies'. Within an individual this viral diversity can reach as 15% (Lukashov and Goudsmit, 1997). The reasons behind such viral diversity include a fast turnover of virions, approximately 109 new virions are produced each day (Ho et al., 1995), and the low fidelity of reverse transcriptase. Reverse transcriptase lacks a proof-reading function, meaning that any nucleotides that are mis-incorporated during DNA synthesis are not corrected (Battula and Loeb, 1976). Studies have shown the mutation rate of HIV RT to be 3.4 x 10-5 mutations per base pair per cycle, which is relatively high compared to other retroviruses (Mansky and Temin, 1995). However, as the rate of recombination, 3-9 crossovers per round of replication (Jetzt et al., 2000), exceeds the mutation rate, recombination is the largest contributing factor in viral evolution (Bocharov et al., 2005).

Intra-strain recombination between variants within the viral quasispecies, as well as interstrain/intersubtype recombination, increases genetic diversity and thereby increases the chance of survival for the virus. Recombination can generate strains capable of evading the host's immune system, strains that are resistant to one or more antiretroviral drugs, or that can replicate faster and more efficiently (Steain et al., 2004). It can also result in the formation of novel genes (Sharp et al., 1996). Recombination can also be a repair mechanism for HIV, allowing viral replication to continue in the presence of a break in a strand of RNA. However, recombination can also result in the emergence of less fit viral strains, as it can break-up favourable combinations of genes and therefore is not always advantageous for the virus (Bretscher et al., 2004).

Studies have shown that multidrug resistant viruses emerge rapidly in the presence of two drugs, due to recombination between strains that were each resistant to a single drug (Moutouh et al., 1996). In vivo, this could allow the emergence of strains that are resistant to many different classes of drugs, and recently intrapatient recombination leading to a multidrug resistant strain has been reported (Weiser et al., 2005). Further, selective pressure placed on the pol region in the presence of anti-retroviral drugs, does not need to be carried across the entire genome as recombination could occur between strains with diverse gag and env regions with an escape mutant in the pol region (Charpentier et al., 2006). Similarly, a study of two patients by van Rij et al. (2003) observed that after the emergence of X4 utilising strains, the R5 and X4 gp120 envelope sequences diverged from each other, whereas the respective gag p17 regions did not. Thus it was proposed that recombination was occurring between the two strains in vivo.

HIV Recombination and Pathogenesis – Biological and Epidemiological Implications 107

Recombination may also produce virus strains that are more successfully transmitted either via sexual contact or perinatal transmission, possibly by increasing the strains affinity for a particular tissue type. A study in Buenos Aires by Thomson et al. (2002b), showed that recombinant viruses predominated in IDUs and heterosexually infected women, whereas subtype B viral strains were more common in men, both heterosexually and homosexually infected (Gao et al., 1996). Studies have also shown that inter-subtype recombinants were more likely to be transmitted via breast milk than subtype C in Tanzania (Koulinska et al., 2006). Further, in Southeast Asia, CRF01\_AE was spread much more rapidly than subtype B, which was also present in the area. In addition, no type E gag or pol gene has been found which may suggest that the recombinant CRF01\_AE virus was more viable and

It is widely recognised that a single individual can become infected with more than one HIV-1 subtype or strain i.e. they harbour a dual infection (Gottlieb et al., 2004; Wang et al., 2000). This circumstance is possible via superinfection or coinfection. In superinfection, new infection takes place with a divergent HIV-1 or HIV-2 strain in already infected individuals. In contrast, coinfection is a concomitant exposure to diverse HIV strains prior to seroconversion, and therefore before the immune system has mounted a response (Steain et

For the majority of patients that have been characterized as being infected with two or more HIV strains, it has generally been assumed that the infections occurred within a very short period of time, if not simultaneously. This is because it was originally thought that establishment of infection with HIV would provide some immunity against re-infection, and that the decrease in the expression of CD4 molecules would make superinfection unlikely (Benson et al., 1993). Furthermore, a study by Otten et al. (1999) examined HIV-2 infection in Pig-tailed Macaques and found that a secondary infection could only be established in the

However, there are to date at least 10 papers examining patients with evidence of superinfection (Chohan et al., 2005; McCutchan et al., 2005; Smith et al., 2005). These include one patient with a triple infection who was thought to have acquired an additional 2 strains through superinfection (van der Kuyl et al., 2005). It was also initially hypothesised that if superinfections were occurring that they would be the result of intravenous inoculation of the virus, with a high dose exposure. However of the reported superinfections, several were

Many of these papers documenting superinfection note that upon acquisition of a second virus, patients tend to experience a more rapid disease progression, with an increase in plasma viral load and a concomitant decrease in CD4+ T cell count (Smith et al., 2005; van der Kuyl et al., 2005). Superinfection has also been reported to lead to recombination between the two infecting strains (McCutchan et al., 2005). For these reasons HIV-infected individuals should be warned that safe-sex practices are still necessary even between seroconcordant couples, to prevent superinfection and the associated disease progression (Steain

Now that superinfection has been demonstrated, it is unknown if these are rare cases or if superinfection is a more common event that is not always detected, and may in part provide some explanation for the large number of recombinant strains seen globally. In cases where

acquired via sexual exposures, including heterosexual contact (Chohan et al., 2005).

consequently the pure type E was eliminated by selective pressures

al., 2004).

et al., 2004).

first 2-4 weeks after the initial infection.

**6. HIV co-infection and superinfection: Pathogenic implications** 

Fig. 6. Schematic representation of the genomic organization of the described CRF genomes. Adapted from the Los Alamos National Laboratory HIV Database (2006b).

Fig. 6. Schematic representation of the genomic organization of the described CRF genomes.

Adapted from the Los Alamos National Laboratory HIV Database (2006b).

Recombination may also produce virus strains that are more successfully transmitted either via sexual contact or perinatal transmission, possibly by increasing the strains affinity for a particular tissue type. A study in Buenos Aires by Thomson et al. (2002b), showed that recombinant viruses predominated in IDUs and heterosexually infected women, whereas subtype B viral strains were more common in men, both heterosexually and homosexually infected (Gao et al., 1996). Studies have also shown that inter-subtype recombinants were more likely to be transmitted via breast milk than subtype C in Tanzania (Koulinska et al., 2006). Further, in Southeast Asia, CRF01\_AE was spread much more rapidly than subtype B, which was also present in the area. In addition, no type E gag or pol gene has been found which may suggest that the recombinant CRF01\_AE virus was more viable and consequently the pure type E was eliminated by selective pressures

## **6. HIV co-infection and superinfection: Pathogenic implications**

It is widely recognised that a single individual can become infected with more than one HIV-1 subtype or strain i.e. they harbour a dual infection (Gottlieb et al., 2004; Wang et al., 2000). This circumstance is possible via superinfection or coinfection. In superinfection, new infection takes place with a divergent HIV-1 or HIV-2 strain in already infected individuals. In contrast, coinfection is a concomitant exposure to diverse HIV strains prior to seroconversion, and therefore before the immune system has mounted a response (Steain et al., 2004).

For the majority of patients that have been characterized as being infected with two or more HIV strains, it has generally been assumed that the infections occurred within a very short period of time, if not simultaneously. This is because it was originally thought that establishment of infection with HIV would provide some immunity against re-infection, and that the decrease in the expression of CD4 molecules would make superinfection unlikely (Benson et al., 1993). Furthermore, a study by Otten et al. (1999) examined HIV-2 infection in Pig-tailed Macaques and found that a secondary infection could only be established in the first 2-4 weeks after the initial infection.

However, there are to date at least 10 papers examining patients with evidence of superinfection (Chohan et al., 2005; McCutchan et al., 2005; Smith et al., 2005). These include one patient with a triple infection who was thought to have acquired an additional 2 strains through superinfection (van der Kuyl et al., 2005). It was also initially hypothesised that if superinfections were occurring that they would be the result of intravenous inoculation of the virus, with a high dose exposure. However of the reported superinfections, several were acquired via sexual exposures, including heterosexual contact (Chohan et al., 2005).

Many of these papers documenting superinfection note that upon acquisition of a second virus, patients tend to experience a more rapid disease progression, with an increase in plasma viral load and a concomitant decrease in CD4+ T cell count (Smith et al., 2005; van der Kuyl et al., 2005). Superinfection has also been reported to lead to recombination between the two infecting strains (McCutchan et al., 2005). For these reasons HIV-infected individuals should be warned that safe-sex practices are still necessary even between seroconcordant couples, to prevent superinfection and the associated disease progression (Steain et al., 2004).

Now that superinfection has been demonstrated, it is unknown if these are rare cases or if superinfection is a more common event that is not always detected, and may in part provide some explanation for the large number of recombinant strains seen globally. In cases where

HIV Recombination and Pathogenesis – Biological and Epidemiological Implications 109

infections. CRF01\_AE, which was originally identified in Thailand appears to circulate in major parts of Asia, particularly Southeast Asia (Figure 5 and 6). Together, CRF01\_AE and other recombinants account for nearly 89%, the highest across the world [Hemelaar et al., 2006]. Since the beginning of HIV pandemic in the last two decades until recently, changes in HIV-1 subtype distribution in Asia have been overwhelming. In Asian countries, the HIV-1 prevalence has been high from the late 1980s to 1990s, with subtype B" (known as the Thai variant of subtype B) being the predominant strain and was most frequently observed amongst IDU [Weniger et al., 1991; Nerurkar et al., 1997]. Concurrently in Thailand and other areas, CRF01\_AE was introduced independently in commercial sex workers [Ou et al., 1993]. Interestingly in the last decade, a gradual yet evident spread of the Thai variant of CRF01\_AE was witnessed in many countries of Asia [Nerurkar et al., 1996]. It was later observed that CRF01\_AE takes over in the HIV-1 epidemic in Southeast Asia, even among IDUs in Thailand, Cambodia, and Vietnam [Nerurkar et al., 1996]. Likewise, countries such as Indonesia and Malaysia demonstrate the predominance of CRF01\_AE and subtype B

**8. Inter-CRF recombination and its possible epidemiologic implications** 

inter-CRF recombinants, and only time will tell regarding epidemiologic.

Among all the HIV-1 subtypes distributed in Asia, CRF01\_AE is reported to play a considerably important role in its epidemic [Hemelaar et al., 2006]. The HIV epidemiology in Asia is bound to be more complex as other recombinant forms are introduced from neighbouring geographic regions, along with the continuing emergence of novel second and third generation recombinant forms of CRF01\_AE in this region. Geographic regions known as recombination hotspots in Asia, including Myanmar and Yunnan province of China appear to have varied and complex forms of HIV-1 recombinants, which emerge continually. Between 2002 and 2004, a novel inter-CRF recombinant has been identified in Yangon, Myanmar, which also appears to be a second class of HIV-1 inter-CRF recombinants comprised of CRF01\_AE and CRF07\_BC [Takebe et al., 2006]. Other Asian region, for instance Macao has first identified the circulation of CRF12\_BF (prevalent in Brazil) among the IDUs, although CRF01\_AE has always being the major HIV-1 strain [Chan et al., 2007a]. This suggests the epidemiologically associated transmission of the current HIV-1 infection in the region and gives clues to the possible initiation of the emergence of novel inter-CRF recombinants between CRF01\_AE and CRF12\_BF. Concurrently in Macao, a diverse form of HIV-1 recombinant has been recently full-length characterized and comprised of CRF12\_BF, CRF14\_BG and subtype G [Chan et al., 2007b]. As the result of the co-circulation of subtypes B and C, two CRFs; CRF07\_BC and CRF08\_BC have emerged in the Yunnan province of China [Piyasirisilp et al., 2000]. An ongoing evolution and emergence of novel recombinant forms of HIV are anticipated in this region, while these two CRFs continue to co-circulate with "pure" subtypes B and C, along with other URF in Yunnan [Yang et al., 2002]. It is predicted that more new recombinant strains between these two CRFs will continue to emerge [Peeters et al., 2000]. With the extensive variability in recombinant breakpoints and crossover points in China, a possible emergence of second and third generation recombinant CRF will continue to give rise to more HIV-1 variants. A recent study has identified approximately 12% of HIV-1 strains found among the IDUs in Southeast Yunnan to be the diverse forms of inter-CRF recombinants between CRF07\_BC and CRF08\_BC [Chan et al., 2007]. This further provides a good insight into

prior to the year 2000.

the second infecting strain is of the same subtype, the differences between the strains may be attributed to the normal quasispecies variation seen within a patient, and therefore may not be recognized as superinfections. Thus in cases where superinfection is detected it is likely to be intersubtype related and thus any quantification of superinfections may be an underestimation (Steain et al., 2004).

It is important to screen for dual infections, as such patients can provide an ideal setting for examining biological and molecular interactions between two viral strains in vivo. Wang et al. (2000), reported the case of a patient who was co-infected with two divergent forms of subtype B, which appeared to be acting in synergy. The two strains were able to segregate based on a differential tropism for monocytes and macrophages. While one of the strains appeared to dominate when co-cultured in peripheral blood mononuclear cells (PBMCs), it was discovered that this strain was only able to productively infect PBMCs when the second viral strains was present, indicating a potential synergistic effect between the two viral strains. In addition, a greater cytopathic effect was observed when the two strains where cocultured, further supporting the idea that a synergistic association of these two viral strains resulted in greater pathogenicity. The patient had acquired the infections via intravenous drug use and progressed rapidly to AIDS, dying within 5 years of infection. This case demonstrates that distinct biological differences exist between strains of the same subtype, and that two strains are able to act in synergy.

## **7. CRFs and pathogenic implications: Asia the "hotbed" of CRFs**

The highly unequal geographic distribution of viral variants is the result of the global variation in the HIV-1 strains, the dynamic nature of the HIV-1 epidemic, and the accidental epidemiologic transmissions. The recombinant HIV-1 strains have been reported from almost all geographic regions of the globe where multiple HIV-1 subtypes have been circulating. Despite this, few HIV-1 geographic "recombination hotspots" have been identified around the world, such as central Myanmar [Vidal et al., 2005], Yunnan province of China [Saksena et al., 2005], Argentina [Renjifo et al., 2001], Brazil [Ball et al., 2003], East Africa [Yang et al., 2003,Renjifo et al., 2004] and more recently Cuba [Wu et al., 2001]. While the predominant viral forms in the global HIV epidemic are subtypes A and C [UNAIDS/WHO, 2006], a different and even more complex HIV genetic diversity has been found in Asia. The HIV spread and its epidemiology in Asia are interesting and closely related to the routes of spread of the epidemic. This is evident from the distribution of subtype C (Figure 5), which was found primarily in India and Africa, and is now spreading to Northern India, Myanmar, and Thailand [Eshleman et al., 2005]. Although the biological aspects that explain this high rate of infection remain unclear, subtype C has dominated the HIV-1 epidemic in India and accounts for almost 97% of infections. Apart from the predominant subtype C, the A/C and B/C inter-subtype recombinants have also been recently identified in North-eastern India [Peeters et al., 2000]. Emergence of A/C recombinants is also consistent with the epidemic observed in Bangladesh [Sanders-Nuell et al., 2007], from where triple recombinants between subtypes A, C and G have been recently reported. Also, it is established that the spread of subtypes B and C, as well as B/C recombinants occurred through the drug route from Eastern Myanmar into Yunnan province of China and moving to north and west into Xinjiang province of China (Figure 5). While other recombinants account for only 4% of the total HIV-1 infection in South and Southeast Asia, the CRF01\_AE has been found to be responsible for 84% of all HIV-1

the second infecting strain is of the same subtype, the differences between the strains may be attributed to the normal quasispecies variation seen within a patient, and therefore may not be recognized as superinfections. Thus in cases where superinfection is detected it is likely to be intersubtype related and thus any quantification of superinfections may be an

It is important to screen for dual infections, as such patients can provide an ideal setting for examining biological and molecular interactions between two viral strains in vivo. Wang et al. (2000), reported the case of a patient who was co-infected with two divergent forms of subtype B, which appeared to be acting in synergy. The two strains were able to segregate based on a differential tropism for monocytes and macrophages. While one of the strains appeared to dominate when co-cultured in peripheral blood mononuclear cells (PBMCs), it was discovered that this strain was only able to productively infect PBMCs when the second viral strains was present, indicating a potential synergistic effect between the two viral strains. In addition, a greater cytopathic effect was observed when the two strains where cocultured, further supporting the idea that a synergistic association of these two viral strains resulted in greater pathogenicity. The patient had acquired the infections via intravenous drug use and progressed rapidly to AIDS, dying within 5 years of infection. This case demonstrates that distinct biological differences exist between strains of the same subtype,

**7. CRFs and pathogenic implications: Asia the "hotbed" of CRFs** 

The highly unequal geographic distribution of viral variants is the result of the global variation in the HIV-1 strains, the dynamic nature of the HIV-1 epidemic, and the accidental epidemiologic transmissions. The recombinant HIV-1 strains have been reported from almost all geographic regions of the globe where multiple HIV-1 subtypes have been circulating. Despite this, few HIV-1 geographic "recombination hotspots" have been identified around the world, such as central Myanmar [Vidal et al., 2005], Yunnan province of China [Saksena et al., 2005], Argentina [Renjifo et al., 2001], Brazil [Ball et al., 2003], East Africa [Yang et al., 2003,Renjifo et al., 2004] and more recently Cuba [Wu et al., 2001]. While the predominant viral forms in the global HIV epidemic are subtypes A and C [UNAIDS/WHO, 2006], a different and even more complex HIV genetic diversity has been found in Asia. The HIV spread and its epidemiology in Asia are interesting and closely related to the routes of spread of the epidemic. This is evident from the distribution of subtype C (Figure 5), which was found primarily in India and Africa, and is now spreading to Northern India, Myanmar, and Thailand [Eshleman et al., 2005]. Although the biological aspects that explain this high rate of infection remain unclear, subtype C has dominated the HIV-1 epidemic in India and accounts for almost 97% of infections. Apart from the predominant subtype C, the A/C and B/C inter-subtype recombinants have also been recently identified in North-eastern India [Peeters et al., 2000]. Emergence of A/C recombinants is also consistent with the epidemic observed in Bangladesh [Sanders-Nuell et al., 2007], from where triple recombinants between subtypes A, C and G have been recently reported. Also, it is established that the spread of subtypes B and C, as well as B/C recombinants occurred through the drug route from Eastern Myanmar into Yunnan province of China and moving to north and west into Xinjiang province of China (Figure 5). While other recombinants account for only 4% of the total HIV-1 infection in South and Southeast Asia, the CRF01\_AE has been found to be responsible for 84% of all HIV-1

underestimation (Steain et al., 2004).

and that two strains are able to act in synergy.

infections. CRF01\_AE, which was originally identified in Thailand appears to circulate in major parts of Asia, particularly Southeast Asia (Figure 5 and 6). Together, CRF01\_AE and other recombinants account for nearly 89%, the highest across the world [Hemelaar et al., 2006]. Since the beginning of HIV pandemic in the last two decades until recently, changes in HIV-1 subtype distribution in Asia have been overwhelming. In Asian countries, the HIV-1 prevalence has been high from the late 1980s to 1990s, with subtype B" (known as the Thai variant of subtype B) being the predominant strain and was most frequently observed amongst IDU [Weniger et al., 1991; Nerurkar et al., 1997]. Concurrently in Thailand and other areas, CRF01\_AE was introduced independently in commercial sex workers [Ou et al., 1993]. Interestingly in the last decade, a gradual yet evident spread of the Thai variant of CRF01\_AE was witnessed in many countries of Asia [Nerurkar et al., 1996]. It was later observed that CRF01\_AE takes over in the HIV-1 epidemic in Southeast Asia, even among IDUs in Thailand, Cambodia, and Vietnam [Nerurkar et al., 1996]. Likewise, countries such as Indonesia and Malaysia demonstrate the predominance of CRF01\_AE and subtype B prior to the year 2000.

## **8. Inter-CRF recombination and its possible epidemiologic implications**

Among all the HIV-1 subtypes distributed in Asia, CRF01\_AE is reported to play a considerably important role in its epidemic [Hemelaar et al., 2006]. The HIV epidemiology in Asia is bound to be more complex as other recombinant forms are introduced from neighbouring geographic regions, along with the continuing emergence of novel second and third generation recombinant forms of CRF01\_AE in this region. Geographic regions known as recombination hotspots in Asia, including Myanmar and Yunnan province of China appear to have varied and complex forms of HIV-1 recombinants, which emerge continually. Between 2002 and 2004, a novel inter-CRF recombinant has been identified in Yangon, Myanmar, which also appears to be a second class of HIV-1 inter-CRF recombinants comprised of CRF01\_AE and CRF07\_BC [Takebe et al., 2006]. Other Asian region, for instance Macao has first identified the circulation of CRF12\_BF (prevalent in Brazil) among the IDUs, although CRF01\_AE has always being the major HIV-1 strain [Chan et al., 2007a]. This suggests the epidemiologically associated transmission of the current HIV-1 infection in the region and gives clues to the possible initiation of the emergence of novel inter-CRF recombinants between CRF01\_AE and CRF12\_BF. Concurrently in Macao, a diverse form of HIV-1 recombinant has been recently full-length characterized and comprised of CRF12\_BF, CRF14\_BG and subtype G [Chan et al., 2007b]. As the result of the co-circulation of subtypes B and C, two CRFs; CRF07\_BC and CRF08\_BC have emerged in the Yunnan province of China [Piyasirisilp et al., 2000]. An ongoing evolution and emergence of novel recombinant forms of HIV are anticipated in this region, while these two CRFs continue to co-circulate with "pure" subtypes B and C, along with other URF in Yunnan [Yang et al., 2002]. It is predicted that more new recombinant strains between these two CRFs will continue to emerge [Peeters et al., 2000]. With the extensive variability in recombinant breakpoints and crossover points in China, a possible emergence of second and third generation recombinant CRF will continue to give rise to more HIV-1 variants. A recent study has identified approximately 12% of HIV-1 strains found among the IDUs in Southeast Yunnan to be the diverse forms of inter-CRF recombinants between CRF07\_BC and CRF08\_BC [Chan et al., 2007]. This further provides a good insight into inter-CRF recombinants, and only time will tell regarding epidemiologic.

HIV Recombination and Pathogenesis – Biological and Epidemiological Implications 111

levels in blood compared with the first infecting virus B1. Except for the excessive recombination between both subtype B strains, there was only minimal evidence that the different HIV-1 strains found in the patient appeared to influence the evolution of each other. While HIV-1 has been constantly exposed to host immune system for eradication of the virus, its replication relies profoundly on host cell machinery. Thus, the HIV-1 fitness is said to be closely related to the host environment (e.g. cellular receptors, intracellular factors and host defence mechanism). Viral diversity gives important impact in the determination of viral load, as well as viral diagnosis. Therefore, HIV-1 diagnosis test, which includes HIV-1 immunoassays have to be competent in detecting all known group M subtypes [Koch et al., ]. Other viral load measurements assays have to be reliable too, for instance polymerase chain reaction-based assays for quantification of the HIV-1 RNA from all known genetic variants of

Genotypic or phenotypic variations within different subtypes are somehow related to any differences in ex vivo fitness. Troyer et a.l [Troyer et al., 2005] provided evidence that increased viral fitness in vivo may be related to a concomitant increase in HIV-1 diversity, and thus serves as a crucial factor in determining disease progression. Furthermore, HIV-1 strains that display viral properties that increase their fitness, for instance subtype C isolates appear to have an extra or third nuclear factor-kappa B (NFκB) element in the long-terminal repeat (LTR). This would enhance transcription in the presence or absence of HIV-1 Tat protein [Hunt et al., 2006]. Another study showed that in comparison to subtype B, subtype C possesses an increased protease activity, and thus augmented cleavage of peptide substrates and possibly improved viral fitness [Velazquez et al., 2001]. Therefore, it is proposed that the increased replicative capacity of subtype C over other HIV-1 subtype isolates suggests its dominance in HIV-1 epidemic. However, in another pair-wise competition study by Arien et al. [Arien et al., 2005] to establish the "pathogenic fitness" (or virulence) of HIV in PBMCs, subtype C seems to have lower fitness when competed with other HIV-1 group M (subtypes A, B, D and CRF01\_AE). These viruses were classified as using either CCR5 or the CXCR4 co-receptor for entry and were competed against the same phenotype to determine the fitness (based on > 2000 competitions). In the same study, it was reported that all HIV-1 group M viruses have a greater fitness than HIV-2 and HIV-1 group O showed the lowest fitness. Thus, with the exception of subtype C, this fitness order seems to reflect the prevalence of HIV in the human population and also the proposed rates of transmission efficiency. It has been reported in 2003 that the CCR5 HIV-1 subtype C isolates were at least 100-fold less fit than any other group M HIV-1 isolates [Ball et al., 2003]. Throughout the disease, subtype C isolates was predicted as preferentially CCR5-tropic and non-syncytium-inducing (NSI). This has absolute difference in infections with isolates of other HIV-1 subtypes, whereby the viruses switch from CCR5 to CXCR4 co-receptor entry during later stage of the disease. Other ex vivo [Ball et al., 2003] and in vivo study [Walker et al., 2005] has implied that subtype C is efficiently transmitted but is less virulent in comparison with other HIV-1 group M isolates. However, among all HIV strains, HIV-1 group M seems to be more virulent and transmissible. It is believed that its progenitor might have been "fitter" for human infection and more adaptive, even after going through the rapid evolution and passage. By contrast, HIV-2 and HIV-1 groups O and N might have limited expansion in the human population, possibly due to poor host adaptation and transmission efficiencies, although the exact reasons for their poor transmissibility and

active establishment in human populations have remained unclear and speculative.

HIV-1 [Swanson et al., 2005].

#### **9. HIV fitness in vivo and in vitro as a consequence of recombination**

Fitness is a parameter defining the replicative adaptation of an organism to its environment [Domingo et al., 1997] as a consequence of the interaction of a multitude of viral and host factors [Quinones-Mateu et al., 2006; Nijhuis et al., 1999 ]. Within a given viral "quasispecies", each clone possesses a fitness denoting to the selection of the viral properties (e.g. activity and stability) in a particular environment. Under a certain selective pressure in a defined microenvironment, viral replication will take place to encode virus that replicates at high rates [331]. Thus, one or more strains possessing better viral properties within a given quasispecies will be positively selected, while unfit variants will be negatively eliminated [331]. The HIV-1 viral factors that affect viral fitness are mainly the biological processes in the virus life cycle: cell entry, genome replication, protein synthesis and processing, and particle assembly and release from cells. The survival of the fittest form of HIV-1 recombinant leads to further viral evolution in a complex population, suggesting a continuous evolving of HIV-1 dynamics, mainly attributed to an incessant process of growth, competition and selection.

As a result of high mutation rate of HIV-1, wide range of sequence possibilities is created. While sometimes it resulted in non-replicative viruses, others may possess varying degree of fitness. Recombinant viruses may have some advantages over the parental strain and thus, may possess important genetic variability for HIV-1 pathogenesis, transmission, diagnosis, treatment and vaccine development. It is undeniable that different biological properties of diverse subtypes will possibly result in transmissibility and pathogenicity variation. During early infection, most subtypes conform to the non-syncytium-inducing CCR5 receptor usage phenotype. However, towards the late infection, these subtypes will shift to the syncytiuminducing CXCR4 receptor usage phenotype. This is true for most but subtype C and D viruses, which do not follow this pattern [336]. In terms of transmission, several studies of vertical transmission have suggested that the maternal HIV-1 subtype is likely to play a role while others disregard this perception [Yang et al., 2003; Tapia et al., 2003].

What is yet to be known is the consistent role of the subtype-associated differences in the efficiency of transmission via different routes. While some studies have reported that subtype D is associated with faster disease progression compared with subtype A [Condra et al., 1995], others have denied the possibilities that HIV genetic subtype determines the rate of disease progression [Alaeus et al., 1999]. Findings of these studies are inconsistent, due to the difference in the study design (sample size, duration of clinical follow-up and the use of surrogate markers of progression) as well as other virus, host and environmental factors. Previous work has also suggested possible biological differences among the HIV-1 subtypes [Jeeninga et al., 2000]. It was reported that the long terminal repeat (LTR) region of CRF01\_AE (the predominant HIV-1 strain in Asia) is much more potent in vitro than the subtype B LTR. When a recombinant CRF01\_AE/B virus was constructed in vitro, it exhibited an intense replication advantage compared to the parental subtype B. This indicated that restrained differences in the LTR promoter activity can exert a significant impact on viral replication kinetics. A recent profound analysis was done by Kozaczynska et al. [Kozaczynska et al., 2007] to describe the study over time of HIV-1 isolates in a patient twice superinfected with HIV-1; an initial infection with a subtype B1 strain, followed by first superinfection with a subtype B2 strain and then with CRF01\_AE. Again, the LTR of CRF01\_AE was found to possess a higher promoter activity, although this was not reflected in the plasma viral load differences. It is remarkable that the later-arriving viruses (strain B2 and CRF01\_AE) replicated at much higher

Fitness is a parameter defining the replicative adaptation of an organism to its environment [Domingo et al., 1997] as a consequence of the interaction of a multitude of viral and host factors [Quinones-Mateu et al., 2006; Nijhuis et al., 1999 ]. Within a given viral "quasispecies", each clone possesses a fitness denoting to the selection of the viral properties (e.g. activity and stability) in a particular environment. Under a certain selective pressure in a defined microenvironment, viral replication will take place to encode virus that replicates at high rates [331]. Thus, one or more strains possessing better viral properties within a given quasispecies will be positively selected, while unfit variants will be negatively eliminated [331]. The HIV-1 viral factors that affect viral fitness are mainly the biological processes in the virus life cycle: cell entry, genome replication, protein synthesis and processing, and particle assembly and release from cells. The survival of the fittest form of HIV-1 recombinant leads to further viral evolution in a complex population, suggesting a continuous evolving of HIV-1 dynamics, mainly attributed to an incessant process of

As a result of high mutation rate of HIV-1, wide range of sequence possibilities is created. While sometimes it resulted in non-replicative viruses, others may possess varying degree of fitness. Recombinant viruses may have some advantages over the parental strain and thus, may possess important genetic variability for HIV-1 pathogenesis, transmission, diagnosis, treatment and vaccine development. It is undeniable that different biological properties of diverse subtypes will possibly result in transmissibility and pathogenicity variation. During early infection, most subtypes conform to the non-syncytium-inducing CCR5 receptor usage phenotype. However, towards the late infection, these subtypes will shift to the syncytiuminducing CXCR4 receptor usage phenotype. This is true for most but subtype C and D viruses, which do not follow this pattern [336]. In terms of transmission, several studies of vertical transmission have suggested that the maternal HIV-1 subtype is likely to play a role

What is yet to be known is the consistent role of the subtype-associated differences in the efficiency of transmission via different routes. While some studies have reported that subtype D is associated with faster disease progression compared with subtype A [Condra et al., 1995], others have denied the possibilities that HIV genetic subtype determines the rate of disease progression [Alaeus et al., 1999]. Findings of these studies are inconsistent, due to the difference in the study design (sample size, duration of clinical follow-up and the use of surrogate markers of progression) as well as other virus, host and environmental factors. Previous work has also suggested possible biological differences among the HIV-1 subtypes [Jeeninga et al., 2000]. It was reported that the long terminal repeat (LTR) region of CRF01\_AE (the predominant HIV-1 strain in Asia) is much more potent in vitro than the subtype B LTR. When a recombinant CRF01\_AE/B virus was constructed in vitro, it exhibited an intense replication advantage compared to the parental subtype B. This indicated that restrained differences in the LTR promoter activity can exert a significant impact on viral replication kinetics. A recent profound analysis was done by Kozaczynska et al. [Kozaczynska et al., 2007] to describe the study over time of HIV-1 isolates in a patient twice superinfected with HIV-1; an initial infection with a subtype B1 strain, followed by first superinfection with a subtype B2 strain and then with CRF01\_AE. Again, the LTR of CRF01\_AE was found to possess a higher promoter activity, although this was not reflected in the plasma viral load differences. It is remarkable that the later-arriving viruses (strain B2 and CRF01\_AE) replicated at much higher

while others disregard this perception [Yang et al., 2003; Tapia et al., 2003].

**9. HIV fitness in vivo and in vitro as a consequence of recombination** 

growth, competition and selection.

levels in blood compared with the first infecting virus B1. Except for the excessive recombination between both subtype B strains, there was only minimal evidence that the different HIV-1 strains found in the patient appeared to influence the evolution of each other. While HIV-1 has been constantly exposed to host immune system for eradication of the virus, its replication relies profoundly on host cell machinery. Thus, the HIV-1 fitness is said to be closely related to the host environment (e.g. cellular receptors, intracellular factors and host defence mechanism). Viral diversity gives important impact in the determination of viral load, as well as viral diagnosis. Therefore, HIV-1 diagnosis test, which includes HIV-1 immunoassays have to be competent in detecting all known group M subtypes [Koch et al., ]. Other viral load measurements assays have to be reliable too, for instance polymerase chain reaction-based assays for quantification of the HIV-1 RNA from all known genetic variants of HIV-1 [Swanson et al., 2005].

Genotypic or phenotypic variations within different subtypes are somehow related to any differences in ex vivo fitness. Troyer et a.l [Troyer et al., 2005] provided evidence that increased viral fitness in vivo may be related to a concomitant increase in HIV-1 diversity, and thus serves as a crucial factor in determining disease progression. Furthermore, HIV-1 strains that display viral properties that increase their fitness, for instance subtype C isolates appear to have an extra or third nuclear factor-kappa B (NFκB) element in the long-terminal repeat (LTR). This would enhance transcription in the presence or absence of HIV-1 Tat protein [Hunt et al., 2006]. Another study showed that in comparison to subtype B, subtype C possesses an increased protease activity, and thus augmented cleavage of peptide substrates and possibly improved viral fitness [Velazquez et al., 2001]. Therefore, it is proposed that the increased replicative capacity of subtype C over other HIV-1 subtype isolates suggests its dominance in HIV-1 epidemic. However, in another pair-wise competition study by Arien et al. [Arien et al., 2005] to establish the "pathogenic fitness" (or virulence) of HIV in PBMCs, subtype C seems to have lower fitness when competed with other HIV-1 group M (subtypes A, B, D and CRF01\_AE). These viruses were classified as using either CCR5 or the CXCR4 co-receptor for entry and were competed against the same phenotype to determine the fitness (based on > 2000 competitions). In the same study, it was reported that all HIV-1 group M viruses have a greater fitness than HIV-2 and HIV-1 group O showed the lowest fitness. Thus, with the exception of subtype C, this fitness order seems to reflect the prevalence of HIV in the human population and also the proposed rates of transmission efficiency. It has been reported in 2003 that the CCR5 HIV-1 subtype C isolates were at least 100-fold less fit than any other group M HIV-1 isolates [Ball et al., 2003]. Throughout the disease, subtype C isolates was predicted as preferentially CCR5-tropic and non-syncytium-inducing (NSI). This has absolute difference in infections with isolates of other HIV-1 subtypes, whereby the viruses switch from CCR5 to CXCR4 co-receptor entry during later stage of the disease. Other ex vivo [Ball et al., 2003] and in vivo study [Walker et al., 2005] has implied that subtype C is efficiently transmitted but is less virulent in comparison with other HIV-1 group M isolates. However, among all HIV strains, HIV-1 group M seems to be more virulent and transmissible. It is believed that its progenitor might have been "fitter" for human infection and more adaptive, even after going through the rapid evolution and passage. By contrast, HIV-2 and HIV-1 groups O and N might have limited expansion in the human population, possibly due to poor host adaptation and transmission efficiencies, although the exact reasons for their poor transmissibility and active establishment in human populations have remained unclear and speculative.

HIV Recombination and Pathogenesis – Biological and Epidemiological Implications 113

the southwest province of Yunnan and almost all provinces reporting HIV cases. In 1998, facing the rapid upsurge in HIV-1 incidence nation-wide, the Chinese government made a concerted effort to strategize the "Middle and Long-term Programming for the Prevention and Control of AIDS" in China. A year later in 1999, several small clinical trails were initiated in Beijing primarily for safety and efficacy testing, sponsored largely by international pharmaceutical companies. The drug regimen tested then consisted of Combivir plus either Indinavir or Abacavir. This small-scale trial period (1999-2001) can be

The second treatment phase started when the cost of imported drugs used for HAART declined significantly and more patients could afford the medications (2001-2003). The population of Chinese patients undergoing therapy for HIV increased, especially in economically developed areas such as Beijing and Shanghai. However, the number of clinical doctors trained to administer these drugs did not expand. Many patients did not have the opportunity to receive comprehensive care, including standardized immunologic and virologic assessments prior to treatment and regularly scheduled follow-up interviews. Some patients judged the efficacy of the medication by a moderation of their symptoms, and consequently decreased their dosage or stopped taking the medicine altogether, without the consent of a physician. Of the patients who initiated treatment in this period, an estimated 25-30 % stopped taking medicine after only one or two months. Whether or not the other patients were able to persist with treatment and return for follow-up interviews is still to be

The third phase of treatment (2003-present) began with the availability of low-priced domestically manufactured and imported generic anti-HIV drugs. This has been undeniably the most beneficial phase in increasing the number of individuals receiving gratis treatment. Nation-wide free ARV treatment started in 2003, part of the China CARES program, consisting of 51 model sites with plans to further expand to 127 counties. However, the bigger hurdle for this ambitious plan has been again the critical shortage of properly trained doctors, nurses and community care workers. Some patients were so anxious to begin taking medicine for HIV that they obtained the necessary drugs without a doctor's prescription. As a consequence, lacking professional guidance and clinical supervision, they used the medicines improperly, leading to the development of a drug resistant virus. In addition, as generic HIV drugs entered the Chinese market from developing countries, some patients began taking medicine without any medical assessment before treatment, and without choosing to register for interviews during treatment. Furthermore, severe side effects associated with generic ARV produced in China led to a large number of patients stopping medication entirely or becoming unwilling to follow doctors' advice and suggestions. As the incidence of HIV infection rises in China, it is anticipated that problems associated with the abuse of ARV will only escalate. It is therefore expected that drug resistant HIV-1 strains

will emerge leading to their high prevalence and transmission over time.

A number of studies in other countries have shown that the prevalence of viruses with drug resistance mutations in acutely or recently infected persons varies between 10 to 20% [Boden et al., 1999; Grant et al., 2002; Little et al., 2002]. Research examining the prevalence and genetic features of drug-resistance strains at national level is lacking in China. Several major institutes in China are combining forces to carry-out genetic studies on viruses collected before and after nation-wide free ARV treatment. Based on preliminary data, it is fairly clear that the prevalence of drug-resistant strains were extremely rare before year 2000.

regarded as the first phase of ARV treatment in China.

determined.

Viral fitness is generally defined as the ability of the virus to replicate within the host and is therefore dependant on host and viral factors [Weber et al., 2003]. Recombination is thought to increase viral fitness. In an HIV-1 recombinant-related fitness study by Njai et al. [Njai et al., 2006], CRF02\_AG isolates demonstrated a higher ex vivo replicative fitness compared to subtypes A and G from the same geographic region in Cameroon, irrespective of the level of CD4+ count and co-receptor tropism. A similar study by Konings et al. [Vijay et al., 2008] showed a 1.4 to 1.9 times higher replication rate increase in the CRF02\_AG strains, in contrast to its progenitor subtypes A and G; an adaptation which implies its broader spread and predominance in West Central Africa. A computer simulation has been developed that mimic the HIV genomic diversification within an infected individual and elucidate the influence of recombination [Vijay et al., 2008]. This study has shown that recombination increases viral fitness regardless of the size of the effective population. In light of these results, it is likely that HIV-1 recombination events in Asia can also contribute to the emergence of viruses for instance, the widespread CRF01\_AE/B inter-subtype recombinants with a biological edge in their host. In vitro studies of the viral fitness and interactions between different viral strains have been assessed with limitation, as only viral replication capacity, defined as "intrinsic capacity of virus to replicate in an ideal environment" [Weber et al., 2003] can be studied in the absence of host selection pressures. As a result, viral replication capacities are compared in vitro between two or more HIV-1 strains in dual infection cultures. This can be achieved by establishing a competition assay, whereby primary strains or recombinant viruses are competed against laboratory strains or parental isolates. In general, two HIV-1 strains are allowed to replicate concurrently for a designated period of time, or as an alternative, HIV-1 recombinant viruses, which are unable to produce new infectious virions are used in a single cycle growth assay, to limit the replication to a single cycle. The experiment is analysed through the measurement of the proportion of each of the initial strains to give a relative replication capacity at the end of the assay. Few studies have taken this approach in order to compare a number of different HIV-1 strains including drug resistance mutants [Weber et al., 2003,Van Maarseveen et al., 2006], isolates from HIVprogressors versus LTNPs [Arien et al., 2005], variable subtypes, as well as different HIV-1 groups or HIV-1 versus HIV-2. To date, none of these studies have been performed on the currently emerging CRF01\_AE/B inter-subtype recombinants from Malaysia, particularly CRF33\_01B. It is therefore important and urgent to identify and understand the biological advantages of these new HIV-1 forms, which presumably will take over the predominance in HIV-1 epidemic in Malaysia.

#### **10. Anti-HIV therapy, drug resistance and its dissemination: An example of China**

In global terms, over the past 15 years the treatment of HIV-1 infection has evolved significantly. In North America and Western Europe, no effective therapy existed until the development and availability of zidovudine (ZDV, AZT) in 1987. In 2005, there are now 26 commercially available antiviral agents (both RT inhibitors [NRTI and NNRTI] and protease inhibitors) to treat HIV-1-infected individuals.

ARV treatment of HIV-1-infected patients in China fell behind that of most developed countries. While highly active antiretroviral therapy (HAART) became widely used in North America and Western Europe in 1996, China was still debating whether or not HIV/AIDS would become a huge epidemic there, despite the large number of IDUs testing positive in

Viral fitness is generally defined as the ability of the virus to replicate within the host and is therefore dependant on host and viral factors [Weber et al., 2003]. Recombination is thought to increase viral fitness. In an HIV-1 recombinant-related fitness study by Njai et al. [Njai et al., 2006], CRF02\_AG isolates demonstrated a higher ex vivo replicative fitness compared to subtypes A and G from the same geographic region in Cameroon, irrespective of the level of CD4+ count and co-receptor tropism. A similar study by Konings et al. [Vijay et al., 2008] showed a 1.4 to 1.9 times higher replication rate increase in the CRF02\_AG strains, in contrast to its progenitor subtypes A and G; an adaptation which implies its broader spread and predominance in West Central Africa. A computer simulation has been developed that mimic the HIV genomic diversification within an infected individual and elucidate the influence of recombination [Vijay et al., 2008]. This study has shown that recombination increases viral fitness regardless of the size of the effective population. In light of these results, it is likely that HIV-1 recombination events in Asia can also contribute to the emergence of viruses for instance, the widespread CRF01\_AE/B inter-subtype recombinants with a biological edge in their host. In vitro studies of the viral fitness and interactions between different viral strains have been assessed with limitation, as only viral replication capacity, defined as "intrinsic capacity of virus to replicate in an ideal environment" [Weber et al., 2003] can be studied in the absence of host selection pressures. As a result, viral replication capacities are compared in vitro between two or more HIV-1 strains in dual infection cultures. This can be achieved by establishing a competition assay, whereby primary strains or recombinant viruses are competed against laboratory strains or parental isolates. In general, two HIV-1 strains are allowed to replicate concurrently for a designated period of time, or as an alternative, HIV-1 recombinant viruses, which are unable to produce new infectious virions are used in a single cycle growth assay, to limit the replication to a single cycle. The experiment is analysed through the measurement of the proportion of each of the initial strains to give a relative replication capacity at the end of the assay. Few studies have taken this approach in order to compare a number of different HIV-1 strains including drug resistance mutants [Weber et al., 2003,Van Maarseveen et al., 2006], isolates from HIVprogressors versus LTNPs [Arien et al., 2005], variable subtypes, as well as different HIV-1 groups or HIV-1 versus HIV-2. To date, none of these studies have been performed on the currently emerging CRF01\_AE/B inter-subtype recombinants from Malaysia, particularly CRF33\_01B. It is therefore important and urgent to identify and understand the biological advantages of these new HIV-1 forms, which presumably will take over the predominance

**10. Anti-HIV therapy, drug resistance and its dissemination: An example of** 

In global terms, over the past 15 years the treatment of HIV-1 infection has evolved significantly. In North America and Western Europe, no effective therapy existed until the development and availability of zidovudine (ZDV, AZT) in 1987. In 2005, there are now 26 commercially available antiviral agents (both RT inhibitors [NRTI and NNRTI] and protease

ARV treatment of HIV-1-infected patients in China fell behind that of most developed countries. While highly active antiretroviral therapy (HAART) became widely used in North America and Western Europe in 1996, China was still debating whether or not HIV/AIDS would become a huge epidemic there, despite the large number of IDUs testing positive in

in HIV-1 epidemic in Malaysia.

inhibitors) to treat HIV-1-infected individuals.

**China** 

the southwest province of Yunnan and almost all provinces reporting HIV cases. In 1998, facing the rapid upsurge in HIV-1 incidence nation-wide, the Chinese government made a concerted effort to strategize the "Middle and Long-term Programming for the Prevention and Control of AIDS" in China. A year later in 1999, several small clinical trails were initiated in Beijing primarily for safety and efficacy testing, sponsored largely by international pharmaceutical companies. The drug regimen tested then consisted of Combivir plus either Indinavir or Abacavir. This small-scale trial period (1999-2001) can be regarded as the first phase of ARV treatment in China.

The second treatment phase started when the cost of imported drugs used for HAART declined significantly and more patients could afford the medications (2001-2003). The population of Chinese patients undergoing therapy for HIV increased, especially in economically developed areas such as Beijing and Shanghai. However, the number of clinical doctors trained to administer these drugs did not expand. Many patients did not have the opportunity to receive comprehensive care, including standardized immunologic and virologic assessments prior to treatment and regularly scheduled follow-up interviews. Some patients judged the efficacy of the medication by a moderation of their symptoms, and consequently decreased their dosage or stopped taking the medicine altogether, without the consent of a physician. Of the patients who initiated treatment in this period, an estimated 25-30 % stopped taking medicine after only one or two months. Whether or not the other patients were able to persist with treatment and return for follow-up interviews is still to be determined.

The third phase of treatment (2003-present) began with the availability of low-priced domestically manufactured and imported generic anti-HIV drugs. This has been undeniably the most beneficial phase in increasing the number of individuals receiving gratis treatment. Nation-wide free ARV treatment started in 2003, part of the China CARES program, consisting of 51 model sites with plans to further expand to 127 counties. However, the bigger hurdle for this ambitious plan has been again the critical shortage of properly trained doctors, nurses and community care workers. Some patients were so anxious to begin taking medicine for HIV that they obtained the necessary drugs without a doctor's prescription. As a consequence, lacking professional guidance and clinical supervision, they used the medicines improperly, leading to the development of a drug resistant virus. In addition, as generic HIV drugs entered the Chinese market from developing countries, some patients began taking medicine without any medical assessment before treatment, and without choosing to register for interviews during treatment. Furthermore, severe side effects associated with generic ARV produced in China led to a large number of patients stopping medication entirely or becoming unwilling to follow doctors' advice and suggestions. As the incidence of HIV infection rises in China, it is anticipated that problems associated with the abuse of ARV will only escalate. It is therefore expected that drug resistant HIV-1 strains will emerge leading to their high prevalence and transmission over time.

A number of studies in other countries have shown that the prevalence of viruses with drug resistance mutations in acutely or recently infected persons varies between 10 to 20% [Boden et al., 1999; Grant et al., 2002; Little et al., 2002]. Research examining the prevalence and genetic features of drug-resistance strains at national level is lacking in China. Several major institutes in China are combining forces to carry-out genetic studies on viruses collected before and after nation-wide free ARV treatment. Based on preliminary data, it is fairly clear that the prevalence of drug-resistant strains were extremely rare before year 2000.

HIV Recombination and Pathogenesis – Biological and Epidemiological Implications 115

prevent widespread of HIV transmission from the high-risk groups to the general population. Comprehensive approaches are necessary, integrating prevention and treatment efforts. Government, NGO, and international organizations bear responsibility to stop this epidemic in China. Scientific communities and pharmaceutical companies both inside and outside China need to work jointly to develop more potent anti-HIV drugs and therapeutics to inhibit viral replication and reduce HIV transmission. We have seen clear evidence in favor of evolution of complex second and third generation recombinant viruses. Continued monitoring and surveillance of these viruses is needed, if an HIV vaccine is to be developed. Moreover, concerted efforts by joint ventures between the state and the private sector are highly needed for developing an HIV vaccine for the ultimate control of HIV and its spread

Alaeus A, Lidman K, Bjorkman A, Giesecke J, Albert J. Similar rate of disease progression

Andersson S, Norrgren H, Dias F, Biberfeld G and Albert J. (1999). Molecular

Arien KK, Abraha A, Quinones-Mateu ME, Kestens L, Vanham G, Arts EJ. The replicative

Arien KK, Troyer RM, Gali Y, Colebunders RL, Arts EJ, Vanham G. Replicative fitness of

Balakrishnan M, Fay PJ and Bambara RA. (2001). The kissing hairpin sequence promotes recombination within the HIV-I 5' leader region. J Biol Chem. 276(39): 36482-92. Ball SC, Abraha A, Collins KR, Marozsan AJ, Baird H, Quinones-Mateu ME, Penn-Nicholson

Ball SC, Abraha A, Collins KR, Marozsan AJ, Baird H, Quinones-Mateu ME, et al.

Battula N and Loeb LA. (1976). On the fidelity of DNA replication. Lack of

Benson RE, Sanfridson A, Ottinger JS, Doyle C and Cullen BR. (1993). Downregulation of

Bessong PO, Larry Obi C, Cilliers T, Choge I, Phoswa M, Pillay C, Papathanasopoulos M

subtype A/G recombinant in West Africa. Virology. 262(2): 312-20.

group O, and HIV-2 isolates. (2005) J Virol. Jul;79(14):8979-90.

1 isolates of subtypes B and C. (2003) J Virol. Jan;77(2):1021-38.

myeloblastosis virus DNA polymerase. J Biol Chem. 251(4): 982-6.

among individuals infected with HIV-1 genetic subtypes A-D. (1999) AIDS. May

characterization of human immunodeficiency virus (HIV)-1 and -2 in individuals from guinea-bissau with single or dual infections: predominance of a distinct HIV-1

fitness of primary human immunodeficiency virus type 1 (HIV-1) group M, HIV-1

historical and recent HIV-1 isolates suggests HIV-1 attenuation over time.

A, Murray M, Richard N, Lobritz M, et al. (2003). Comparing the ex vivo fitness of CCR5-tropic human immunodeficiency virus type 1 isolates of subtypes B and C. J

Comparing the ex vivo fitness of CCR5-tropic human immunodeficiency virus type

exodeoxyribonuclease activity and error-correcting function in avian

cell-surface CD4 expression by simian immunodeficiency virus Nef prevents viral

and Morris L. (2005). Characterization of human immunodeficiency virus type 1 from a previously unexplored region of South Africa with a high HIV prevalence.

in the most populous nation of the world.

(2005)AIDS Oct 14;19(15):1555-64.

super infection. J Exp Med. 177(6): 1561-6.

AIDS Res Hum Retroviruses. 21(1): 103-9.

Virol. 77(2): 1021-38.

28;13(8):901-7.

**12. References** 

Between year 2001 and 2003, however, drug-resistant strains began to emerge and in some areas the prevalence is as high as 5%. Beginning in 2004, there was a significant increase in the prevalence of drug-resistant drugs across entire China, coinciding with the nation-wide free ARV treatment. Some areas have reported 20-30% drug-resistant strains specifically against NNRTI (unpublished data), and some areas were reported to have as high as 60% drug-resistant strains. The significant increase in the prevalence of drug-resistance could be due to the selection of the cohort and the time from transmission to resistance testing. However, it has clearly shown that resistance tests should be recommended routinely for patients with new infection.

The widespread use of antiretroviral drugs has led to the development and subsequent transmission of drug-resistant HIV-1 and the transmission of drug-resistant viruses has been documented through vertical, sexual, and parenteral routes [Erice et al., 1993; Masquelier et al., 1993; Veenestra et al., 1995]. Patients who are infected with drug-resistant HIV-1 and initiate antiretroviral therapy show poorer treatment responses than patients who are infected with wild-type (WT) viruses [Grant et al., 2002; Little et al., 2002]. Also, in the absence of selection pressures exerted by drugs, some transmitted drug-resistance mutations may persist for months before reversion to a more replication-competent variant. Even when these drug resistant mutations are no longer detectable by population-based nucleotide-sequence, they can persist in the reservoir of latently infected CD4+ memory T cells and may rapidly reemerge under the selective pressure provided by antiretroviral treatment [Wong et al., 1997; Finzi et al., 1997]. In subjects who acquired drug-resistant virus during primary infection, plasma HIV RNA is not suppressed as readily by potent antiretroviral therapy. The slower response to the treatment and the limited viral suppression may facilitate the selection of variants with greater drug resistance.

Thus, given the current spread and changing trends in HIV epidemiology in China, it is extremely urgent to understand the prevalence of drug-resistant strains in China and its changing patterns over time. Otherwise, we will face insurmountable challenges in tailoring our ARV regimens to elicit optimal therapeutic responses. In addition, the recombination between drug resistance HIV-1 strains in the Asia-pacific can cause epidemiologic shift, which may eventually compromise effective drug treatments currently available in these countries, including China. Since, at present, China and India are the economic hubs of Asia the human trafficking may lead to effective dispersal of such recombinant viruses. Therefore, well-coordinated international approaches are needed for surveillance and monitoring of the emergence of new drug resistance recombinant viruses.

## **11. Conclusions**

It is hard to predict how the future of HIV epidemic will shape-up, since a number of complicating factors appear to unfold, including the possible effects of government intervention and unexpected changes (for the better or for the worse) in the behavior of affected populations. However, it is likely that the number of HIV infections is now on the rise, it is expected that the total number of HIV infections in China and India will surpass rest of the world, if no effective countermeasures are taken. Nonetheless, recombination between HIV-1 strains in geographical areas where multiple subtypes circulate will continue to shape future HIV epidemic through the generation of fitter strains capable of transmitting and dispersing in human populations fatser. China, India and other developing countries provide the right medium for this scenario. These countries stand at a critical juncture to prevent widespread of HIV transmission from the high-risk groups to the general population. Comprehensive approaches are necessary, integrating prevention and treatment efforts. Government, NGO, and international organizations bear responsibility to stop this epidemic in China. Scientific communities and pharmaceutical companies both inside and outside China need to work jointly to develop more potent anti-HIV drugs and therapeutics to inhibit viral replication and reduce HIV transmission. We have seen clear evidence in favor of evolution of complex second and third generation recombinant viruses. Continued monitoring and surveillance of these viruses is needed, if an HIV vaccine is to be developed. Moreover, concerted efforts by joint ventures between the state and the private sector are highly needed for developing an HIV vaccine for the ultimate control of HIV and its spread in the most populous nation of the world.

#### **12. References**

114 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Between year 2001 and 2003, however, drug-resistant strains began to emerge and in some areas the prevalence is as high as 5%. Beginning in 2004, there was a significant increase in the prevalence of drug-resistant drugs across entire China, coinciding with the nation-wide free ARV treatment. Some areas have reported 20-30% drug-resistant strains specifically against NNRTI (unpublished data), and some areas were reported to have as high as 60% drug-resistant strains. The significant increase in the prevalence of drug-resistance could be due to the selection of the cohort and the time from transmission to resistance testing. However, it has clearly shown that resistance tests should be recommended routinely for

The widespread use of antiretroviral drugs has led to the development and subsequent transmission of drug-resistant HIV-1 and the transmission of drug-resistant viruses has been documented through vertical, sexual, and parenteral routes [Erice et al., 1993; Masquelier et al., 1993; Veenestra et al., 1995]. Patients who are infected with drug-resistant HIV-1 and initiate antiretroviral therapy show poorer treatment responses than patients who are infected with wild-type (WT) viruses [Grant et al., 2002; Little et al., 2002]. Also, in the absence of selection pressures exerted by drugs, some transmitted drug-resistance mutations may persist for months before reversion to a more replication-competent variant. Even when these drug resistant mutations are no longer detectable by population-based nucleotide-sequence, they can persist in the reservoir of latently infected CD4+ memory T cells and may rapidly reemerge under the selective pressure provided by antiretroviral treatment [Wong et al., 1997; Finzi et al., 1997]. In subjects who acquired drug-resistant virus during primary infection, plasma HIV RNA is not suppressed as readily by potent antiretroviral therapy. The slower response to the treatment and the limited viral

suppression may facilitate the selection of variants with greater drug resistance.

monitoring of the emergence of new drug resistance recombinant viruses.

Thus, given the current spread and changing trends in HIV epidemiology in China, it is extremely urgent to understand the prevalence of drug-resistant strains in China and its changing patterns over time. Otherwise, we will face insurmountable challenges in tailoring our ARV regimens to elicit optimal therapeutic responses. In addition, the recombination between drug resistance HIV-1 strains in the Asia-pacific can cause epidemiologic shift, which may eventually compromise effective drug treatments currently available in these countries, including China. Since, at present, China and India are the economic hubs of Asia the human trafficking may lead to effective dispersal of such recombinant viruses. Therefore, well-coordinated international approaches are needed for surveillance and

It is hard to predict how the future of HIV epidemic will shape-up, since a number of complicating factors appear to unfold, including the possible effects of government intervention and unexpected changes (for the better or for the worse) in the behavior of affected populations. However, it is likely that the number of HIV infections is now on the rise, it is expected that the total number of HIV infections in China and India will surpass rest of the world, if no effective countermeasures are taken. Nonetheless, recombination between HIV-1 strains in geographical areas where multiple subtypes circulate will continue to shape future HIV epidemic through the generation of fitter strains capable of transmitting and dispersing in human populations fatser. China, India and other developing countries provide the right medium for this scenario. These countries stand at a critical juncture to

patients with new infection.

**11. Conclusions** 


HIV Recombination and Pathogenesis – Biological and Epidemiological Implications 117

Coffin JM. (1979). Structure, replication, and recombination of retrovirus genomes: some

Condra JH, Schleif WA, Blahy OM, Gabryelski LJ, Graham DJ, Quintero JC, et al. In vivo

D'Souza V and Summers MF. (2005). How retroviruses select their genomes. Nat Rev

Damond F, Worobey M, Campa P, Farfara I, Colin G, Matheron S, Brun-Vezinet F,

Dang Q, Chen J, Unutmaz D, Coffin JM, Pathak VK, Powell D, KewalRamani VN, Maldarelli

Delgado E, Thomson MM, Villahermosa ML, Sierra M, Ocampo A, Miralles C, Rodriguez-

Domingo E, Holland JJ. RNA virus mutations and fitness for survival. (1997). Annu Rev

Dowling WE, Kim B, Mason CJ, Wasunna KM, Alam U, Elson L, Birx DL, Robb ML,

Duesberg PH. (1968). Physical properties of Rous Sarcoma Virus RNA. Proc Natl Acad Sci U

Erice, A., et al., Brief report: primary infection with zidovudine-resistant human

Esteves A, Parreira R, Piedade J, Venenno T, Franco M, Germano de Sousa J, Patricio L,

Finzi, D., et al., Identification of a reservoir for HIV-1 in patients on highly active

Gao F, Bailes E, Robertson DL, Chen Y, Rodenburg CM, Michael SF, Cummins LB, Arthur

Gao F, Vidal N, Li Y, Trask SA, Chen Y, Kostrikis LG, Ho DD, Kim J, Oh MD, Choe K, et al.

Gao F, Yue L, White A, Pappas B, Barchue J, Hanson A, Greene B, Sharp P, Shaw G and

Garcia PM, Kalish LA, Pitt J, Minkoff H, Quinn TC, Burchett SK, Kornegay J, Jackson B,

immunodeficiency virus type 1. N Engl J Med, 1993. 328:1163-5.

Portugal. AIDS Res Hum Retroviruses. 19(6): 511-7.

antiretroviral therapy. Science, 1997. 278:1295-300.

troglodytes troglodytes. Nature. 397(6718): 436-41.

AIDS Res Hum Retroviruses. 17(8): 675-88.

west Africa. Nature. 358(6386): 495-9.

and cell-mediated pathways. Proc Natl Acad Sci U S A. 101(2): 632-7. De Sa Filho DJ, Sucupira MC, Caseiro MM, Sabino EC, Diaz RS and Janini LM. (2006).

Perez R, Diz-Aren J, Ojea-de Castro R, Losada E, et al. (2002).

emergence of HIV-1 variants resistant to multiple protease inhibitors. (1995)

Robertson DL and Simon F. (2004). Identification of a highly divergent HIV type 2 and proposal for a change in HIV type 2 classification. AIDS Res Hum

F and Hu WS. (2004). Nonrandom HIV-1 infection and double infection via direct

Identification of two HIV type 1 circulating recombinant forms in Brazil. AIDS Res

McCutchan FE and Carr JK. (2002). Forty-one near full-length HIV-1 sequences from Kenya reveal an epidemic of subtype A and A-containing recombinants.

Brum P, Costa A and Canas-Ferreira WF. (2003). Spreading of HIV- 1 subtype G and envB/gagG recombinant strains among injecting drug users in Lisbon,

LO, Peeters M, Shaw GM, et al. (1999). Origin of HIV-1 in the chimpanzee Pan

(2001). Evidence of two distinct subsubtypes within the HIV-1 subtype A radiation.

Hahn B. (1992). Human infection by genetically diverse SIVSM-related HIV-2 in

Moye J, Hanson C, et al. (1999). Maternal levels of plasma human

unifying hypotheses. J Gen Virol. 42(1): 1-26.

Nature. Apr 6;374(6522):569-71.

Microbiol. 3(8): 643-55.

Retroviruses. 20(6): 666-72.

Hum Retroviruses. 22(1): 1-13.

Microbiol;51:151-78.

AIDS. 16(13): 1809-20.

S A. 60(4): 1511-8.


Bobkov AF, Kazennova EV, Selimova LM, Khanina TA, Ryabov GS, Bobkova MR,

Bocharov G, Ford NJ, Edwards J, Breinig T, Wain-Hobson S and Meyerhans A. (2005). A

Boden, D., et al., HIV-1 drug resistance in newly infected individuals. Jama, 1999. 282:1135-

Bretscher MT, Althaus CL, Muller V and Bonhoeffer S. (2004). Recombination in HIV and the evolution of drug resistance: for better or for worse? Bioessays. 26(2): 180-8. Carr JK, Foley BT, Leitner T, Salminen M, Korber B and McCutchan F. (1998). Reference

Casado G, Thomson MM, Delgado E, Sierra M, Vazquez-De Parga E, Perez- Alvarez L,

Chan DP, Tsui SK, Ip P, Lee S, Lam C, Lau I, et al. Analysis of the full-length genome

Chan DP, Tsui SK, Ip P, Lee S, Lam C, Lau I, et al. Identification of a cluster of HIV-1

Chan DP, Tsui SK, Ip P, Lee S, Lam C, Lau I, et al. Identification of a cluster of HIV-1

Charpentier C, Nora T, Tenaillon O, Clavel F and Hance AJ. (2006). Extensive recombination

Chen Z, Luckay A, Sodora DL, Telfer P, Reed P, Gettie A, Kanu JM, Sadek RF, Yee J, Ho DD,

Chohan B, Lavreys L, Rainwater SM and Overbaugh J. (2005). Evidence for frequent

single feral sooty mangabey troop. J Virol. 70(6): 3617-27.

International AIDS Society conference; 2007; Sydney, Australia.

Society conference; 2007; Sydney, Australia.

Society conference; 2007; Sydney, Australia.

74(2): 191-6.

Group. III-10-19.

2472-82.

Virol. 79(16): 10701-8.

41.

Gen Virol. 86(Pt 11): 3109-18.

Sukhanova AL, Kravchenko AV, Ladnaya NN, Weber JN, et al. (2004). Temporal trends in the HIV-1 epidemic in Russia: predominance of subtype A. J Med Virol.

genetic-algorithm approach to simulating human immunodeficiency virus evolution reveals the strong impact of multiply infected cells and recombination. J

Sequences Representing the Principal Genetic Diversity of HIV-1 in the Pandemic. In Human Retroviruses and AIDS 1998. Korber B, KC, Foley B, Hahn B, McCutchan F, Mellors JW, and Sodroski J. Los Alamos, NM, Theoretical Biology and Biophysics

Ocampo A and Najera R. (2003). Near full-length genome characterization of an HIV type 1 CRF05\_DF virus from Spain. AIDS Res Hum Retroviruses. 19(8): 719-25.

sequence of HIV-1 BF recombinant from an injection drug user in Macao. 4th

CRF12\_BF in a population of injection drug users in Macao. 4th International AIDS

CRF12\_BF in a population of injection drug users in Macao. 4th International AIDS

among human immunodeficiency virus type 1 quasispecies makes an important contribution to viral diversity in individual patients. Journal of Virology. 80(5):

et al. (1997). Human immunodeficiency virus type 2 (HIV-2) seroprevalence and characterization of a distinct HIV-2 genetic subtype from the natural range of simian immunodeficiency virus-infected sooty mangabeys. J Virol. 71(5): 3953-60. Chen Z, Telfier P, Gettie A, Reed P, Zhang L, Ho DD and Marx PA. (1996). Genetic

characterization of new West African simian immunodeficiency virus SIVsm: geographic clustering of household-derived SIV strains with human immunodeficiency virus type 2 subtypes and genetically diverse viruses from a

reinfection with human immunodeficiency virus type 1 of a different subtype. J


HIV Recombination and Pathogenesis – Biological and Epidemiological Implications 119

Jetzt AE, Yu H, Klarmann GJ, Ron Y, Preston BD and Dougherty JP. (2000). High rate of

Jung A, Maier R, Vartanian JP, Bocharov G, Jung V, Fischer U, Meese E, Wain- Hobson S

Kalish ML, Baldwin A, Raktham S, Wasi C, Luo CC, Schochetman G, Mastro TD, Young N,

Koch WH, Sullivan PS, Roberts C, Francis K, Downing R, Mastro TD, et al. Evaluation of

Korber B, Muldoon M, Theiler J, Gao F, Gupta R, Lapedes A, Hahn BH, Wolinsky S and

Koulinska IN, Ndung'u T, Mwakagile D, Msamanga G, Kagoma C, Fawzi W, Essex M and

Lal RB, Chakrabarti S and Yang C. (2005). Impact of genetic diversity of HIV-1 on diagnosis,

Lau KA, Wang B, Saksena NK. (2007). Emerging trends of HIV epidemiology in Asia. AIDS

Laukkanen T, Carr JK, Janssens W, Liitsola K, Gotte D, McCutchan FE, Op de Coul E,

Lemey P, Pybus OG, Wang B, Saksena NK, Salemi M and Vandamme AM. (2003). Tracing

Liitsola K, Tashkinova I, Laukkanen T, Korovina G, Smolskaja T, Momot O, Mashkilleyson

Little, S.J., et al., Antiretroviral-drug resistance among patients recently infected with HIV.

Thailand: implications for HIV vaccine trials. AIDS. 9(8): 851-7.

and G.(2006) J Med Virol. May;78(5):523-34.

of group M viral variants. (2001) J Clin Microbiol. Mar;39(3):1017-20. Konings FA, Burda ST, Urbanski MM, Zhong P, Nadas A, Nyambi PN. Human

Virol. 74(3): 1234-40.

418(6894): 144.

288(5472): 1789-96.

Aug 23;4(1):59.

269(1): 95-104.

6588-92.

Rev. Oct-Dec;9(4):218-29. Review.

N Engl J Med, 2002. 347:385-94.

drug users in Kaliningrad. AIDS. 12(14): 1907-19.

314.

recombination throughout the human immunodeficiency virus type 1 genome. J

and Meyerhans A. (2002). Multiply infected spleen cells in HIV patients. Nature.

Vanichseni S, Rubsamen-Waigmann H, et al. (1995). The evolving molecular epidemiology of HIV-1 envelope subtypes in injecting drug users in Bangkok,

United States-licensed human immunodeficiency virus immunoassays for detection

immunodeficiency virus type 1 (HIV-1) circulating recombinant form 02\_AG (CRF02\_AG) has a higher in vitro replicative capacity than its parental subtypes A

Bhattacharya T. (2000). Timing the ancestor of the HIV-1 pandemic strains. Science.

Renjifo B. (2001). A new human immunodeficiency virus type 1 circulating recombinant form from Tanzania. AIDS Res Hum Retroviruses. 17(5): 423-31. Kozaczynska K, Cornelissen M, Reiss P, Zorgdrager F, van der Kuyl AC. HIV-1 sequence

evolution in vivo after superinfection with three viral strains. (2007) Retrovirology.

antiretroviral therapy & vaccine development. Indian J Med Res. 121(4): 287-

Cornelissen M, Heyndrickx L, van der Groen G, et al. (2000). Virtually full-length subtype F and F/D recombinant HIV-1 from Africa and South America. Virology.

the origin and history of the HIV-2 epidemic. Proc Natl Acad Sci U S A. 100(11):

N, Chaplinskas S, Brummer-Korvenkontio H, Vanhatalo J, et al. (1998). HIV-1 genetic subtype A/B recombinant strain causing an explosive epidemic in injecting

immunodeficiency virus type 1 RNA and the risk of perinatal transmission. Women and Infants Transmission Study Group. N Engl J Med. 341(6): 394-402.


Gottlieb GS, Nickle DC, Jensen MA, Wong KG, Grobler J, Li F, Liu SL, Rademeyer C, Learn

Grant, R.M., et al., Time trends in primary HIV-1 drug resistance among recently infected

Gutierrez M, Tajada P, Alvarez A, De Julian R, Baquero M, Soriano V and Holguin A. (2004).

among immigrant sex workers in Madrid, Spain. J Med Virol. 74(4): 521-7. Harris ME, Serwadda D, Sewankambo N, Kim B, Kigozi G, Kiwanuka N, Phillips JB,

Hemelaar J, Gouws E, Ghys PD, Osmanov S. Global and regional distribution of HIV-1 genetic subtypes and recombinants in 2004. (2006) AIDS. Oct 24;20(16):W13-23. Hierholzer J, Montano S, Hoelscher M, Negrete M, Hierholzer M, Avila MM, Carrillo MG,

Hirsch VM, Olmsted RA, Murphey-Corb M, Purcell RH and Johnson PR. (1989). An African primate lentivirus (SIVsm) closely related to HIV-2. Nature. 339(6223): 389-92. Ho DD, Neumann AU, Perelson AS, Chen W, Leonard JM, Markowitz M. Rapid turnover

Hsu TW and Taylor JM. (1982). Effect of aphidicolin on avian sarcoma virus replication. J

Hu WS and Temin HM. (1990a). Genetic consequences of packaging two RNA genomes in

Hu WS and Temin HM. (1990b). Retroviral recombination and reverse transcription.

Huet T, Cheynier R, Meyerhans A, Roelants G and Wain-Hobson S. (1990). Genetic

Hunt PW, Harrigan PR, Huang W, Bates M, Williamson DW, McCune JM, et al. Prevalence

Jeeninga RE, Hoogenkamp M, Armand-Ugon M, de Baar M, Verhoef K, Berkhout B.

Detectable Viremia. (2006) J Infect Dis. Oct 1;194(7):926-30.

predominate. AIDS Res Hum Retroviruses. 18(17): 1281-90.

20(8): 885-8.

18(18): 1339-50.

Virol. 44(2): 493-8.

Apr;74(8):3740-51.

Natl Acad Sci U S A. 87(4): 1556-60.

Science. 250(4985): 1227-33.

126.

9.

progression. Lancet. 363(9409): 619-22.

persons. Jama, 2002. 288:181-8.

immunodeficiency virus type 1 RNA and the risk of perinatal transmission. Women and Infants Transmission Study Group. N Engl J Med. 341(6): 394-402. Gomez-Carrillo M, Quarleri JF, Rubio AE, Carobene MG, Dilernia D, Carr JK and Salomon

H. (2004). Drug resistance testing provides evidence of the globalization of HIV type 1: a new circulating recombinant form. AIDS Research & Human Retroviruses.

GH, Karim SS, et al. (2004). Dual HIV-1 infection associated with rapid disease

Prevalence of HIV-1 non-B subtypes, syphilis, HTLV, and hepatitis B and C viruses

Wabwire F, Meehen M, Lutalo T, et al. (2002). Among 46 near full length HIV type 1 genome sequences from Rakai District, Uganda, subtype D and AD recombinants

Russi JC, Vinoles J, Alava A, et al. (2002). Molecular Epidemiology of HIV Type 1 in Ecuador, Peru, Bolivia, Uruguay, and Argentina. AIDS Res Hum Retroviruses.

of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 1995; 373:123-

one retroviral particle: pseudodiploidy and high rate of genetic recombination. Proc

organization of a chimpanzee lentivirus related to HIV-1. Nature. 345(6273): 356-

of CXCR4 Tropism among Antiretroviral-Treated HIV-1-Infected Patients with

Functional differences between the long terminal repeat transcriptional promoters of human immunodeficiency virus type 1 subtypes A through G. (2000) J Virol.


HIV Recombination and Pathogenesis – Biological and Epidemiological Implications 121

Njai HF, Gali Y, Vanham G, Clybergh C, Jennes W, Vidal N, et al. The predominance of

Otten RA, Ellenberger DL, Adams DR, Fridlund CA, Jackson E, Pieniazek D and Rayfield

Ou CY, Takebe Y, Weniger BG, Luo CC, Kalish ML, Auwanit W, et al. Independent

Parreira R, Padua E, Piedade J, Venenno T, Paixao MT and Esteves A. (2005). Genetic

Peeters M. (2000). Recombinant HIV sequences: Their role in the global epidemic. In HIV

Perez-Alvarez L, Carmona R, Munoz M, Delgado E, Thomson MM, Contreras G, Pedreira

Piyasirisilp S, McCutchan FE, Carr JK, Sanders-Buell E, Liu W, Chen J, et al. A recent

Preston BD, Poiesz BJ, Loeb LA. Fidelity oh HIV-1 reverse transcriptase. Science 1988;

Quinones-Mateu ME, Arts EJ. Virus fitness: concept, quantification, and application to HIV population dynamics. (2006) Curr Top Microbiol Immunol ;299:83-140. Quinones-Mateu ME, Ball SC, Marozsan AJ, Torre VS, Albright JL, Vanham G, et al. A dual

Renjifo B, Fawzi W, Mwakagile D, Hunter D, Msamanga G, Spiegelman D, Garland M,

Robertson DL, Anderson J, P., Bradac JA, Carr JK, Foley B, Funkhouser RK, Gao F, Hahn

to HIV-1 subtype A or D. AIDS. 18(12): 1629-1636.

Spain: study of genotypic resistance. Antivir Ther. 8(4): 355-60.

nemestrina (pig-tailed macaque) model. J Infect Dis. 180(3): 673-84.

Thailand. (1993). Lancet. May 8;341(8854):1171-4.

Retrovirology. Jul 3;3(1):40.

77(1): 8-16.

Dec;74(23):11286-95.

Oct;74(19):9222-33.

242:1168-1171.

39-54.

Human Immunodeficiency Virus type 1 (HIV-1) circulating recombinant form 02 (CRF02\_AG) in West Central Africa may be related to its replicative fitness. (2006)

MA. (1999). Identification of a window period for susceptibility to dual infection with two distinct human immunodeficiency virus type 2 isolates in a Macaca

introduction of two major HIV-1 genotypes into distinct high-risk populations in

analysis of human immunodeficiency virus type 1 nef in Portugal: subtyping, identification of mosaic genes, and amino acid sequence variability. J Med Virol.

Sequence Compendium 2000. Kuiken CL, Foley B, Hahn B et al.. Los Alamos, Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, NM.

JD, Rodriguez Real R, Vazquez de Parga E, Medrano L, et al. (2003). High incidence of non-B and recombinant HIV-1 strains in newly diagnosed patients in Galicia,

outbreak of human immunodeficiency virus type 1 infection in southern China was initiated by two highly homogeneous, geographically separated strains, circulating recombinant form AE and a novel BC recombinant. (2000). J Virol.

infection/competition assay shows a correlation between ex vivo human immunodeficiency virus type 1 fitness and disease progression. (2000) J Virol.

Kagoma C, Kim A, Chaplin B, et al. (2001). Differences in perinatal transmission among human immunodeficiency virus type 1 genotypes. J Hum Virol. 4(1): 16-25. Renjifo B, Gilbert P, Chaplin B, Msamanga G, Mwakagile D, Fawzi W, Group TVaHS and

Essex M. (2004). Preferential in-utero transmission of HIV-1 subtype C as compared

BH, Kuiken C, Learn GH, et al. (1999). HIV-1 Nomenclature Proposal. In Human Retroviruses and AIDS 1999. Kuiken, CL, Foley, B, Hahn, B et al. . Los Alamos,


Lukashov VV and Goudsmit J. (1997). Evolution of the human immunodeficiency virus type

Mamadou S, Vidal N, Montavon C, Ben A, Djibo A, Rabiou S, Soga G, Delaporte E, Mboup S

Mansky LM and Temin HM. (1995). Lower in vivo mutation rate of human

Masquelier, B., et al., Primary infection with zidovudine-resistant HIV. N Engl J Med, 1993.

McCutchan FE, Hegerich PA, Brennan TP, Phanuphak P, Singharaj P, Jugsudee A, Berman

McCutchan FE, Hoelscher M, Tovanabutra S, Piyasirisilp S, Sanders-Buell E, Ramos G,

Meloni ST, Kim B, Sankale JL, Hamel DJ, Tovanabutra S, Mboup S, McCutchan FE and

Michel N, Allespach I, Venzke S, Fackler OT and Keppler OT. (2005). The Nef protein of

Moutouh L, Corbeil J and Richman DD. (1996). Recombination leads to the rapid emergence

Negroni M and Buc H. (2001). Mechanisms of retroviral recombination. Annu Rev Genet. 35:

Nerurkar VR, Nguyen HT, Dashwood WM, Hoffmann PR, Yin C, Morens DM, et al. HIV

Nijhuis M, Schuurman R, de Jong D, Erickson J, Gustchina E, Albert J, et al. Increased fitness

mutations during suboptimal therapy. (1999) AIDS. Dec 3;13(17):2349-59.

Vietnam. (1996) AIDS Res Hum Retroviruses. Jun 10;12(9):841-3.

circulating in West Africa: sub-subtype A3. J Virol. 78(22): 12438-45. Meloni ST, Sankale JL, Hamel DJ, Eisen G, Gueye-Ndiaye A, Mboup S and Kanki PJ. (2004b).

to the subtype consensus. J Virol. 71(9): 6332-8.

reverse transcriptase. J Virol. 69(8): 5087-94.

Thailand. AIDS Res Hum Retroviruses. 8(11): 1887-95.

in Senegal from 1988 to 2001. J Virol. 78(22): 12455-61.

19(1): 77-82.

329:1123-4.

11693-704.

817-23.

275-302.

U S A. 93(12): 6106-11.

1 subtype-specific V3 domain is confined to a sequence space with a fixed distance

and Peeters M. (2003). Emergence of complex and diverse CRF02- AG/CRF06-cpx recombinant HIV type 1 strains in Niger, West Africa. AIDS Res Hum Retroviruses.

immunodeficiency virus type 1 than that predicted from the fidelity of purified

PW, Gray AM, Fowler AK and Burke DS. (1992). Genetic variants of HIV-1 in

Jagodzinski L, Polonis V, Maboko L, Mmbando D, et al. (2005). Indepth analysis of a heterosexually acquired human immunodeficiency virus type 1 superinfection: evolution, temporal fluctuation, and intercompartment dynamics from the seronegative window period through 30 months post-infection. J Virol. 79(18):

Kanki PJ. (2004a). Distinct human immunodeficiency virus type 1 subtype A virus

Molecular epidemiology of human immunodeficiency virus type 1 sub-subtype A3

human immunodeficiency virus establishes superinfection immunity by a dual strategy to downregulate cell-surface CCR5 and CD4. Curr Biol. 15(8): 714-23. Mokili JL, Rogers M, Carr JK, Simmonds P, Bopopi JM, Foley BT, Korber BT, Birx DL and

McCutchan FE. (2002). Identification of a novel clade of human immunodeficiency virus type 1 in Democratic Republic of Congo. AIDS Res Hum Retroviruses. 18(11):

of HIV-1 dually resistant mutants under selective drug pressure. Proc Natl Acad Sci

type 1 subtype E in commercial sex workers and injection drug users in southern

of drug resistant HIV-1 protease as a result of acquisition of compensatory


HIV Recombination and Pathogenesis – Biological and Epidemiological Implications 123

Thomson MM, Perez-Alvarez L and Najera R. (2002b). Molecular epidemiology of HIV-1

Tovanabutra S, Beyrer C, Sakkhachornphop S, Razak MH, Ramos GL, Vongchak T,

Tovanabutra S, Polonis V, De Souza M, Trichavaroj R, Chanbancherd P, Kim B, Sanders-

Troyer RM, Collins KR, Abraha A, Fraundorf E, Moore DM, Krizan RW, et al. Changes in

Van der Kuyl AC, Kozaczynska K, van den Burg R, Zorgdrager F, Back N, Jurriaans S,

van Maarseveen NM, Huigen MC, de Jong D, Smits AM, Boucher CA, Nijhuis M. A novel

van Rij RP, Worobey M, Visser JA and Schuitemaker H. (2003). Evolution of R5 and X4

Veenstra, J., et al., Transmission of zidovudine-resistant human immunodeficiency virus

Vidal N, Mulanga C, Bazepeo SE, Lepira F, Delaporte E and Peeters M. (2006). Identification

Vidal N, Peeters M, Mulanga-Kabeya C, Nzilambi N, Robertson D, Ilunga W, Sema H,

Vijay NN, Vasantika, Ajmani R, Perelson AS, Dixit NM. Recombination increases human

Visawapoka U, Tovanabutra S, Currier JR, Cox JH, Mason CJ, Wasunna M,

recombinant of HIV-1 is found in Thailand. AIDS. 15(8): 1063-5.

2002. AIDS Res Hum Retroviruses. 20(5): 465-75.

progression. (2005) J Virol.Jul;79(14):9006-18.

http://www.unaids.org/epi/2010/index.asp.

England Journal of Medicine. 352(24): 2557-9.

recombination. Virology. 314(1): 451-9.

Central Africa. J Virol. 74(22): 10498-507.

Hum Retroviruses. 22(7): 695-702.

UNAIDS. (2010). AIDS-epidemic update 2010. From

May;133(2):185-94.

22;98(11):6062-7.

Retroviruses. 22(2): 182-7.

Jun;89(Pt 6):1467-77.

Infect Dis. 2(8): 461-71.

genetic forms and its significance for vaccine development and therapy. Lancet

Rungruengthanakit K, Saokhieo P, Tejafong K, Kim B, et al. (2004). The changing molecular epidemiology of HIV type 1 among northern Thai drug users, 1999 to

Buell E, Nitayaphan S, Brown A, Robb MR, et al. (2001). First CRF01\_AE/B

human immunodeficiency virus type 1 fitness and genetic diversity during disease

Berkhout B, Reiss P and Cornelissen M. (2005). Triple HIV-1 infection. New

real-time PCR assay to determine relative replication capacity for HIV-1 protease variants and/or reverse transcriptase variants. (2006) J Virol Methods.

human immunodeficiency virus type 1 gag sequences in vivo: evidence for

type 1 variants following deliberate injection of blood from a patient with AIDS: characteristics and natural history of the virus. Clin Infect Dis, 1995. 21:556-60. Velazquez-Campoy A, Todd MJ, Vega S, Freire E. Catalytic efficiency and vitality of HIV-1

proteases from African viral subtypes. (2001) Proc Natl Acad Sci U S A. May

and molecular characterization of subsubtype A4 in central Africa. AIDS Res Hum

Tshimanga K, Bongo B and Delaporte E. (2000). Unprecedented degree of human immunodeficiency virus type 1 (HIV-1) group M genetic diversity in the Democratic Republic of Congo suggests that the HIV-1 pandemic originated in

immunodeficiency virus fitness, but not necessarily diversity. (2008) J Gen Virol.

Ponglikitmongkol M, Dowling WE, Robb ML, Birx DL, et al. (2006). Circulating and unique recombinant forms of HIV type 1 containing subsubtype A2. AIDS Res

NM., Theoretical Biology and Biophysics Group, Los Alamos National Laboratory.: 492-505.


Saksena NK, Wang B and Dyer WB. (2001). Biological and Molecular Mechanisms in

Saksena NK, Wang B, Steain MC, Yang RG and Zhang LQ. (2005). Snapshot of HIV

Sanders-Buell E, Saad MD, Abed AM, Bose M, Todd CS, Strathdee SA, Botros BA, Safi N,

Sharp PM, Robertson DL and Hahn BH. (1995). Cross-species transmission and

Smith DM, Wong JK, Hightower GK, Ignacio CC, Koelsch KK, Daar ES, Richman DD and

Soares EA, Martinez AM, Souza TM, Santos AF, Da Hora V, Silveira J, Bastos FI, Tanuri A

Songok EM, Lihana RW, Kiptoo MK, Genga IO, Kibaya R, Odhiambo F, Kobayashi K, Ago

Steain MC, Wang B, Dwyer DE and Saksena NK. (2004). HIV-1 co-infection, superinfection

Steain MC, Wang B, Yang C, Shi YP, Nahlen B, Lal RB and Saksena NK. (2005). HIV type 1

Swanson P, de Mendoza C, Joshi Y, Golden A, Hodinka RL, Soriano V, et al. Impact of

Takebe Y. Inter-CRF Recombinants: A New Class of HIV-1 Recombinants and Its

Tapia N, Franco S, Puig-Basagoiti F, Menendez C, Alonso PL, Mshinda H, et al. Influence of

Tatt ID, Barlow KL, Clewley JP, Gill ON and Parry JV. (2004). Surveillance of HIV- 1

Progression of HIV Disease. AIDS reviews. 3(3): 133-44.

pathogenesis in China. Cell Res. 15(11-12): 953-61.

HIV and SIV. Nature. 383(6601): 586-7.

and recombination. Sexual Health. 1(4): 239-250.

JAMA. 292(10): 1177-8.

Suppl 4(4): S81-6.

161-5.

21(10): 882-5.

Microbiol. Aug;43(8):3860-8.

Defic Syndr. 36(5): 1092-9.

Infections; 2006; Denver, Colorado, USA.

(2003) J Gen Virol. Mar;84(Pt 3):607-13.

492-505.

NM., Theoretical Biology and Biophysics Group, Los Alamos National Laboratory.:

Earhart KC, Scott PT, Michael N, McCutchan FE. (2007). A nascent HIV type 1 epidemic among injecting drug users in Kabul, Afghanistan is dominated by complex AD recombinant strain, CRF35\_AD. (2007). AIDS Res Hum Retroviruses. Jun;23(6):834-9. Erratum in: AIDS Res Hum Retroviruses. 2007 Jul;23(7):953-4. Sharp PM, Bailes E, Stevenson M, Emerman M and Hahn BH. (1996). Gene acquisition in

recombination of 'AIDS' viruses. Philos Trans R Soc Lond B Biol Sci. 349(1327): 41-7.

Little SJ. (2004). Incidence of HIV superinfection following primary infection.

and Soares MA. (2005). HIV-1 subtype C dissemination in southern Brazil. AIDS. 19

Y, Ndembi N, Okoth F, et al. (2003). Identification of env CRF-10 among HIV variants circulating in rural western Kenya. AIDS Res Hum Retroviruses. 19(2):

sequence diversity and dual infections in Kenya. AIDS Res Hum Retroviruses.

human immunodeficiency virus type 1 (HIV-1) genetic diversity on performance of four commercial viral load assays: LCx HIV RNA Quantitative, AMPLICOR HIV-1 MONITOR v1.5, VERSANT HIV-1 RNA 3.0, and NucliSens HIV-1 QT. (20035)J Clin

Epidemiological Implications. 13th Conference on Retroviruses and Opportunistic

human immunodeficiency virus type 1 subtype on mother-to-child transmission.

subtypes among heterosexuals in England and Wales, 1997-2000. J Acquir Immune


**5** 

*India* 

**New Therapeutics** 

*Indian institute of Science,* 

**Insulin-Like Growth Factor System in HIV/AIDS:** 

One of the important regulators for growth and development of the human body is the endocrine system. The endocrine system is composed of glands that secrete hormones into the circulatory system, which are then distributed throughout the body, regulating the function of tissues and maintaining homeostasis. Among these hormones are the insulin-like growth factors (IGF), similar in molecular structure to insulin and playing an important role

A complex network of molecules, including its binding proteins, proteases and receptors, which together comprise the 'IGF system', modulates the biological function of the IGFs. This system comprises the following components (Figure 1): (i) Two peptide hormones, IGF-1 and - 2, (ii) type 1 and type 2 IGF receptors, (iii) six IGF-binding proteins (IGFBP; numbered 1-6) and (iv) IGFBP proteases. IGF-1 and -2 are small signalling peptides (~7.5 kDa) that stimulate action by binding to specific cell surface receptors (IGF-1R) evoking subsequent response inside the cell. Six soluble IGF binding proteins, the IGFBPs, which range in size from 22-31 kDa and share overall sequence and structural homology with each other, regulate the activity of the IGFs. IGFBPs bind strongly to IGFs (KD ~ 300-700 pM) to ensure that the majority of circulating IGF in the blood stream is sequestered and at the tissue level inhibit the action of IGFs by blocking their access to the receptors. Proteolysis of the IGFBPs dissociates IGFs from the complex, enabling them to bind and activate the cell surface receptors (Figure 1). In tissues, IGFs form a binary complex with IGFBPs, whereas circulating IGFs are associated in ternary complexes containing IGFBP-3 (and IGFBP-5) and a third protein known as the acid-labile subunit (ALS). The ternary complex has a molecular mass of 150 kDa. The most abundant IGF-

In recent years, the IGF system in general and IGFBPs in particular have become the focus as clinically important targets of cancer therapeutics (Chan et al., 1998; Harrison et al., 1996; LeRoith & Roberts, 2003; Ma et al., 1999; Rosenzweig & Atreya, 2010; Wu et al., 2004; Yu et al., 1999). Different strategies have been proposed to inhibit cancer growth by blocking IGF-I-R binding and function (recently reviewed by (Rosenzweig & Atreya, 2010)). In this regard, the therapeutic potential of the IGFBPs in inhibiting IGF-1/IGF-2 activity and thereby inhibiting cancer cell growth has been demonstrated. Notably, the IGFBPs do not

bind insulin and thus do not interfere with insulin-insulin receptor interactions.

in cell growth, proliferation, differentiation (LeRoith & Roberts, 2003).

binding protein in the circulation is IGFBP-3 followed by IGFBP-2.

**1. Introduction** 

**A Structure Based Approach to the Design of** 

Monalisa Swain, Harsha Balaram and Hanudatta S. Atreya


## **Insulin-Like Growth Factor System in HIV/AIDS: A Structure Based Approach to the Design of New Therapeutics**

Monalisa Swain, Harsha Balaram and Hanudatta S. Atreya *Indian institute of Science, India* 

#### **1. Introduction**

124 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Walker PR, Pybus OG, Rambaut A and Holmes EC. (2005). Comparative population

Walker PR, Pybus OG, Rambaut A, Holmes EC. Comparative population dynamics of HIV-1

Wang B, Lal RB, Dwyer DE, Miranda-Saksena M, Boadle R, Cunningham AL and Saksena

Weber J, Rangel HR, Chakraborty B, Tadele M, Martinez MA, Martinez-Picado J, et al. A

Wei X, Ghosh SK, Taylor ME, Johnson VA, Emini EA, Deutsch P, et al. Viral dynamics in human immunodeficiency virus type 1 infection. Nature 1995; 373:117-122. Weiss RA, Mason WS and Vogt PK. (1973). Genetic recombinants and heterozygotes derived

Weniger BG, Limpakarnjanarat K, Ungchusak K, Thanprasertsuk S, Choopanya K,

Wong, J.K., et al., Recovery of replication-competent HIV despite prolonged suppression of

Wu W, Blumberg BM, Fay PJ and Bambara RA. (1995). Strand transfer mediated by human

Wu Y and Marsh JW. (2003). Early transcription from nonintegrated DNA in human

Yang C, Li M, Newman RD, Shi YP, Ayisi J, van Eijk AM, et al. Genetic diversity of HIV-1 in

Yang C, Li M, Newman RD, Shi YP, Ayisi J, van Eijk AM, Otieno J, Misore AO, Steketee RW,

specific differences in mother-to-child transmission. AIDS. 17(11): 1667-74. Yang C, Li M, Shi YP, Winter J, van Eijk AM, Ayisi J, Hu DJ, Steketee R, Nahlen BL and Lal

Yang R, Xia X, Kusagawa S, Zhang C, Ben K, Takebe Y. On-going generation of multiple

Yu H, Jetzt AE, Ron Y, Preston BD and Dougherty JP. (1998). The nature of human immunodeficiency virus type 1 strand transfers. J Biol Chem. 273(43): 28384-91. Zhu T, Korber BT, Nahmias AJ, Hooper E, Sharp PM and Ho DD. (1998). An African HIV-1

epidemic growth. Infect Genet Evol. 5(3): 199-208.

(2005) Infect Genet Evol. Apr;5(3):199-208.

(2003) J Gen Virol. Aug;84(Pt 8):2217-28.

plasma viremia. Science, 1997. 278:1291-5.

results in mis-incorporation. J Biol Chem. 270(1): 325-32.

immunodeficiency virus infection. J Virol. 77(19): 10376-82.

(1991) AIDS;5 Suppl 2:S71-85.

AIDS. Jul 25;17(11):1667-74.

Hum Retroviruses. 20(5): 565-74.

AIDS. Jul 5;16(10):1401-7.

391(6667): 594-7.

274(1): 105-19.

dynamics of HIV-1 subtypes B and C: subtype-specific differences in patterns of

subtypes B and C: subtype-specific differences in patterns of epidemic growth.

NK. (2000). Molecular and biological interactions between two HIV- 1 strains from a coinfected patient reveal the first evidence in favor of viral synergism. Virology.

novel TaqMan real-time PCR assay to estimate ex vivo human immunodeficiency virus type 1 fitness in the era of multi-target (pol and env) antiretroviral therapy.

from endogenous and exogenous avian RNA tumor viruses. Virology. 52(2): 535-52.

Vanichseni S, et al. The epidemiology of HIV infection and AIDS in Thailand.

immunodeficiency virus reverse transcriptase in vitro is promoted by pausing and

western Kenya: subtype-specific differences in mother-to-child transmission. (2003)

Nahlen BL, et al. (2003a). Genetic diversity of HIV-1 in western Kenya: subtype-

RB. (2004). Genetic diversity and high proportion of intersubtype recombinants among HIV type 1-infected pregnant women in Kisumu, western Kenya. AIDS Res

forms of HIV-1 intersubtype recombinants in the Yunnan Province of China. (2002)

sequence from 1959 and implications for the origin of the epidemic. Nature.

One of the important regulators for growth and development of the human body is the endocrine system. The endocrine system is composed of glands that secrete hormones into the circulatory system, which are then distributed throughout the body, regulating the function of tissues and maintaining homeostasis. Among these hormones are the insulin-like growth factors (IGF), similar in molecular structure to insulin and playing an important role in cell growth, proliferation, differentiation (LeRoith & Roberts, 2003).

A complex network of molecules, including its binding proteins, proteases and receptors, which together comprise the 'IGF system', modulates the biological function of the IGFs. This system comprises the following components (Figure 1): (i) Two peptide hormones, IGF-1 and - 2, (ii) type 1 and type 2 IGF receptors, (iii) six IGF-binding proteins (IGFBP; numbered 1-6) and (iv) IGFBP proteases. IGF-1 and -2 are small signalling peptides (~7.5 kDa) that stimulate action by binding to specific cell surface receptors (IGF-1R) evoking subsequent response inside the cell. Six soluble IGF binding proteins, the IGFBPs, which range in size from 22-31 kDa and share overall sequence and structural homology with each other, regulate the activity of the IGFs. IGFBPs bind strongly to IGFs (KD ~ 300-700 pM) to ensure that the majority of circulating IGF in the blood stream is sequestered and at the tissue level inhibit the action of IGFs by blocking their access to the receptors. Proteolysis of the IGFBPs dissociates IGFs from the complex, enabling them to bind and activate the cell surface receptors (Figure 1). In tissues, IGFs form a binary complex with IGFBPs, whereas circulating IGFs are associated in ternary complexes containing IGFBP-3 (and IGFBP-5) and a third protein known as the acid-labile subunit (ALS). The ternary complex has a molecular mass of 150 kDa. The most abundant IGFbinding protein in the circulation is IGFBP-3 followed by IGFBP-2.

In recent years, the IGF system in general and IGFBPs in particular have become the focus as clinically important targets of cancer therapeutics (Chan et al., 1998; Harrison et al., 1996; LeRoith & Roberts, 2003; Ma et al., 1999; Rosenzweig & Atreya, 2010; Wu et al., 2004; Yu et al., 1999). Different strategies have been proposed to inhibit cancer growth by blocking IGF-I-R binding and function (recently reviewed by (Rosenzweig & Atreya, 2010)). In this regard, the therapeutic potential of the IGFBPs in inhibiting IGF-1/IGF-2 activity and thereby inhibiting cancer cell growth has been demonstrated. Notably, the IGFBPs do not bind insulin and thus do not interfere with insulin-insulin receptor interactions.

Insulin-Like Growth Factor System in HIV/AIDS:

**1.2 Role of IGF system in HIV & AIDS** 

A Structure Based Approach to the Design of New Therapeutics 127

the N- and C-terminal domains in IGFBPs are essential for IGF-1/2 binding (Clemmons, 2001; Kibbey et al., 2006; Siwanowicz et al., 2005). The central 'linker' domain which is structurally disordered has been proposed to be site where most of the post-translational modifications take place. This is also the region where proteases act to cleave the IGFBPs. IGFBP levels are regulated by proteolysis following their secretion from the cell, which dissociates the IGFBP-IGF complex resulting in an increase in IGF-1/2 available for interacting with the IGF-1R (Bunn & Fowlkes, 2003) (Figure 1). This is evidenced from the differential effects of IGFBP-3 in tumor vs. normal prostate cells, wherein IGF-1 bio-

In addition to its involvement in various cancers, the IGF-system has also been implicated in diabetes, uremic cachexia, muscle wasting in congestive heart failure (CHF), aging, human immunodeficiency virus (HIV), acquired immunodeficiency syndrome (AIDS), and AIDS cachexia (Hambrecht et al., 2002). It is now well understood that the growth hormone (GH)/IGF axis is significantly affected in HIV and AIDS patients (Meininger & Grinspoon, 2001). Patients with HIV infection or AIDS are known to have multiple growth hormone (GH)/IGF axis related defects which include: abnormal GH secretion, profound decrease in serum levels of IGF- I and IGF- II, abnormal post-translational modifications of IGF binding proteins, increased concentration of IGFBP-2 and reduced IGFBP-1/-3 concentration (Meininger & Grinspoon, 2001). In patients with AIDS wasting syndrome acquired GH resistance has been found to result in increased GH concentrations which occurs as a function of weight loss and loss of lean body mass. Conversely in patients with HIV lipodystrophy syndrome GH concentrations are reduced due to increased abdominal visceral adiposity. The GH/IGF axis has also been implicated in HIV associated osteopenia in patients with HIV-1-infection but without any symptoms of AIDS-associated wasting (Stagi et al., 2004). Thus, due to its significant role IGF levels are closely monitored in HIV/AIDS patients and help to track disease progression (see review by Congote 2005).

In addition to changes in IGF levels, the concentration of IGFBPs has also been observed to vary in patients with AIDs or HIV infection compared to healthy individuals. It has been observed that the levels of IGFBP-3 decrease whereas those of IGFBP-1 and -2 increase in HIV/AIDS patients (Congote, 2005). The levels of IGF-1/2 and those of IGFBP-1, -2 and -3 in HIV-infected children and adults vary throughout the course of the disease. Administration of GH significantly increases the levels of IGFBP-3 in all HIV infected patients except for patients with AIDS wasting (Mynarcik et al., 1999). Disease progression is associated with decrease IGF-2 levels and increase in IGFBP-2 & IGFBP-3 protease activity, infact IGFBP-2 levels are one of the first parameters to increase after HIV infection, before the development of AIDS (Helle et al., 2001) . The elevated IGFBP-3 protease activity is somewhat restored in patients undergoing antiretroviral therapy treatment (Helle et al., 2001). The proteolysis of IGFBP-3 causes IGF to be released, which is captured by IGFBP-2. It has been hypothesized that the low IGF and high IGFBP-2 levels found in HIV infection may contribute to enhanced lymphocyte apoptosis. This may in turn lead to immune dysfunction in patients. In another study, IGFBP-1 was observed to be highly phosphorylated and IGFBP-3 ternary

availability is increased via IGFBP proteolysis (Miyamoto et al., 2004).

**1.2.1 Involvement of growth hormone/IGF axis in AIDS** 

**1.2.2 Role of IGF-binding proteins in HIV & AIDS** 

Fig. 1. Illustration of the IGF-system and its components.

#### **1.1 IGF-binding proteins (IGFBPs)**

While extensive studies have been carried out on the role of IGFs in different biological systems and under diseased conditions, a molecular-level understanding of IGF-IGFBP interactions is lacking. The three-dimensional (3D) structures have not yet been determined for any of the full-length IGFBPs. Based on sequence analysis, it is now understood that all IGFBPs contain three structural domains of nearly equal size (Firth & Baxter, 2002; Krywicki & Yee, 1992; LeRoith & Roberts, 2003; Rosenzweig, 2004). The N-and C-terminal domains are highly conserved in sequence across the IGFBPs. They contain 16-18 cysteine residues, forming 8-9 disulfide bonds. Their disulphide bonding indicates that the IGFBPs are thyroglobulin type-1 domain homologues. In recent years, structural studies have been carried out on individual domains in IGFBP-1, -2, -4, -5 and -6 (Kalus et al., 1998; Kibbey et al., 2006; Kuang et al., 2006; Sala et al., 2005; Sitar et al., 2006; Siwanowicz et al., 2005). Studies involving site directed mutagenesis have identified key residues in IGFBPs that are required for binding the IGFs (Clemmons, 2001). These studies have also revealed that both the N- and C-terminal domains in IGFBPs are essential for IGF-1/2 binding (Clemmons, 2001; Kibbey et al., 2006; Siwanowicz et al., 2005). The central 'linker' domain which is structurally disordered has been proposed to be site where most of the post-translational modifications take place. This is also the region where proteases act to cleave the IGFBPs.

IGFBP levels are regulated by proteolysis following their secretion from the cell, which dissociates the IGFBP-IGF complex resulting in an increase in IGF-1/2 available for interacting with the IGF-1R (Bunn & Fowlkes, 2003) (Figure 1). This is evidenced from the differential effects of IGFBP-3 in tumor vs. normal prostate cells, wherein IGF-1 bioavailability is increased via IGFBP proteolysis (Miyamoto et al., 2004).

## **1.2 Role of IGF system in HIV & AIDS**

126 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Fig. 1. Illustration of the IGF-system and its components.

While extensive studies have been carried out on the role of IGFs in different biological systems and under diseased conditions, a molecular-level understanding of IGF-IGFBP interactions is lacking. The three-dimensional (3D) structures have not yet been determined for any of the full-length IGFBPs. Based on sequence analysis, it is now understood that all IGFBPs contain three structural domains of nearly equal size (Firth & Baxter, 2002; Krywicki & Yee, 1992; LeRoith & Roberts, 2003; Rosenzweig, 2004). The N-and C-terminal domains are highly conserved in sequence across the IGFBPs. They contain 16-18 cysteine residues, forming 8-9 disulfide bonds. Their disulphide bonding indicates that the IGFBPs are thyroglobulin type-1 domain homologues. In recent years, structural studies have been carried out on individual domains in IGFBP-1, -2, -4, -5 and -6 (Kalus et al., 1998; Kibbey et al., 2006; Kuang et al., 2006; Sala et al., 2005; Sitar et al., 2006; Siwanowicz et al., 2005). Studies involving site directed mutagenesis have identified key residues in IGFBPs that are required for binding the IGFs (Clemmons, 2001). These studies have also revealed that both

**1.1 IGF-binding proteins (IGFBPs)** 

## **1.2.1 Involvement of growth hormone/IGF axis in AIDS**

In addition to its involvement in various cancers, the IGF-system has also been implicated in diabetes, uremic cachexia, muscle wasting in congestive heart failure (CHF), aging, human immunodeficiency virus (HIV), acquired immunodeficiency syndrome (AIDS), and AIDS cachexia (Hambrecht et al., 2002). It is now well understood that the growth hormone (GH)/IGF axis is significantly affected in HIV and AIDS patients (Meininger & Grinspoon, 2001). Patients with HIV infection or AIDS are known to have multiple growth hormone (GH)/IGF axis related defects which include: abnormal GH secretion, profound decrease in serum levels of IGF- I and IGF- II, abnormal post-translational modifications of IGF binding proteins, increased concentration of IGFBP-2 and reduced IGFBP-1/-3 concentration (Meininger & Grinspoon, 2001). In patients with AIDS wasting syndrome acquired GH resistance has been found to result in increased GH concentrations which occurs as a function of weight loss and loss of lean body mass. Conversely in patients with HIV lipodystrophy syndrome GH concentrations are reduced due to increased abdominal visceral adiposity. The GH/IGF axis has also been implicated in HIV associated osteopenia in patients with HIV-1-infection but without any symptoms of AIDS-associated wasting (Stagi et al., 2004). Thus, due to its significant role IGF levels are closely monitored in HIV/AIDS patients and help to track disease progression (see review by Congote 2005).

#### **1.2.2 Role of IGF-binding proteins in HIV & AIDS**

In addition to changes in IGF levels, the concentration of IGFBPs has also been observed to vary in patients with AIDs or HIV infection compared to healthy individuals. It has been observed that the levels of IGFBP-3 decrease whereas those of IGFBP-1 and -2 increase in HIV/AIDS patients (Congote, 2005). The levels of IGF-1/2 and those of IGFBP-1, -2 and -3 in HIV-infected children and adults vary throughout the course of the disease. Administration of GH significantly increases the levels of IGFBP-3 in all HIV infected patients except for patients with AIDS wasting (Mynarcik et al., 1999). Disease progression is associated with decrease IGF-2 levels and increase in IGFBP-2 & IGFBP-3 protease activity, infact IGFBP-2 levels are one of the first parameters to increase after HIV infection, before the development of AIDS (Helle et al., 2001) . The elevated IGFBP-3 protease activity is somewhat restored in patients undergoing antiretroviral therapy treatment (Helle et al., 2001). The proteolysis of IGFBP-3 causes IGF to be released, which is captured by IGFBP-2. It has been hypothesized that the low IGF and high IGFBP-2 levels found in HIV infection may contribute to enhanced lymphocyte apoptosis. This may in turn lead to immune dysfunction in patients. In another study, IGFBP-1 was observed to be highly phosphorylated and IGFBP-3 ternary

Insulin-Like Growth Factor System in HIV/AIDS:

production (Congote, 2005; Rao et al., 2010).

thereby serving as potent therapeutics.

2010a).

**1.4.1 Structural characterization of IGFBP-2 and its fragments** 

consistent with those observed in the individual domains.

growth of the tumor is associated with increased IGF-1 levels.

A Structure Based Approach to the Design of New Therapeutics 129

malignancy. In the case of cancer cachexia, which includes muscle wasting and anorexia, the

Interestingly, it was found that administration of lGF-I in complex with IGFBP-3, but not free IGF-I, is a potent stimulator of muscle protein synthesis in rats with chronic under nutrition (Zdanowicz & Teichberg, 2003). In contrast to free IGF-1, significantly higher dosage levels can be used. Administration of IGF-1 in this form increases the bioavailability of IGF-1. Moreover, a high dosage level of this complex does not result in hypoglycemic condition owing to the fact that IGF-1/IGFBP-3 complex does not interact with the insulin receptor. In the case of cancer cachexia, the IGF-1/IGFBP-3 complex fails to alter tumor growth, but improves the tumor-host nutritional state by improving food intake, attenuating weight loss and improving glucose metabolism (Wang et al., 2000). Thus treatment with IGF-1/IGFBP-3 complex seems to be a promising approach to improve whole-body glucose uptake and glucose tolerance, while increasing hepatic glucose

**1.4 Structural studies of IGFBP-IGF interactions for developing potent therapeutics**  In order to improve the efficacy of treatment involving the administration of IGF-1/IGFBP-3 complex discussed above, it is important to understand the structural aspects of IGF-IGFBP interactions. Such studies help in enhancing the binding affinity of IGF-1 to IGFBP-3 and also aid in engineering IGFBPs to be protease resistant. We discuss below a thermodynamic approach that we have adopted to study IGF-IGFBP interactions. This brings out the utility of such studies in designing new forms of IGFBPs with enhanced IGF binding affinity

While structures of individual domains are known, 3D structures of full-length IGFBPs and/or their complex with IGF-1 have not yet been determined. This has been due to the difficulty in expressing full-length IGFBPs at milligram quantity levels required for X-raycrystallography or NMR spectroscopy. All IGFBPs contain 16-18 cysteines bridged by disulphide bonds, which makes them difficult to be expressed in bacterial systems. These proteins thus tend to precipitate inside bacterial cells resulting in inclusion bodies. We recently reported the first high-yield expression and structural characterization of functional full-length recombinant human IGFBP-2 (rhIGFBP-2) in *E. Coli* (Swain et al., 2010a). Figure 2 shows the 2D [15N-1H] NMR spectrum of bacterially expressed IGFBP-2. A good dispersion of peaks seen in the spectrum indicates a well-folded conformation of the protein. The secondary structural content estimated based on the NMR spectrum was found to be

In cysteine rich proteins, a key requirement is to conserve the pattern of intra-molecular disulphide bonds required for the protein function. Often scrambling (mis-pairing) of disulphide bonds result during purification resulting in heterogeneity of conformations. In our study, we employed an efficient denaturing-refolding protocol. This involved first denaturing the protein in presence of the a reducing agent such as β-mercaptoethanol or DTT followed by slow removal of the denaturing agent and the reducing agent through dialysis resulting in a unique pattern of intra-molecular disulphide bonds. This is evident from the single set of peaks seen in the 2D NMR spectrum shown in Figure 2 (Swain et al.,

complexes were formed with reduced ability in AIDS patients with wasting (Frost et al., 1996; Gelato & Frost, 1997).

#### **1.3 IGF-system in treatment of HIV & AIDS**

Given the significant role played by GH, IGFs and IGFBPs in HIV and AIDs, different approaches for treatment based on the GH/IGF-system have been proposed. We discuss here the two approaches, which underscore the importance of this system.

#### **1.3.1 Treatment with growth hormone**

In our body carbohydrates are the main source of energy needed for survival. Insufficient carbohydrate intake causes the body to burn the reserved fats followed by burning the muscle for energy which leads to loss of lean body mass (LBM). Muscle wasting or Wasting Syndrome (WS) is the most frequent problem in the patients with HIV infection and results in significant loss of body mass (Dudgeon et al., 2006). There are a variety of reasons why patients continue to lose weight, including: loss of appetite, increased metabolism, altered hormone levels, increased cytokine production which produces more fats than proteins. This is further aggravated by different drugs which cause nausea resulting in decreased food intake by patients and poor nutrient absorption which are necessary to maintain body mass. HIV-associated adipose redistribution syndrome (HARS) is an HIV-associated disorder characterized by excess truncal fat, including visceral adipose tissue (VAT). Muscle wasting, and particularly loss of metabolically active lean tissue, contributes to increased mortality, accelerated disease progression, and impairment of strength and functional status. The effect of treatment with protein anabolic agents, including GH, IGF-I, testosterone, nandrolonedecanoate, oxandrolone, and oxymetholone, have been studied in patients with HIV associated wasting. These studies have demonstrated that this treatment can increase lean body mass (LBM) and in some cases provide functional benefits and improvements in quality of life (Mulligan & Schambelan, 2002; Spinola-Castro et al., 2008). The immunologic effects of recombinant human growth hormone (rhGH), recombinant human insulin-like growth factor-1 (IGF-1), or their combination, in patients with moderately advanced HIV infection has been studied. The treatment with a combination of rhGH/rhIGF-1 and low dose of rhGH is reasonably well tolerated, resulting in increased body weight and modest improvements in HIV-specific immune function (Lee et al., 1996; Nguyen et al., 1998). Patients treated with rhGH sustain losses in VAT and truncal fat with no effect on subcutaneous fat in the abdomen or limbs. It has also been observed that nonhigh-density lipoprotein cholesterol (non-HDL-C) decreases significantly with rhGH treatment (Grunfeld et al., 2007).

#### **1.3.2 Treatment with IGF-1 and IGF: IGFBP complexes**

The observation that improved muscle mass, but not linear growth is associated with normalized IGF-1 concentration suggests that IGF-1 may be a potential therapeutic strategy to improve lean body mass in HIV-infected children (Chantry et al., 2008). Treatment with low dose recombinant IGF-1 produces significant, but transient, nitrogen retention. Alternate routes of IGF-1 administration or co-administration with GH prevents attenuation of IGF-1 action (Lieberman et al., 1994). However, administration of IGF-1 has its own set of problems. It causes lowering of glucose levels, which restricts its dosage, and thereby it's anabolic potential. Further, high levels of IGF-1 are warning signs for the increased risk of

complexes were formed with reduced ability in AIDS patients with wasting (Frost et al.,

Given the significant role played by GH, IGFs and IGFBPs in HIV and AIDs, different approaches for treatment based on the GH/IGF-system have been proposed. We discuss

In our body carbohydrates are the main source of energy needed for survival. Insufficient carbohydrate intake causes the body to burn the reserved fats followed by burning the muscle for energy which leads to loss of lean body mass (LBM). Muscle wasting or Wasting Syndrome (WS) is the most frequent problem in the patients with HIV infection and results in significant loss of body mass (Dudgeon et al., 2006). There are a variety of reasons why patients continue to lose weight, including: loss of appetite, increased metabolism, altered hormone levels, increased cytokine production which produces more fats than proteins. This is further aggravated by different drugs which cause nausea resulting in decreased food intake by patients and poor nutrient absorption which are necessary to maintain body mass. HIV-associated adipose redistribution syndrome (HARS) is an HIV-associated disorder characterized by excess truncal fat, including visceral adipose tissue (VAT). Muscle wasting, and particularly loss of metabolically active lean tissue, contributes to increased mortality, accelerated disease progression, and impairment of strength and functional status. The effect of treatment with protein anabolic agents, including GH, IGF-I, testosterone, nandrolonedecanoate, oxandrolone, and oxymetholone, have been studied in patients with HIV associated wasting. These studies have demonstrated that this treatment can increase lean body mass (LBM) and in some cases provide functional benefits and improvements in quality of life (Mulligan & Schambelan, 2002; Spinola-Castro et al., 2008). The immunologic effects of recombinant human growth hormone (rhGH), recombinant human insulin-like growth factor-1 (IGF-1), or their combination, in patients with moderately advanced HIV infection has been studied. The treatment with a combination of rhGH/rhIGF-1 and low dose of rhGH is reasonably well tolerated, resulting in increased body weight and modest improvements in HIV-specific immune function (Lee et al., 1996; Nguyen et al., 1998). Patients treated with rhGH sustain losses in VAT and truncal fat with no effect on subcutaneous fat in the abdomen or limbs. It has also been observed that nonhigh-density lipoprotein cholesterol (non-HDL-C) decreases significantly with rhGH

The observation that improved muscle mass, but not linear growth is associated with normalized IGF-1 concentration suggests that IGF-1 may be a potential therapeutic strategy to improve lean body mass in HIV-infected children (Chantry et al., 2008). Treatment with low dose recombinant IGF-1 produces significant, but transient, nitrogen retention. Alternate routes of IGF-1 administration or co-administration with GH prevents attenuation of IGF-1 action (Lieberman et al., 1994). However, administration of IGF-1 has its own set of problems. It causes lowering of glucose levels, which restricts its dosage, and thereby it's anabolic potential. Further, high levels of IGF-1 are warning signs for the increased risk of

here the two approaches, which underscore the importance of this system.

1996; Gelato & Frost, 1997).

**1.3 IGF-system in treatment of HIV & AIDS** 

**1.3.1 Treatment with growth hormone** 

treatment (Grunfeld et al., 2007).

**1.3.2 Treatment with IGF-1 and IGF: IGFBP complexes** 

malignancy. In the case of cancer cachexia, which includes muscle wasting and anorexia, the growth of the tumor is associated with increased IGF-1 levels.

Interestingly, it was found that administration of lGF-I in complex with IGFBP-3, but not free IGF-I, is a potent stimulator of muscle protein synthesis in rats with chronic under nutrition (Zdanowicz & Teichberg, 2003). In contrast to free IGF-1, significantly higher dosage levels can be used. Administration of IGF-1 in this form increases the bioavailability of IGF-1. Moreover, a high dosage level of this complex does not result in hypoglycemic condition owing to the fact that IGF-1/IGFBP-3 complex does not interact with the insulin receptor. In the case of cancer cachexia, the IGF-1/IGFBP-3 complex fails to alter tumor growth, but improves the tumor-host nutritional state by improving food intake, attenuating weight loss and improving glucose metabolism (Wang et al., 2000). Thus treatment with IGF-1/IGFBP-3 complex seems to be a promising approach to improve whole-body glucose uptake and glucose tolerance, while increasing hepatic glucose production (Congote, 2005; Rao et al., 2010).

## **1.4 Structural studies of IGFBP-IGF interactions for developing potent therapeutics**

In order to improve the efficacy of treatment involving the administration of IGF-1/IGFBP-3 complex discussed above, it is important to understand the structural aspects of IGF-IGFBP interactions. Such studies help in enhancing the binding affinity of IGF-1 to IGFBP-3 and also aid in engineering IGFBPs to be protease resistant. We discuss below a thermodynamic approach that we have adopted to study IGF-IGFBP interactions. This brings out the utility of such studies in designing new forms of IGFBPs with enhanced IGF binding affinity thereby serving as potent therapeutics.

## **1.4.1 Structural characterization of IGFBP-2 and its fragments**

While structures of individual domains are known, 3D structures of full-length IGFBPs and/or their complex with IGF-1 have not yet been determined. This has been due to the difficulty in expressing full-length IGFBPs at milligram quantity levels required for X-raycrystallography or NMR spectroscopy. All IGFBPs contain 16-18 cysteines bridged by disulphide bonds, which makes them difficult to be expressed in bacterial systems. These proteins thus tend to precipitate inside bacterial cells resulting in inclusion bodies. We recently reported the first high-yield expression and structural characterization of functional full-length recombinant human IGFBP-2 (rhIGFBP-2) in *E. Coli* (Swain et al., 2010a). Figure 2 shows the 2D [15N-1H] NMR spectrum of bacterially expressed IGFBP-2. A good dispersion of peaks seen in the spectrum indicates a well-folded conformation of the protein. The secondary structural content estimated based on the NMR spectrum was found to be consistent with those observed in the individual domains.

In cysteine rich proteins, a key requirement is to conserve the pattern of intra-molecular disulphide bonds required for the protein function. Often scrambling (mis-pairing) of disulphide bonds result during purification resulting in heterogeneity of conformations. In our study, we employed an efficient denaturing-refolding protocol. This involved first denaturing the protein in presence of the a reducing agent such as β-mercaptoethanol or DTT followed by slow removal of the denaturing agent and the reducing agent through dialysis resulting in a unique pattern of intra-molecular disulphide bonds. This is evident from the single set of peaks seen in the 2D NMR spectrum shown in Figure 2 (Swain et al., 2010a).

Insulin-Like Growth Factor System in HIV/AIDS:

A Structure Based Approach to the Design of New Therapeutics 131

Fig. 3. TEM images of IGFBP-2249-289 nanotubes under non-reducing conditions

Bacterial expression of functional full length human IGFBP-2 opens up new avenues to carry out structure-based functional studies in this protein family. One promising application is to generate/engineer antibodies against human IGFBP-2 and use it for detection of IGFBP-2 in HIV/AIDS patients. As mentioned above, it is now established that IGFBP-2 levels are significantly elevated in HIV/AIDS patients (Congote, 2005) and hence IGFBP-2 can be used a bio-marker for diagnosing or tracking the progression of this disease. In recent years, several bio-markers have been proposed or developed for monitoring HIV infection. These include: CD4 count (Smurzynski et al., 2010), TNF-alpha receptor type 2 as a useful serum marker for metabolic dysfunction (Gelato et al., 2002), fibroblast growth factor-21 (FGF21) (Domingo et al., 2010), levels of iron bound and iron-related proteins in urine to identify HIV-infected children at risk of developing HIVAN and HIV-HUS (Soler-Garcia et al., 2009), plasma levels of high sensitivity C reactive protein (hsCRP), interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1) (Padilla et al., 2011), vascular cell adhesion molecule-1 (sVCAM-1) and plasminogen activator inhibitor-1 (PAI-1) (Padilla et al., 2011). Elements of the IGF system have also been found to be promising bio-markers. However, the detection of proteins by antibodies is the most efficient and sensitive method. This will serve to detect/monitor variations in IGFBP-2 levels in patients with HIV/AIDS. Further, once the structural details of IGFBP2-IGFBP-2 antibody interactions are defined, the antibodies can be engineered to have tight binding to

Structural studies of individual domains of the IGFBPs in free and complexed form with IGF-1 has provided considerable insights into their interactions (Kalus et al., 1998; Kibbey et al., 2006; Kuang et al., 2006; Sala et al., 2005; Sitar et al., 2006; Siwanowicz et al., 2005). As mentioned above, IGFBPs contain three structural domains of nearly equal size (these are denoted as N-terminal, middle or L-domain and C-terminal domains, respectively). It is

**1.4.2 IGFBP-2 as a biomarker for monitoring disease progression** 

IGFBP-2 which in turn will enhance the sensitivity of IGFBP-2 detection.

**1.4.3 Structural features of IGF-IGFBP interactions** 

Fig. 2. Two dimensional [15N-1H] HSQC NMR spectrum of full length hIGFBP-2 (33 kDa) which correlates the polypeptide backbone 15N chemical shift with its directly attached 1H. The spectrum was acquired at 288 K at 1H resonance frequency of 700 MHz with a ~1 mM sample concentration. A good dispersion of peaks indicates a well-folded conformation of the protein.

In order to understand the mechanistic aspects of IGF-IGFBP interactions, we have undertaken the study of different domains and fragments of IGFBP-2. Biochemical studies reveal that removal of 41 residues (249-289) from the C-terminal tail of full-length hIGFBP-2 (hereafter denoted as IGFBP-2249-289) significantly increases the rate of IGF dissociation, in turn abolishing the ability of the truncated protein to effectively bind IGF (Kibbey et al., 2006). Wild type IGFBP-2 249-289 contains two cysteines. However, due to an artifact of cloning full length IGFBP-2 and subsequently the C-terminal polypeptide IGFBP-2249-289, our recombinant species all have an additional cysteine at position 281. This resulted in three cysteines in IGFBP-2 249-289 raising the possibility of forming dimers or higher order aggregates. In the presence of reducing agents such as β-mercaptoethanol (which are known to reduce disulphide bonds) the protein remained as a monomer. However, upon removal of β-mercaptoethanol by dialysis and/or ultrafiltration, it was found that the polypeptide self-assembled spontaneously into soluble nanotubes several micrometers long (Swain et al., 2010b) (see Figure 3). These tubular structures were studied using different biophysical techniques such as transmission electron microscopy (TEM), NMR spectroscopy, fluorescence and circular dichroism (CD). The observation that formation/dissociation of such nanotubes is reversible (they exchange between monomeric and polymeric forms in presence/absence of reducing agents) and their high mechanical stability due to covalent interaction between the individual components offers new avenues for designing novel IGFBP-based self-assembling nanotubes for biomedical applications.

Fig. 2. Two dimensional [15N-1H] HSQC NMR spectrum of full length hIGFBP-2 (33 kDa) which correlates the polypeptide backbone 15N chemical shift with its directly attached 1H. The spectrum was acquired at 288 K at 1H resonance frequency of 700 MHz with a ~1 mM sample concentration. A good dispersion of peaks indicates a well-folded conformation of

In order to understand the mechanistic aspects of IGF-IGFBP interactions, we have undertaken the study of different domains and fragments of IGFBP-2. Biochemical studies reveal that removal of 41 residues (249-289) from the C-terminal tail of full-length hIGFBP-2 (hereafter denoted as IGFBP-2249-289) significantly increases the rate of IGF dissociation, in turn abolishing the ability of the truncated protein to effectively bind IGF (Kibbey et al., 2006). Wild type IGFBP-2 249-289 contains two cysteines. However, due to an artifact of cloning full length IGFBP-2 and subsequently the C-terminal polypeptide IGFBP-2249-289, our recombinant species all have an additional cysteine at position 281. This resulted in three cysteines in IGFBP-2 249-289 raising the possibility of forming dimers or higher order aggregates. In the presence of reducing agents such as β-mercaptoethanol (which are known to reduce disulphide bonds) the protein remained as a monomer. However, upon removal of β-mercaptoethanol by dialysis and/or ultrafiltration, it was found that the polypeptide self-assembled spontaneously into soluble nanotubes several micrometers long (Swain et al., 2010b) (see Figure 3). These tubular structures were studied using different biophysical techniques such as transmission electron microscopy (TEM), NMR spectroscopy, fluorescence and circular dichroism (CD). The observation that formation/dissociation of such nanotubes is reversible (they exchange between monomeric and polymeric forms in presence/absence of reducing agents) and their high mechanical stability due to covalent interaction between the individual components offers new avenues for designing novel

IGFBP-based self-assembling nanotubes for biomedical applications.

the protein.

Fig. 3. TEM images of IGFBP-2249-289 nanotubes under non-reducing conditions

#### **1.4.2 IGFBP-2 as a biomarker for monitoring disease progression**

Bacterial expression of functional full length human IGFBP-2 opens up new avenues to carry out structure-based functional studies in this protein family. One promising application is to generate/engineer antibodies against human IGFBP-2 and use it for detection of IGFBP-2 in HIV/AIDS patients. As mentioned above, it is now established that IGFBP-2 levels are significantly elevated in HIV/AIDS patients (Congote, 2005) and hence IGFBP-2 can be used a bio-marker for diagnosing or tracking the progression of this disease. In recent years, several bio-markers have been proposed or developed for monitoring HIV infection. These include: CD4 count (Smurzynski et al., 2010), TNF-alpha receptor type 2 as a useful serum marker for metabolic dysfunction (Gelato et al., 2002), fibroblast growth factor-21 (FGF21) (Domingo et al., 2010), levels of iron bound and iron-related proteins in urine to identify HIV-infected children at risk of developing HIVAN and HIV-HUS (Soler-Garcia et al., 2009), plasma levels of high sensitivity C reactive protein (hsCRP), interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1) (Padilla et al., 2011), vascular cell adhesion molecule-1 (sVCAM-1) and plasminogen activator inhibitor-1 (PAI-1) (Padilla et al., 2011). Elements of the IGF system have also been found to be promising bio-markers. However, the detection of proteins by antibodies is the most efficient and sensitive method. This will serve to detect/monitor variations in IGFBP-2 levels in patients with HIV/AIDS. Further, once the structural details of IGFBP2-IGFBP-2 antibody interactions are defined, the antibodies can be engineered to have tight binding to IGFBP-2 which in turn will enhance the sensitivity of IGFBP-2 detection.

#### **1.4.3 Structural features of IGF-IGFBP interactions**

Structural studies of individual domains of the IGFBPs in free and complexed form with IGF-1 has provided considerable insights into their interactions (Kalus et al., 1998; Kibbey et al., 2006; Kuang et al., 2006; Sala et al., 2005; Sitar et al., 2006; Siwanowicz et al., 2005). As mentioned above, IGFBPs contain three structural domains of nearly equal size (these are denoted as N-terminal, middle or L-domain and C-terminal domains, respectively). It is

Insulin-Like Growth Factor System in HIV/AIDS:

interactions.

A Structure Based Approach to the Design of New Therapeutics 133

evaluate the extent of change in stability of the protein complex upon mutation of key residues in IGFBP. The residues chosen were those that have been verified experimentally to be involved in binding IGFs. A large body of work has been carried out in the past wherein different site-specific mutants, deletion mutants and/or truncated forms of the IGFBPs have been tested for their IGF-1 binding activities (Clemmons, 2001). Many of these residues are known to be conserved across all six IGFBPs. With this information in hand, our objective is to map on the 3D structures of IGFBP mutations that are known to destabilize IGF-IGFBP

In recent years, computational methods have been proposed to design specific mutants with enhanced ligand binding affinity (Sammond et al., 2007). These are structure-based methods that systematically predict single mutations at protein-protein interfaces which enhance binding affinities. This is based on the hypothesis that increasing the buried hydrophobic surface area or reducing buried hydrophilic surface area leads to enhanced affinity if steric clashes are avoided and all polar groups buried in the core of the protein have a hydrogen bond partner. In the 3D structures of IGFBPs in complex with IGF-1, we mutated residues, which are known to be involved in binding IGF-1, and evaluated the resulting change in thermodynamic stability (via free-energy change). We observed that mutation of residues important for binding IGF-1 increases the free energy of the complex resulting in the destabilization and weakening of IGF-IGFBP interaction. This implies that thermodynamic stability of the IGF-IGFBP complex can be used as an indicator of IGF-1 binding affinity. The study was carried out in 3 stages: first, the change in free energy of the IGF-IGFBP complex upon mutation of residues in IGFBP was predicted. This was carried out using the program I-MUTANT (Capriotti et al., 2005). Next, based on these results, the structure of the complex containing residues in IGFBP that resulted in lowering or increasing the stability of the complex was structurally modeled using the software ROSETTA-DOCK (Lyskov & Gray, 2008). Finally, the modeled structures were subjected to energy minimization followed by 10 ns MD simulations using the program GROMACS (Van der Spoel et al., 2005). In order to understand the structural basis of increased or decreased thermodynamic stability of the IGF-IGFBP complex, the hydrophobic and polar surface areas accessible were evaluated using the program NACCSESS (Hubbard et al.,1993). Two protein complexes of IGFBP with IGF-1 and two uncomplexed forms of the same protein were used for the analysis namely, IGFBP5 N- terminal and IGFBP1 C-terminal having PDB ID 1H59, 1BOE (representing the complexed form) and 2DSQ, 1ZT3 (representing the uncomplexed form), respectively. These two proteins were chosen due to the fact that high-resolution structures of IGF-bound and

unbound forms are currently only available for these proteins.

The free energy calculations were done as follows:

Using I-MUTANT 2.0, each residue within the predicted binding sites and conserved regions of N- and C-domain of IGFBP-4 and IGFBP-1, respectively, was mutated to the 19 other possible amino acids at that position and the change in free energy for each mutation was identified based on the G value (defined below) predicted by the software. Based on this, 5 residues from the N-domain of IGFBP5 and 2 residues from the IGFBP1 that gave the highest number of stable predictions among all the 19 possible substitutions were identified.

Overall G mutation [ G mutation ] –[ G mutation ] *complex uncomplexed*

**1.4.4.1 Energy calculations** 

now established that both N- and C-domains in IGFBPs are involved in binding IGF-1 with the central domain structurally disordered. High-resolution 3D structures are available for the following IGFBP domains in uncomplexed form: (i) N-terminal domain of IGFBP-1, (ii) C-terminal domain of IGFBP-2, (iii) N-terminal domain of IGFBP-5 and (iv) C-terminal domain of IGFBP-6. In IGF-bound form, structures are available for N-terminal domain of IGFBP-4 and -5 and C-terminal domain of IGFBP-1.

The relative affinities of IGFs vary for the different IGFBPs with IGFBP-1,3,4 having higher affinities for IGF-1 compared to IGF-2 and vice-versa for IGFBP-2,5,6 (Kiefer et al., 1992; Roghani et al., 1991). The salient features of these structural interactions are: (i) the individual domains of different IGFBPs are similar in structure with root mean square deviation (RMSD) < 2-3 Å; (ii) the structures of N- and C-domains of IGFBPs in free and in complex with IGFs are similar indicating that the domains do no undergo a significant conformational change upon binding; (iii) there is a cooperativity between the N- and C-domains of IGFBPs in binding IGF (Kuang et al., 2007). That is, binding of IGF-1 to one of the domains enhances its binding to the other domain. This is presumably due to conformation change or stabilization of IGF-1 upon binding to one domain, which renders its conformation suitable for binding the other domain; (iv) the individual domains bind IGFs with much lower affinity than the full-length protein; (v) the IGF-receptor binding sites of IGFs are masked upon binding IGFBPs. This explains why IGFs do not bind the receptor in IGFBP bound form; (vi) upon binding IGFBP the structurally flexible or disordered regions of IGFs are stabilized. Figure 4 illustrates the mode of IGF binding to IGFBP along with structures of N- and C-domains of IGFBP-5 and IGFBP-1, respectively, in complexed and uncomplexed forms.

Fig. 4. Three-dimensional structures of IGFBPs in complexed and uncomplexed forms. (a) A ternary complex consisting of the N-domain of IGFBP-4 (orange), C-domain of IGFBP-4 (grey) bound to IGF-1 (light blue). The two domains clasp IGF-1 binding it tightly and blocking its interaction with IGF-1 receptor. (b) Superimposition of 3D the structures of the N-terminal domain of IGFBP-5 in complex and uncomplexed form (RMSD = 1.9Å) and (c) Superimposition of the 3D structures of the C-terminal domain of IGFBP-1 in complex and uncomplexed form (RMSD = 1.3 Å). The low RMSD values indicate that the conformation of IGFBPs do not change significantly upon binding IGF-1.

#### **1.4.4 Improving IGF-IGFBP interaction**

One of the goals of our work is to engineer IGFBPs in order to improve their IGF binding affinity. This will be useful in therapeutics discussed above, which involves the administration of IGF-IGFBP complex rather than free IGF-1 alone. Towards this end, we have carried out structure-based thermodynamic studies of IGFBP in complex with IGF-1 to

now established that both N- and C-domains in IGFBPs are involved in binding IGF-1 with the central domain structurally disordered. High-resolution 3D structures are available for the following IGFBP domains in uncomplexed form: (i) N-terminal domain of IGFBP-1, (ii) C-terminal domain of IGFBP-2, (iii) N-terminal domain of IGFBP-5 and (iv) C-terminal domain of IGFBP-6. In IGF-bound form, structures are available for N-terminal domain of

The relative affinities of IGFs vary for the different IGFBPs with IGFBP-1,3,4 having higher affinities for IGF-1 compared to IGF-2 and vice-versa for IGFBP-2,5,6 (Kiefer et al., 1992; Roghani et al., 1991). The salient features of these structural interactions are: (i) the individual domains of different IGFBPs are similar in structure with root mean square deviation (RMSD) < 2-3 Å; (ii) the structures of N- and C-domains of IGFBPs in free and in complex with IGFs are similar indicating that the domains do no undergo a significant conformational change upon binding; (iii) there is a cooperativity between the N- and C-domains of IGFBPs in binding IGF (Kuang et al., 2007). That is, binding of IGF-1 to one of the domains enhances its binding to the other domain. This is presumably due to conformation change or stabilization of IGF-1 upon binding to one domain, which renders its conformation suitable for binding the other domain; (iv) the individual domains bind IGFs with much lower affinity than the full-length protein; (v) the IGF-receptor binding sites of IGFs are masked upon binding IGFBPs. This explains why IGFs do not bind the receptor in IGFBP bound form; (vi) upon binding IGFBP the structurally flexible or disordered regions of IGFs are stabilized. Figure 4 illustrates the mode of IGF binding to IGFBP along with structures of N- and C-domains of IGFBP-5 and IGFBP-1,

Fig. 4. Three-dimensional structures of IGFBPs in complexed and uncomplexed forms. (a) A ternary complex consisting of the N-domain of IGFBP-4 (orange), C-domain of IGFBP-4 (grey) bound to IGF-1 (light blue). The two domains clasp IGF-1 binding it tightly and blocking its interaction with IGF-1 receptor. (b) Superimposition of 3D the structures of the N-terminal domain of IGFBP-5 in complex and uncomplexed form (RMSD = 1.9Å) and (c) Superimposition of the 3D structures of the C-terminal domain of IGFBP-1 in complex and uncomplexed form (RMSD = 1.3 Å). The low RMSD values indicate that the conformation of

One of the goals of our work is to engineer IGFBPs in order to improve their IGF binding affinity. This will be useful in therapeutics discussed above, which involves the administration of IGF-IGFBP complex rather than free IGF-1 alone. Towards this end, we have carried out structure-based thermodynamic studies of IGFBP in complex with IGF-1 to

IGFBP-4 and -5 and C-terminal domain of IGFBP-1.

respectively, in complexed and uncomplexed forms.

(a (b (c

IGFBPs do not change significantly upon binding IGF-1.

**1.4.4 Improving IGF-IGFBP interaction** 

evaluate the extent of change in stability of the protein complex upon mutation of key residues in IGFBP. The residues chosen were those that have been verified experimentally to be involved in binding IGFs. A large body of work has been carried out in the past wherein different site-specific mutants, deletion mutants and/or truncated forms of the IGFBPs have been tested for their IGF-1 binding activities (Clemmons, 2001). Many of these residues are known to be conserved across all six IGFBPs. With this information in hand, our objective is to map on the 3D structures of IGFBP mutations that are known to destabilize IGF-IGFBP interactions.

In recent years, computational methods have been proposed to design specific mutants with enhanced ligand binding affinity (Sammond et al., 2007). These are structure-based methods that systematically predict single mutations at protein-protein interfaces which enhance binding affinities. This is based on the hypothesis that increasing the buried hydrophobic surface area or reducing buried hydrophilic surface area leads to enhanced affinity if steric clashes are avoided and all polar groups buried in the core of the protein have a hydrogen bond partner. In the 3D structures of IGFBPs in complex with IGF-1, we mutated residues, which are known to be involved in binding IGF-1, and evaluated the resulting change in thermodynamic stability (via free-energy change). We observed that mutation of residues important for binding IGF-1 increases the free energy of the complex resulting in the destabilization and weakening of IGF-IGFBP interaction. This implies that thermodynamic stability of the IGF-IGFBP complex can be used as an indicator of IGF-1 binding affinity. The study was carried out in 3 stages: first, the change in free energy of the IGF-IGFBP complex upon mutation of residues in IGFBP was predicted. This was carried out using the program I-MUTANT (Capriotti et al., 2005). Next, based on these results, the structure of the complex containing residues in IGFBP that resulted in lowering or increasing the stability of the complex was structurally modeled using the software ROSETTA-DOCK (Lyskov & Gray, 2008). Finally, the modeled structures were subjected to energy minimization followed by 10 ns MD simulations using the program GROMACS (Van der Spoel et al., 2005). In order to understand the structural basis of increased or decreased thermodynamic stability of the IGF-IGFBP complex, the hydrophobic and polar surface areas accessible were evaluated using the program NACCSESS (Hubbard et al.,1993). Two protein complexes of IGFBP with IGF-1 and two uncomplexed forms of the same protein were used for the analysis namely, IGFBP5 N- terminal and IGFBP1 C-terminal having PDB ID 1H59, 1BOE (representing the complexed form) and 2DSQ, 1ZT3 (representing the uncomplexed form), respectively. These two proteins were chosen due to the fact that high-resolution structures of IGF-bound and unbound forms are currently only available for these proteins.

#### **1.4.4.1 Energy calculations**

Using I-MUTANT 2.0, each residue within the predicted binding sites and conserved regions of N- and C-domain of IGFBP-4 and IGFBP-1, respectively, was mutated to the 19 other possible amino acids at that position and the change in free energy for each mutation was identified based on the G value (defined below) predicted by the software. Based on this, 5 residues from the N-domain of IGFBP5 and 2 residues from the IGFBP1 that gave the highest number of stable predictions among all the 19 possible substitutions were identified. The free energy calculations were done as follows:

Overall G mutation [ G mutation ] –[ G mutation ] *complex uncomplexed*

Insulin-Like Growth Factor System in HIV/AIDS:

IGFBP complex.

IGF-IGFBP complex.

A Structure Based Approach to the Design of New Therapeutics 135

Figure 6 illustrates mutation of residue G-57 of the N-domain of IGFBP-5, resulting in enhancement of binding with IGF. Thus, if carried out this mutation will strengthen the IGF-

Fig. 6. Thermodynamic analysis of mutations in IGFBP which enhance IGF-IGFBP

**1.4.4.2 Structural basis for increase/decrease IGF-binding affinity in mutant IGFBP** 

Exposed hydrophobic surface area at interface Exposed hydrophobic surface area of IGF 1 Exposed hydrophobic surface area of IGFBP –

 

interactions. The G (mutation) values (defined above) are shown for G57 of IGFBP-5. Its mutation to several of the other 19 amino acid types results in increased stabilization of the

In order to understand the structural basis of our findings above, the structure of IGF-IGFBP complex containing the mutations (L70Q and G57K) were constructed using ROSETTA-DOCK software and subjected to energy minimization and MD simulations. The solvent accessible areas of hydrophobic residues at the interface were then evaluated as follows:

Exposed hydrophobic surface area of IGF IGFBP Complex

 In general, a large exposure of hydrophobic surface area in the binding interface of two proteins (that is, an increase in solvent accessibility of non-polar residues) is known to result in destabilization of protein interactions (Jones et al., 2008; Sammond et al., 2007; Vallone et al., 1998). On the other hand, decreases in hydrophobic surface area at the binding interface upon mutation indicates that the complex formed is more stable than the wild type. Figure 7

G (mutation) is in general the free energy change of a given structure (complexed or uncomplexed) upon mutation of a given residue. The value of G (mutation) either in complex or uncomplexed form (indicated in the subscript above) was obtained from I-MUTANT 2.0 by specifying the desired residue to be mutated. Thus, the overall G (mutation) for a desired mutation depends on the free energies of both complexed and uncomplexed forms. A positive value of the overall G (mutation) indicates that the particular mutation stabilizes the complex, whereas a negative value for the overall G (mutation) indicates de-stabilization.

Figure 5 shows an example of two mutations (one in the N-domain of IGFBP-5 and the second in the C-domain of IGFBP-1), which show de-stabilization upon mutation to any of the other 19 amino acids. This indicates that these residues are very important for binding and mutation of these residues lowers IGF-binding affinity. The lowering of binding strengths upon mutation of these residues has been verified experimentally in the past. For instance L-70 was mutated to Glu in one study (Imai et al., 2000) and C226 was mutated to Tyr in another study (Brinkman et al., 1991). Since cysteines are highly conserved across IGFBPs and are involved in extensive intra-molecular disulphide bonds, their mutation to any other amino acid causes a reduction of IGF-1 binding. This is corroborated by the thermodynamics analysis presented here.

Fig. 5. Thermodynamic analysis of mutations in IGFBP which cause de-stabilization or weakening of the IGF-IGFBP complex. The G (mutation) values (defined above) are shown for two residues: L-70 of IGFBP-5 and C-226 of IGFBP-5 which are highly conserved across all IGFBPs and involved in binding IGF-1. Their mutation to any of the other 19 amino acid types results in de-stabilization of the interaction of IGFBP with IGF-1. Thus, these residues are important for binding IGF-1.

G (mutation) is in general the free energy change of a given structure (complexed or uncomplexed) upon mutation of a given residue. The value of G (mutation) either in complex or uncomplexed form (indicated in the subscript above) was obtained from I-MUTANT 2.0 by specifying the desired residue to be mutated. Thus, the overall G (mutation) for a desired mutation depends on the free energies of both complexed and uncomplexed forms. A positive value of the overall G (mutation) indicates that the particular mutation stabilizes the complex, whereas a negative value for the overall G

Figure 5 shows an example of two mutations (one in the N-domain of IGFBP-5 and the second in the C-domain of IGFBP-1), which show de-stabilization upon mutation to any of the other 19 amino acids. This indicates that these residues are very important for binding and mutation of these residues lowers IGF-binding affinity. The lowering of binding strengths upon mutation of these residues has been verified experimentally in the past. For instance L-70 was mutated to Glu in one study (Imai et al., 2000) and C226 was mutated to Tyr in another study (Brinkman et al., 1991). Since cysteines are highly conserved across IGFBPs and are involved in extensive intra-molecular disulphide bonds, their mutation to any other amino acid causes a reduction of IGF-1 binding. This is corroborated by the

Fig. 5. Thermodynamic analysis of mutations in IGFBP which cause de-stabilization or weakening of the IGF-IGFBP complex. The G (mutation) values (defined above) are shown for two residues: L-70 of IGFBP-5 and C-226 of IGFBP-5 which are highly conserved across all IGFBPs and involved in binding IGF-1. Their mutation to any of the other 19 amino acid types results in de-stabilization of the interaction of IGFBP with IGF-1. Thus,

(mutation) indicates de-stabilization.

thermodynamics analysis presented here.

these residues are important for binding IGF-1.

Figure 6 illustrates mutation of residue G-57 of the N-domain of IGFBP-5, resulting in enhancement of binding with IGF. Thus, if carried out this mutation will strengthen the IGF-IGFBP complex.

Fig. 6. Thermodynamic analysis of mutations in IGFBP which enhance IGF-IGFBP interactions. The G (mutation) values (defined above) are shown for G57 of IGFBP-5. Its mutation to several of the other 19 amino acid types results in increased stabilization of the IGF-IGFBP complex.

#### **1.4.4.2 Structural basis for increase/decrease IGF-binding affinity in mutant IGFBP**

In order to understand the structural basis of our findings above, the structure of IGF-IGFBP complex containing the mutations (L70Q and G57K) were constructed using ROSETTA-DOCK software and subjected to energy minimization and MD simulations. The solvent accessible areas of hydrophobic residues at the interface were then evaluated as follows:

> Exposed hydrophobic surface area at interface Exposed hydrophobic surface area of IGF 1 Exposed hydrophobic surface area of IGFBP – Exposed hydrophobic surface area of IGF IGFBP Complex

In general, a large exposure of hydrophobic surface area in the binding interface of two proteins (that is, an increase in solvent accessibility of non-polar residues) is known to result in destabilization of protein interactions (Jones et al., 2008; Sammond et al., 2007; Vallone et al., 1998). On the other hand, decreases in hydrophobic surface area at the binding interface upon mutation indicates that the complex formed is more stable than the wild type. Figure 7

Insulin-Like Growth Factor System in HIV/AIDS:

light at the end of tunnel is getting brighter.

**3. Acknowledgements** 

of Science, Bangalore.

994, ISSN 0888-8809

W306-W310, ISSN 0305-1048

**4. References** 

A Structure Based Approach to the Design of New Therapeutics 137

various stages of the disease with different manifestations. The IGF system in general has been extensively studied in this context. While the system is complex in nature with many different proteins interacting to form a regulatory network, the key players have been the IGFs themselves and their binding proteins, the IGFBPs. The recent finding that administration of IGF-1: IGFBP-3 complexes improves whole body glucose uptake is a promising step towards treatment of HIV-associated wasting. In this context, it is important to understand the structural basis of IGF-IGFBP interactions in general, which has been elusive due to the difficulty in producing functional human IGFBPs in large quantities. In our laboratory, we have been successful for the first time in producing bacterially expressed human IGFBP-2 in soluble, functional and monomeric form. This opens up new avenues to study IGF-IGFBP interactions at the atomic level. Further, human IGFBP-2 antibodies can now be generated and used for detection of IGFBP-2 in HIV patients. High-resolution structural studies of IGF in complex with IGFBP will help us to design improved IGFBP species with improve interactions and enhanced binding affinity. All six IGFBPs in the human body are similar in nature as far as their interaction with IGF is concerned. Thus, findings from one IGFBP may be extended to other IGFBPs as well. The available structures of individual domains of IGFBPs are thus very helpful and will aid in future unraveling in detail, of the modes of the interaction of the full length proteins with the IGFs. Efforts are underway in this direction in our laboratory. While much work still needs to be done, the

The facilities provided by NMR Research Centre at IISc supported by Department of Science and Technology (DST), India is gratefully acknowledged. HSA acknowledges support from DST-SERC research award. MS acknowledges fellowship from Council of Scientific and Industrial Research (CSIR) This project is in collaboration with Prof. S. A. Rosenzweig, Medical University of South Carolina, USA and Prof. P. Kondaiah, MRDG, Indian Institute

Brinkman, A.; Kortleve, D. J.; Zwarthoff, E. C. & Drop, S. L. S. (1991). Mutations in the c-

Bunn, R. C. & Fowlkes, J. L. (2003). Insulin-like growth factor binding protein proteolysis. *Trends in Endocrinology and Metabolism,* Vol. 14, pp. 176-181, ISSN 1043-2760 Capriotti, E.; Fariselli, P. & Casadio, R. (2005). I-Mutant2.0: predicting stability changes upon

Chan, J. M.; Stampfer, M. J.; Giovannucci, E.; Gann, P. H.; Ma, J.; Wilkinson, P.; Hennekens,

risk: A prospective study. *Science,* Vol. 279, pp. 563-566, ISSN 0036-8075 Chantry, C. J.; Hughes, M. D.; Alvero, C.; Cervia, J. S.; Hodge, J.; Borum, P.; Moye, J. &

terminal part of insulin-like growth factor (IGF)-binding protein-1 result in dimer formation and loss of IGF binding capacity. *Molecular Endocrinology,* Vol. 5, pp. 987-

mutation from the protein sequence or structure. *Nucleic Acids Research,* Vol. 33, pp.

C. H. & Pollak, M. (1998). Plasma insulin-like growth factor I and prostate cancer

Team, P. (2008). Insulin-like growth factor-1 and lean body mass in HIV-infected

shows the result of analyzing the solvent accessible areas of the two mutations described above.

Fig. 7. The change in hydrophobic surface area of the IGF-IGFBP-5 interfaces upon mutation. G57K is a stabilizing mutation for the IGF-IGFBP-5 complex due to a large decrease in non-polar surface area at the interface for IGF-1. On the other hand, the L70Q mutation renders the IGF-IGFBP complex unstable due to a large exposure of hydrophobic surface area in IGFBP-5.

In the case of L70Q mutation, the exposed hydrophobic surface areas of both IGFBP-5 and IGF-1 are increased at the interface. This together results in weakening of the complex and hence the IGF-binding affinity of IGFBP-5 is reduced. In the case of G57K, the exposed surface area of IGF-1 at the interface is reduced considerably while that of IGFBP-5 increases slightly. Significantly, the overall change in surface areas is favorable for enhanced binding of IGF-1 with IGFBP-5. This underscores the importance of thermodynamic studies in evaluating ligand binding affinities in this class of proteins.

## **2. Conclusion**

AIDS is a debilitating disease with more than 25 million people having succumbed since the start of the epidimic. In order to combat this disease, multiple approaches have been proposed and needs to be taken. New findings related to diagnosis and disease progression continue to emerge. A key finding in the past decade, which is now well established, is the involvement of the hormonal peptides IGF-1 and IGF-2 and the IGF-binding proteins in various stages of the disease with different manifestations. The IGF system in general has been extensively studied in this context. While the system is complex in nature with many different proteins interacting to form a regulatory network, the key players have been the IGFs themselves and their binding proteins, the IGFBPs. The recent finding that administration of IGF-1: IGFBP-3 complexes improves whole body glucose uptake is a promising step towards treatment of HIV-associated wasting. In this context, it is important

to understand the structural basis of IGF-IGFBP interactions in general, which has been elusive due to the difficulty in producing functional human IGFBPs in large quantities. In our laboratory, we have been successful for the first time in producing bacterially expressed human IGFBP-2 in soluble, functional and monomeric form. This opens up new avenues to study IGF-IGFBP interactions at the atomic level. Further, human IGFBP-2 antibodies can now be generated and used for detection of IGFBP-2 in HIV patients. High-resolution structural studies of IGF in complex with IGFBP will help us to design improved IGFBP species with improve interactions and enhanced binding affinity. All six IGFBPs in the human body are similar in nature as far as their interaction with IGF is concerned. Thus, findings from one IGFBP may be extended to other IGFBPs as well. The available structures of individual domains of IGFBPs are thus very helpful and will aid in future unraveling in detail, of the modes of the interaction of the full length proteins with the IGFs. Efforts are underway in this direction in our laboratory. While much work still needs to be done, the light at the end of tunnel is getting brighter.

## **3. Acknowledgements**

136 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

shows the result of analyzing the solvent accessible areas of the two mutations described

Fig. 7. The change in hydrophobic surface area of the IGF-IGFBP-5 interfaces upon mutation. G57K is a stabilizing mutation for the IGF-IGFBP-5 complex due to a large decrease in non-polar surface area at the interface for IGF-1. On the other hand, the L70Q mutation renders the IGF-IGFBP complex unstable due to a large exposure of hydrophobic

evaluating ligand binding affinities in this class of proteins.

In the case of L70Q mutation, the exposed hydrophobic surface areas of both IGFBP-5 and IGF-1 are increased at the interface. This together results in weakening of the complex and hence the IGF-binding affinity of IGFBP-5 is reduced. In the case of G57K, the exposed surface area of IGF-1 at the interface is reduced considerably while that of IGFBP-5 increases slightly. Significantly, the overall change in surface areas is favorable for enhanced binding of IGF-1 with IGFBP-5. This underscores the importance of thermodynamic studies in

AIDS is a debilitating disease with more than 25 million people having succumbed since the start of the epidimic. In order to combat this disease, multiple approaches have been proposed and needs to be taken. New findings related to diagnosis and disease progression continue to emerge. A key finding in the past decade, which is now well established, is the involvement of the hormonal peptides IGF-1 and IGF-2 and the IGF-binding proteins in

above.

surface area in IGFBP-5.

**2. Conclusion** 

The facilities provided by NMR Research Centre at IISc supported by Department of Science and Technology (DST), India is gratefully acknowledged. HSA acknowledges support from DST-SERC research award. MS acknowledges fellowship from Council of Scientific and Industrial Research (CSIR) This project is in collaboration with Prof. S. A. Rosenzweig, Medical University of South Carolina, USA and Prof. P. Kondaiah, MRDG, Indian Institute of Science, Bangalore.

## **4. References**


Insulin-Like Growth Factor System in HIV/AIDS:

505, ISSN 0167-7799

895X

0167-6806

6558-6572, ISSN 0261-4189

A Structure Based Approach to the Design of New Therapeutics 139

Imai, Y.; Moralez, A.; Andag, U.; Clarke, J. B.; Busby, W. H. & Clemmons, D. R. (2000).

Jones, D. S.; Silverman, A. P. & Cochran, J. R. (2008). Developing therapeutic proteins by

Kalus, W.; Zweckstetter, M.; Renner, C.; Sanchez, Y.; Georgescu, J.; Grol, M.; Demuth, D.;

Kibbey, M. M.; Jameson, M. J.; Eaton, E. M. & Rosenzweig, S. A. (2006). Insulin-like growth

Kiefer, M. C.; Schmid, C.; Waldvogel, M.; Schlapfer, I.; Futo, E.; Masiarz, F. R.; Green, K.;

Krywicki, R. F. & Yee, D. (1992). The insulin-like growth factor family of ligands, receptors,

Kuang, Z. H.; Yao, S. G.; Keizer, D. W.; Wang, C. C.; Bach, L. A.; Forbes, B. E.; Wallace, J. C.

Kuang, Z. H.; Yao, S. G.; McNeil, K. A.; Thompson, J. A.; Bach, L. A.; Forbes, B. E.; Wallace, J.

Lee, P. D. K.; Pivarnik, J. M.; Bukar, J. G.; Muurahainen, N.; Berry, P. S.; Skolnik, P. R.;

LeRoith, D. & Roberts, C. T. (2003). The insulin-like growth factor system and cancer. *Cancer* 

Lieberman, S. A.; Butterfield, G. E.; Harrison, D. & Hoffman, A. R. (1994). Anabolic effects of

Lyskov, S. & Gray, J. J. (2008). The RosettaDock server for local proteinprotein docking.

*Nucleic Acids Research,* Vol. 36, pp. W233-W238, ISSN 0305-1048

recombinant insulin-like growth factor-1 in cachexia patients with the acquiredimmunodeficiency-syndrome. *Journal of Clinical Endocrinology & Metabolism,* Vol. 78,

*of Biological Chemistry,* Vol. 267, pp. 12692-12699, ISSN 0021-9258

*Molecular Biology,* Vol. 364, pp. 690-704, ISSN 0022-2836

46, pp. 13720-13732, ISSN 0006-2960

Vol. 81, pp. 2968-2975, ISSN 0021-972X

pp. 404-410, ISSN 0021-972X

*Letters,* Vol. 195, pp. 127-137, ISSN 0304-3835

*Biological Chemistry,* Vol. 275, pp. 18188-18194, ISSN 0021-9258

Substitutions for hydrophobic amino acids in the N-terminal domains of IGFBP-3 and-5 markedly reduce IGF-I binding and alter their biologic actions. *Journal of* 

engineering ligand-receptor interactions. *Trends in Biotechnology,* Vol. 26, pp. 498-

Schumacher, R.; Dony, C.; Lang, K. & Holak, T. A. (1998). Structure of the IGFbinding domain of the insulin-like growth factor-binding protein-5 (IGFBP-5): Implications for IGF and IGF-I receptor interactions. *EMBO Journal,* Vol. 17, pp.

factor binding protein-2: Contributions of the C-terminal domain to insulin-like growth factor-1 binding. *Molecular Pharmacology,* Vol. 69, pp. 833-845, ISSN 0026-

Barr, P. J. & Zapf, J. (1992). Characterization of recombinant human insulin-like growth factor binding protein-4, protein-5, and protein-6 produced in yeast. *Journal* 

and binding-proteins. *Breast Cancer Research and Treatment,* Vol. 22, pp. 7-19, ISSN

& Norton, R. S. (2006). Structure, dynamics and heparin binding of the C-terminal domain of insulin-like growth factor-binding protein-2 (IGFBP-2). *Journal of* 

C. & Norton, R. S. (2007). Cooperativity of the N- and C-terminal domains of insulin-like growth factor (IGF) binding protein 2 in IGF binding. *Biochemistry,* Vol.

Nerad, J. L.; Kudsk, K. A.; Jackson, L.; Ellis, K. J. & Gesundheit, N. (1996). A randomized, placebo-controlled trial of combined insulin-like growth factor I and low dose growth hormone therapy for wasting associated with human immunodeficiency virus infection. *Journal of Clinical Endocrinology & Metabolism,*

children. *Jaids-Journal of Acquired Immune Deficiency Syndromes,* Vol. 48, pp. 437-443, ISSN 1525-4135


Clemmons, D. R. (2001). Use of mutagenesis to probe IGF-binding protein

Congote, L. F. (2005). Monitoring insulin-like growth factors in HIV infection and AIDS.

Domingo, P.; Gallego-Escuredo, J. M.; Domingo, J. C.; Gutierrez, M. D.; Mateo, M. G.;

Firth, S. M. & Baxter, R. C. (2002). Cellular actions of the insulin-like growth factor binding

Frost, R. A.; Fuhrer, J.; Steigbigel, R.; Mariuz, P.; Lang, C. H. & Gelato, M. C. (1996). Wasting

Gelato, M. C. & Frost, R. A. (1997). IGFBP-3 - Functional and structural implications in aging and wasting syndromes. *Endocrine,* Vol. 7, pp. 81-85, ISSN 0969-711X Gelato, M. C.; Mynarcik, D. & McNurlan, M. A. (2002). Soluble tumour necrosis factor alpha

Hambrecht, R.; Schulze, P. C.; Gielen, S.; Linke, A.; Mobius-Winkler, S.; Yu, J. T.; Kratzsch, J.;

Harrison, L. E.; Blumberg, D.; Berman, R.; Ng, B.; Hochwald, S.; Brennan, M. F. & Burt, M.

Helle, S. I.; Ueland, T.; Ekse, D.; Froland, S. S.; Holly, J. M. P.; Lonning, P. E. & Aukrust, P.

Hubbard, S. J. & Thoronton, J. M. (1993). Computer program, Department of Biochemistry

and Molecular biology, University College London

proteins. *Endocrine Reviews,* Vol. 23, pp. 824-854, ISSN 0163-769X

*Clinica Chimica Acta,* Vol. 361, pp. 30-53, ISSN 0009-8981

Vol. 7, pp. 299-310, ISSN 1464-2662

39, pp. 1175-1181, ISSN 0735-1097

227-233, ISSN 0021-972X

514, ISSN 0300-0664

1525-4135

0022-4804

ISSN 1525-4135

769X

children. *Jaids-Journal of Acquired Immune Deficiency Syndromes,* Vol. 48, pp. 437-443,

structure/function relationships. *Endocrine Reviews,* Vol. 22, pp. 800-817, ISSN 0163-

Fernandez, I.; Vidal, F.; Giralt, M. & Villarroya, F. (2010). Serum FGF21 levels are elevated in association with lipodystrophy, insulin resistance and biomarkers of liver injury in HIV-1-infected patients. *Aids,* Vol. 24, pp. 2629-2637, ISSN 0269-9370 Dudgeon, W. D.; Phillips, K. D.; Carson, J. A.; Brewer, R. B.; Durstine, J. L. & Hand, G. A.

(2006). Counteracting muscle wasting in HIV-infected individuals. *Hiv Medicine,*

in the acquired immune deficiency syndrome is associated with multiple defects in the serum insulin-like growth factor system. *Clinical Endocrinology,* Vol. 44, pp. 501-

receptor 2, a serum marker of resistance to the anabolic actions of growth hormone in subjects with HIV disease. *Clinical Science,* Vol. 102, pp. 85-90, ISSN 0143-5221 Grunfeld, C.; Thompson, M.; Brown, S. J.; Richmond, G.; Lee, D.; Muurahainen, N. & Kotler,

D. P. (2007). Recombinant human growth hormone to treat HIV-associated adipose redistribution syndrome - 12-Week induction and 24-week maintenance therapy. *Jaids-Journal of Acquired Immune Deficiency Syndromes,* Vol. 45, pp. 286-297, ISSN

Baldauf, G.; Busse, M. W.; Schubert, A.; Adams, V. & Schuler, G. (2002). Reduction of insulin-like growth factor-1 expression in the skeletal muscle of noncachectic patients with chronic heart failure. *Journal of the American College of Cardiology,* Vol.

(1996). Effect of human growth hormone on human pancreatic carcinoma growth, protein, and cell cycle kinetics. *Journal of Surgical Research,* Vol. 61, pp. 317-322, ISSN

(2001). The insulin-like growth factor system in human immunodeficiency virus infection: Relations to immunological parameters, disease progression, and antiretroviral therapy. *Journal of Clinical Endocrinology & Metabolism,* Vol. 86, pp.


Insulin-Like Growth Factor System in HIV/AIDS:

0022-2836

2126

A Structure Based Approach to the Design of New Therapeutics 141

Sammond, D. W.; Eletr, Z. M.; Purbeck, C.; Kimple, R. J.; Siderovski, D. P. & Kuhlman, B.

Sitar, T.; Popowicz, G. M.; Siwanowicz, I.; Huber, R. & Holak, T. A. (2006). Structural basis

Siwanowicz, I.; Popowicz, G. M.; Wisniewska, M.; Huber, R.; Kuenkele, K. P.; Lang, K.;

Smurzynski, M.; Wu, K. L.; Benson, C. A.; Bosch, R. J.; Collier, A. C. & Koletar, S. L. (2010).

Soler-Garcia, A. A.; Johnson, D.; Hathout, Y. & Ray, P. E. (2009). Iron-Related Proteins:

Stagi, S.; Bindi, G.; Galluzzi, F.; Galli, L.; Salti, R. & de Martino, M. (2004). Changed bone

Swain, M.; Slomiany, M. G.; Rosenzweig, S. A. & Atreya, H. S. (2010a). High-yield bacterial

Swain, M.; Thirupathi, R.; Krishnarjuna, B.; Eaton, E. M.; Kibbey, M. M.; Rosenzweig, S. A. &

Vallone, B.; Miele, A. E.; Vecchini, P.; Chiancone, E. & Brunori, M. (1998). Free energy of

Van der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E. & Berendsen, H. J. C.

*Chemistry,* Vol. 280, pp. 29812-29819, ISSN 0021-9258

*America,* Vol. 103, pp. 13028-13033, ISSN 0027-8424

*E Metabologia,* Vol. 52, pp. 818-832, ISSN 0004-2730

Vol. 55, pp. 117-127, ISSN 1525-4135

699, ISSN 0300-0664

195-200, ISSN 0003-9861

pp. 6103-6107, ISSN 0027-8424

26, pp. 1701-1718, ISSN 0192-8651

7345

factor-binding protein-1 isolated from human amniotic fluid. *Journal of Biological* 

(2007). Structure-based protocol for identifying mutations that enhance proteinprotein binding affinities. *Journal of Molecular Biology,* Vol. 371, pp. 1392-1404, ISSN

for the inhibition of insulin-like growth factors by insulin-like growth factorbinding proteins. *Proceedings of the National Academy of Sciences of the United States of* 

Engh, R. A. & Holak, T. A. (2005). Structural basis for the regulation of insulin-like growth factors by IGF binding proteins. *Structure,* Vol. 13, pp. 155-167, ISSN 0969-

Relationship Between CD4(+) T-Cell Counts/HIV-1 RNA Plasma Viral Load and AIDS-Defining Events Among Persons Followed in the ACTG Longitudinal Linked Randomized Trials Study. *Jaids-Journal of Acquired Immune Deficiency Syndromes,*

Candidate Urine Biomarkers in Childhood HIV-Associated Renal Diseases. *Clinical Journal of the American Society of Nephrology,* Vol. 4, pp. 763-771, ISSN 1555-9041 Spinola-Castro, A. M.; Siviero-Miachon, A. A.; Da Silva, M. T. N. & Guerra-Junior, G. (2008).

The use of growth hormone to treat endocrine-metabolic disturbances in Acquired Immunodeficiency Syndrome (Aids) patients. *Arquivos Brasileiros De Endocrinologia* 

status in human immunodeficiency virus type 1 (HIV-1) perinatally infected children is related to low serum free IGF-1. *Clinical Endocrinology,* Vol. 61, pp. 692-

expression and structural characterization of recombinant human insulin-like growth factor binding protein-2. *Archives of Biochemistry and Biophysics,* Vol. 501, pp.

Atreya, H. S. (2010b). Spontaneous and reversible self-assembly of a polypeptide fragment of insulin-like growth factor binding protein-2 into fluorescent nanotubular structures. *Chemical Communications,* Vol. 46, pp. 216-218, ISSN 1359-

burying hydrophobic residues in the interface between protein subunits. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 95,

(2005). GROMACS: Fast, flexible, and free. *Journal of Computational Chemistry,* Vol.


Ma, J.; Pollak, M. N.; Giovannucci, E.; Chan, J. M.; Tao, Y. Z.; Hennekens, C. H. & Stampfer,

Meininger, G. & Grinspoon, S. (2001). Regulation of the growth hormone/insulin-like

Miyamoto, S.; Yano, K.; Sugimoto, S.; Ishii, G.; Hasebe, T.; Endoh, Y.; Kodama, K.; Goya, M.;

Mynarcik, D. C.; Frost, R. A.; Lang, C. H.; DeCristofaro, K.; McNurlan, M. A.; Garlick, P. J.;

Nguyen, B. Y.; Clerici, M.; Venzon, D. J.; Bauza, S.; Murphy, W. J.; Longo, D. L.; Baseler, M.;

Padilla, S.; Masia, M.; Garcia, N.; Jarrin, I.; Tormo, C. & Gutierrez, F. (2011). Early changes in

Rao, M. N.; Mulligan, K.; Tai, V.; Wen, M. J.; Dyachenko, A.; Weinberg, M.; Li, X. J.; Lang, T.;

Roghani, M.; Lassarre, C.; Zapf, J.; Povoa, G. & Binoux, M. (1991). 2 Insulin-like growth-

Rosenzweig, S. A. (2004). What's new in the IGF-binding proteins? *Growth Hormone & Igf* 

Rosenzweig, S. A. & Atreya, H. S. (2010). Defining the pathway to insulin-like growth factor

Sala, A.; Capaldi, S.; Campagnoli, M.; Faggion, B.; Labo, S.; Perduca, M.; Romano, A.;

*Metabolism,* Vol. 95, pp. 4361-4366, ISSN 0021-972X

Vol. 73, pp. 658-666, ISSN 0021-972X

*Research,* Vol. 14, pp. 329-336, ISSN 1096-6374

*Cancer Institute,* Vol. 91, pp. 620-625, ISSN 0027-8874

Vol. 85, pp. 151-159, ISSN 0167-5273

2144

ISSN 1077-9450

0269-9370

0006-2952

2334

M. J. (1999). Prospective study of colorectal cancer risk in men and plasma levels of insulin-like growth factor (IGF)-I and IGF-binding protein-3. *Journal of the National* 

growth factor-I axis in HIV disease. *Endocrinologist,* Vol. 11, pp. 188-195, ISSN 1051-

Chiba, T. & Ochiai, A. (2004). Matrix metalloproteinase-7 facilitates insulin-like growth factor bioavailability through its proteinase activity on insulin-like growth factor binding protein 3. *Cancer Research,* Vol. 64, pp. 665-671, ISSN 0008-5472 Mulligan, K. & Schambelan, M. (2002). Anabolic treatment with GH, IGF-I, or anabolic

steroids in patients with HIV-associated wasting. *International Journal of Cardiology,*

Steigbigel, R. T.; Fuhrer, J.; Ahnn, S. & Gelato, M. C. (1999). Insulin-like growth factor system in patients with HIV infection: Effect of exogenous growth hormone administration. *Journal of Acquired Immune Deficiency Syndromes,* Vol. 22, pp. 49-55,

Gesundheit, N.; Broder, S.; Shearer, G. & Yarchoan, R. (1998). Pilot study of the immunologic effects of recombinant human growth hormone and recombinant insulin-like growth factor in HIV-infected patients. *Aids,* Vol. 12, pp. 895-904, ISSN

inflammatory and pro-thrombotic biomarkers in patients initiating antiretroviral therapy with abacavir or tenofovir. *Bmc Infectious Diseases,* Vol. 11, pp. ISSN 1471-

Grunfeld, C.; Schwarz, J. M. & Schambelan, M. (2010). Effects of Insulin-Like Growth Factor (IGF)-I/IGF-Binding Protein-3 Treatment on Glucose Metabolism and Fat Distribution in Human Immunodeficiency Virus-Infected Patients with Abdominal Obesity and Insulin Resistance. *Journal of Clinical Endocrinology &* 

factor (IGF)-binding proteins are responsible for the selective affinity for IGF-II if cerebrospinal-fluid binding-protein. *Journal of Clinical Endocrinology & Metabolism,*

system targeting in cancer. *Biochemical Pharmacology,* Vol. 80, pp. 1115-1124, ISSN

Carrizo, M. E.; Valli, M.; Visai, L.; Minchiotti, L.; Galliano, M. & Monaco, H. L. (2005). Structure and properties of the C-terminal domain of insulin-like growth factor-binding protein-1 isolated from human amniotic fluid. *Journal of Biological Chemistry,* Vol. 280, pp. 29812-29819, ISSN 0021-9258


**6** 

*Canada* 

**Cellular Restriction Factors: Exploiting the** 

Jenna N. Kelly, Jessica G.K. Tong, Clayton J. Hattlmann,

Matthew W. Woods and Stephen D. Barr *The University of Western Ontario London,* 

**Body's Antiviral Proteins to Combat HIV-1/AIDS** 

In 1983, when researchers first isolated HIV-1 from an AIDS patient, few imagined that it foretold a worldwide pandemic (Broder & Gallo, 1984, Barre-Sinoussi et al., 1983). More than 25 years later, 65 million people have been infected with HIV-1; nearly half of these people have died of AIDS, and despite many scientific advances we are still without an efficacious vaccine (Merson, 2006). The majority of HIV-1 infections and deaths have occurred in developing countries, with sub-Saharan Africa accounting for over 38 million HIV-1 infections alone. Sadly, the number of new infections currently exceeds our ability to treat everyone infected with the virus, and in the hardest-hit countries the social and

After HIV-1 was isolated, a blood test to screen patients and the blood supply quickly followed, as did research on its structure and pathogenesis. Many assumed that a vaccine would be available in a few years, and excitement increased further with the licensing of the first effective drug against HIV-1, zidovudine (AZT) (Fischl et al., 1987). However, researchers soon discovered that the virus was highly resilient, and HIV-1 quickly developed resistance to AZT (Poli et al., 1989, Richman et al., 1994). Over the next few years, a number of new antiretroviral drugs were developed that attacked the virus in different ways, and it was at this time that a new approach to therapy ensued. Highly active antiretroviral therapy (HAART) combined three or more different drugs to reduce HIV-1 replication, and significantly improved the prognosis of HIV-1-infected individuals (Richman et al., 2009). However, HAART was not a cure, and many patients were resistant to at least one of the antiretroviral drugs. In addition, the drugs were highly toxic, making

During this time, the vaccine field was also hard at work, trying to develop a safe and effective HIV-1 vaccine. Most initial vaccine approaches focused on the HIV-1 envelope protein (gp120), and aimed to induce an antibody response to gp120. AIDSVAX was the first vaccine candidate of this type, and was developed by a U.S. pharmaceutical company called VaxGen (Flynn et al., 2005, Pitisuttithum et al., 2006). An alternative approach, developed by Merck, aimed to induce a T-cell response to HIV-1 using a recombinant adenovirus vector expressing HIV-1 Gag, Pol and Nef proteins (Shiver et al., 2002). Unfortunately, results from

**1. Introduction** 

**1.1 Overview of HIV-1/AIDS therapies** 

economic backlash has been profound.

adherence to treatment difficult.


## **Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS**

Jenna N. Kelly, Jessica G.K. Tong, Clayton J. Hattlmann, Matthew W. Woods and Stephen D. Barr *The University of Western Ontario London, Canada* 

## **1. Introduction**

142 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Wang, W.; Iresjo, B. M.; Karlsson, L. & Svanberg, E. (2000). Provision of rhIGF-I/IGFBP-3

Wu, X. F.; Zhao, H.; Do, K. A.; Johnson, M. M.; Dong, Q.; Hong, W. K. & Spitz, M. R. (2004).

Yu, H.; Spitz, M. R.; Mistry, J.; Gu, J.; Hong, W. K. & Wu, X. F. (1999). Plasma levels of

Zdanowicz, M. M. & Teichberg, S. (2003). Effects of insulin-like growth factor-1/binding

model. *Clinical Nutrition,* Vol. 19, pp. 127-132, ISSN 0261-5614

*the National Cancer Institute,* Vol. 91, pp. 151-156, ISSN 0027-8874

*Research,* Vol. 10, pp. 3988-3995, ISSN 1078-0432

228, pp. 891-897, ISSN 1535-3702

complex attenuated development of cancer cachexia in an experimental tumor

Serum levels of insulin growth factor (IGF-I) and IGF-binding protein predict risk of second primary tumors in patients with head and neck cancer. *Clinical Cancer* 

insulin-like growth factor-I and lung cancer risk: a case-control analysis. *Journal of* 

protein-3 complex on muscle atrophy in rats. *Experimental Biology and Medicine,* Vol.

## **1.1 Overview of HIV-1/AIDS therapies**

In 1983, when researchers first isolated HIV-1 from an AIDS patient, few imagined that it foretold a worldwide pandemic (Broder & Gallo, 1984, Barre-Sinoussi et al., 1983). More than 25 years later, 65 million people have been infected with HIV-1; nearly half of these people have died of AIDS, and despite many scientific advances we are still without an efficacious vaccine (Merson, 2006). The majority of HIV-1 infections and deaths have occurred in developing countries, with sub-Saharan Africa accounting for over 38 million HIV-1 infections alone. Sadly, the number of new infections currently exceeds our ability to treat everyone infected with the virus, and in the hardest-hit countries the social and economic backlash has been profound.

After HIV-1 was isolated, a blood test to screen patients and the blood supply quickly followed, as did research on its structure and pathogenesis. Many assumed that a vaccine would be available in a few years, and excitement increased further with the licensing of the first effective drug against HIV-1, zidovudine (AZT) (Fischl et al., 1987). However, researchers soon discovered that the virus was highly resilient, and HIV-1 quickly developed resistance to AZT (Poli et al., 1989, Richman et al., 1994). Over the next few years, a number of new antiretroviral drugs were developed that attacked the virus in different ways, and it was at this time that a new approach to therapy ensued. Highly active antiretroviral therapy (HAART) combined three or more different drugs to reduce HIV-1 replication, and significantly improved the prognosis of HIV-1-infected individuals (Richman et al., 2009). However, HAART was not a cure, and many patients were resistant to at least one of the antiretroviral drugs. In addition, the drugs were highly toxic, making adherence to treatment difficult.

During this time, the vaccine field was also hard at work, trying to develop a safe and effective HIV-1 vaccine. Most initial vaccine approaches focused on the HIV-1 envelope protein (gp120), and aimed to induce an antibody response to gp120. AIDSVAX was the first vaccine candidate of this type, and was developed by a U.S. pharmaceutical company called VaxGen (Flynn et al., 2005, Pitisuttithum et al., 2006). An alternative approach, developed by Merck, aimed to induce a T-cell response to HIV-1 using a recombinant adenovirus vector expressing HIV-1 Gag, Pol and Nef proteins (Shiver et al., 2002). Unfortunately, results from

Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 145

Since these discoveries, several new cellular restriction factors have been identified, including a number of factors that restrict HIV-1 replication (Chakrabarti & Simon, 2010). Many of these restriction factors are up-regulated in response to type I interferons (IFNs), which are typically activated in the presence of viruses by pattern recognition receptors (PRRs), such as Toll-like receptors or retinoic acid induced gene (RIG)-like receptors (Kumagai et al., 2008). Following secretion, type I IFNs bind to the interferon α/β receptor (IFNAR) on the cell surface and induce signalling through the Janus Kinases/Signal Transducers and Activators of Transcription (Jak/Stat) pathway. This leads to the activation of hundreds of IFN-responsive genes that restrict viral replication, including cellular

Type I IFNs potently inhibit early and late stages of the HIV-1 lifecycle, and systemic administration of IFNα reduces HIV-1 plasma viremia *in vivo* (Meylan et al., 1993, Tavel et al., 2010). During viral infection, plasmacytoid dendritic cells (pDCs) are the main producers of IFNα, however the capacity of pDCs to produce IFNα is impaired during acute HIV-1 infection, and this DC subtype appears to be depleted in chronic HIV-1 infection (Borrow & Bhardwaj, 2008, Soumelis et al., 2001). In addition, the HIV-1 accessory proteins Vpr and Vif can degrade interferon regulatory factor 3 (IRF-3), which plays a critical role in type I IFN induction (Okumura et al., 2008). Since cellular restriction factors are the 'effector' proteins of the IFN response, differences in the ability of pDCs to produce IFNα may contribute to differences in HIV-1 replication and disease progression among patients. Understanding the molecular mechanisms behind these HIV-1 restriction factors, may lead to the development

Fig. 2. The interferon response. Interferon (IFN) binds to the interferon receptor at the plasma membrane, activating the Janus Kinases/Signal Transducers and Activators of Transcription (Jak/Stat) pathway. This results in the up-regulation of hundreds of IFNresponsive genes, including cellular restriction factors that prevent HIV-1 replication.

The HIV-1 lifecycle offers a multitude of steps that can be targeted by cellular HIV-1 restriction factors (Figure 3). The lifecycle begins when the HIV-1 envelope protein (gp120) binds to the host cell via its CD4 receptor, and following a conformational change, it binds to either the CXCR4 or CCR5 chemokine co-receptor (Deng et al., 1996). This interaction facilitates viral and cell membrane fusion, which is followed by the release of the viral core into the cell cytoplasm. In the cytoplasm, the HIV-1 capsid protein is lost in a process called uncoating, and the single-stranded RNA genome is reverse-transcribed into doublestranded cDNA. Reverse transcription is carried out by the virion-associated reverse

restriction factors (Baum & Garcia-Sastre, 2010) (Figure 2).

of drugs that mimic or promote their activities.

**1.3 The HIV-1 lifecycle** 

both of these trials were disappointing, and neither approach provided protection from HIV-1 infection (Buchbinder et al., 2008, McElrath et al., 2008). Moreover, the Merck vaccine actually seemed to suppress the immune response, due to pre-existing immunity to the adenovirus vector (Priddy et al., 2008, Roberts et al., 2006).

The third and largest trial was recently performed in Thailand, and aimed to induce both a T-cell and antibody response to HIV-1. In the study, 16,000 Thai men and women received either placebo or vaccine injections, and were subsequently monitored for HIV-1 infection over a 3 year period (Rerks-Ngarm et al., 2009). The vaccine group received injections of a recombinant canarypox vector vaccine ALVAC-HIV (Sanofi Pasteur), plus booster injections of a recombinant gp120 subunit vaccine AIDSVAX B/E (Global Solutions for Infectious Diseases). Results from this trial showed a modest benefit among vaccine recipients, with a vaccine efficacy of 26-30%. However, vaccination did not affect the levels of viremia or CD4+ T cell counts of infected individuals, and many were disappointed with the results (Rerks-Ngarm et al., 2009).

Over the last decade, several host proteins have been identified that are capable of inhibiting HIV-1 replication (Figure 1). These so-called 'cellular-restriction factors' are a new arm of the innate immune system, and inhibit stages of the HIV-1 lifecycle that are not targeted by current AIDS therapies. Our understanding of how cellular restriction factors target HIV-1 replication is far from compete, but research in this area may provide a new avenue for future AIDS therapies (Barr, 2010).

Fig. 1. HIV-1/AIDS timeline. Timeline documenting major events in the HIV-1/AIDS pandemic, as well as the identification of several key HIV-1 cellular restriction factors.

#### **1.2 Cellular restriction factors**

Cellular restriction factors are host proteins that can inhibit specific steps in the lifecycle of a virus. The concept of cellular restriction factors first emerged in the 1970's, when researchers identified strains of inbred mice that were resistant to Friend murine leukemia virus (MLV) induced leukemia (Lilly, 1967, Pincus et al., 1971). Interestingly, these studies showed that mice with certain 'Friend virus susceptibility' (Fv) loci, could inhibit MLV replication *in vitro* and were subsequently resistant to leukemia. The *Fv1* and *Fv4* genes were particularly interesting, and were later shown to encode host proteins that resembled virus components. The *Fv1* gene encoded a protein that was similar to an endogenous retroviral Gag protein, and was shown to inhibit a post-entry stage of MLV replication (Ikeda et al., 1985). In contrast, the *Fv4* gene encoded a protein that resembled *env* (envelope) sequences in a specific strain of MLV, which obstructed binding of wild-type MLV to target cells (Pryciak & Varmus, 1992).

Since these discoveries, several new cellular restriction factors have been identified, including a number of factors that restrict HIV-1 replication (Chakrabarti & Simon, 2010). Many of these restriction factors are up-regulated in response to type I interferons (IFNs), which are typically activated in the presence of viruses by pattern recognition receptors (PRRs), such as Toll-like receptors or retinoic acid induced gene (RIG)-like receptors (Kumagai et al., 2008). Following secretion, type I IFNs bind to the interferon α/β receptor (IFNAR) on the cell surface and induce signalling through the Janus Kinases/Signal Transducers and Activators of Transcription (Jak/Stat) pathway. This leads to the activation of hundreds of IFN-responsive genes that restrict viral replication, including cellular restriction factors (Baum & Garcia-Sastre, 2010) (Figure 2).

Type I IFNs potently inhibit early and late stages of the HIV-1 lifecycle, and systemic administration of IFNα reduces HIV-1 plasma viremia *in vivo* (Meylan et al., 1993, Tavel et al., 2010). During viral infection, plasmacytoid dendritic cells (pDCs) are the main producers of IFNα, however the capacity of pDCs to produce IFNα is impaired during acute HIV-1 infection, and this DC subtype appears to be depleted in chronic HIV-1 infection (Borrow & Bhardwaj, 2008, Soumelis et al., 2001). In addition, the HIV-1 accessory proteins Vpr and Vif can degrade interferon regulatory factor 3 (IRF-3), which plays a critical role in type I IFN induction (Okumura et al., 2008). Since cellular restriction factors are the 'effector' proteins of the IFN response, differences in the ability of pDCs to produce IFNα may contribute to differences in HIV-1 replication and disease progression among patients. Understanding the molecular mechanisms behind these HIV-1 restriction factors, may lead to the development of drugs that mimic or promote their activities.

Fig. 2. The interferon response. Interferon (IFN) binds to the interferon receptor at the plasma membrane, activating the Janus Kinases/Signal Transducers and Activators of Transcription (Jak/Stat) pathway. This results in the up-regulation of hundreds of IFNresponsive genes, including cellular restriction factors that prevent HIV-1 replication.

#### **1.3 The HIV-1 lifecycle**

144 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

both of these trials were disappointing, and neither approach provided protection from HIV-1 infection (Buchbinder et al., 2008, McElrath et al., 2008). Moreover, the Merck vaccine actually seemed to suppress the immune response, due to pre-existing immunity to the

The third and largest trial was recently performed in Thailand, and aimed to induce both a T-cell and antibody response to HIV-1. In the study, 16,000 Thai men and women received either placebo or vaccine injections, and were subsequently monitored for HIV-1 infection over a 3 year period (Rerks-Ngarm et al., 2009). The vaccine group received injections of a recombinant canarypox vector vaccine ALVAC-HIV (Sanofi Pasteur), plus booster injections of a recombinant gp120 subunit vaccine AIDSVAX B/E (Global Solutions for Infectious Diseases). Results from this trial showed a modest benefit among vaccine recipients, with a vaccine efficacy of 26-30%. However, vaccination did not affect the levels of viremia or CD4+ T cell counts of infected individuals, and many were disappointed with the results

Over the last decade, several host proteins have been identified that are capable of inhibiting HIV-1 replication (Figure 1). These so-called 'cellular-restriction factors' are a new arm of the innate immune system, and inhibit stages of the HIV-1 lifecycle that are not targeted by current AIDS therapies. Our understanding of how cellular restriction factors target HIV-1 replication is far from compete, but research in this area may provide a new avenue for

Fig. 1. HIV-1/AIDS timeline. Timeline documenting major events in the HIV-1/AIDS pandemic, as well as the identification of several key HIV-1 cellular restriction factors.

Cellular restriction factors are host proteins that can inhibit specific steps in the lifecycle of a virus. The concept of cellular restriction factors first emerged in the 1970's, when researchers identified strains of inbred mice that were resistant to Friend murine leukemia virus (MLV) induced leukemia (Lilly, 1967, Pincus et al., 1971). Interestingly, these studies showed that mice with certain 'Friend virus susceptibility' (Fv) loci, could inhibit MLV replication *in vitro* and were subsequently resistant to leukemia. The *Fv1* and *Fv4* genes were particularly interesting, and were later shown to encode host proteins that resembled virus components. The *Fv1* gene encoded a protein that was similar to an endogenous retroviral Gag protein, and was shown to inhibit a post-entry stage of MLV replication (Ikeda et al., 1985). In contrast, the *Fv4* gene encoded a protein that resembled *env* (envelope) sequences in a specific strain of MLV, which obstructed binding of wild-type MLV to target cells (Pryciak

adenovirus vector (Priddy et al., 2008, Roberts et al., 2006).

(Rerks-Ngarm et al., 2009).

future AIDS therapies (Barr, 2010).

**1.2 Cellular restriction factors** 

& Varmus, 1992).

The HIV-1 lifecycle offers a multitude of steps that can be targeted by cellular HIV-1 restriction factors (Figure 3). The lifecycle begins when the HIV-1 envelope protein (gp120) binds to the host cell via its CD4 receptor, and following a conformational change, it binds to either the CXCR4 or CCR5 chemokine co-receptor (Deng et al., 1996). This interaction facilitates viral and cell membrane fusion, which is followed by the release of the viral core into the cell cytoplasm. In the cytoplasm, the HIV-1 capsid protein is lost in a process called uncoating, and the single-stranded RNA genome is reverse-transcribed into doublestranded cDNA. Reverse transcription is carried out by the virion-associated reverse

Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 147

Fig. 3. The HIV-1 lifecycle. The HIV-1 envelope protein (Env) binds to the CD4+ receptor

TRIM5α belongs to the tripartite motif-containing (TRIM) family of proteins, of which there are currently 77 identified members (Nisole et al., 2005). Although the *TRIM5* gene gives rise to several isoforms through differential splicing, TRIM5α is the only isoform with potent anti-HIV-1 activity. All TRIM proteins have a conserved RBCC motif, which consists of a RING domain, one or two B-box domains and a predicted coiled-coil region. The majority of TRIM proteins, including TRIM5α, have a C-terminal B30.2 domain. The **R**eally **I**nteresting **N**ew **G**ene (RING) domain has intrinsic E3 ligase activity, and together with an E1 activating enzyme and an E2 conjugating enzyme it can transfer ubiquitin or ubiquitin-like molecules to target proteins (Ozato et al., 2008). This modification can alter a protein's halflife, subcellular localization or interaction with other proteins. Importantly, two RING domain cysteine residues (C15 and C18) are essential for the E3 ligase activity of the RING domain (C15 and C18). These two residues are also required by rhTRIM5α for restricting

and CXCR4/CCR5 co-receptor on the host cell. The viral core is released into the cytoplasm, where the RNA genome is reverse-transcribed into double-stranded cDNA. The cDNA is imported into the nucleus, where it integrates into the host genome. The viral genes are transcribed and the RNA is exported to the cytoplasm, where it is translated into protein. The viral proteins traffic to the membrane where they assemble

and bud out of the host cell.

**2.1.2 TRIM5α: Structure and function** 

HIV-1 replication (Diaz-Griffero et al., 2006).

transcriptase enzyme, and precedes the formation of the multimeric pre-integration complex. This complex, which consists of both host and viral proteins, is transported along microtubules to the nucleus, where the HIV-1 integrase enzyme facilitates integration of the viral cDNA into the host genome. Following integration, HIV-1 transcription occurs from the 5' long terminal repeat (LTR) promoter, and leads to the synthesis of spliced HIV-1 RNA and unspliced HIV-1 genomic RNA. The RNA is exported into the cytoplasm where the main structural protein of HIV-1, the Gag polyprotein, is translated along with other viral proteins. The Gag protein oligomerizes and traffics to the cell membrane, where it forms higher-order structures and assembles into virions with other viral proteins. Cellular proteins are also involved in assembly, particularly Tsg101 and AIP1/ALIX, which participate in the budding and release of immature, non-infectious virions from the cell membrane (Garrus et al., 2001, Strack et al., 2003). As budding occurs, the Gag protein is cleaved into its four structural domains (matrix, capsid, nucleocapsid and p6) by the viral protease, generating mature infectious viral particles that are released from the cell membrane (Ganser-Pornillos et al., 2008, Ono, 2009). The following sections review the main HIV-1 cellular restriction factors in the order of their point of attack in the lifecycle, beginning with capsid uncoating and ending with viral release.

## **2. Lifecycle target: HIV-1 capsid uncoating**

## **2.1 TRIM5α**

#### **2.1.1 TRIM5α: History and background**

For years, researchers have been aware of a barrier to HIV-1 infection in Old world monkey (OWM) cells, however they have only recently begun to understand the nature of it. Early in the AIDS epidemic, the discovery that the host-range of HIV-1 was limited to humans and apes suggested that other primates may have an internal mechanism to combat HIV-1 infection (Alter et al., 1984, Gajdusek et al., 1985, Lusso et al., 1988). A large number of mammalian cell lines were tested for susceptibility to HIV-1 infection, including cells derived from humans, OWMs (monkeys of African and Asian origin) and New world monkeys (NWM) (monkeys of Central and South American origin) (Hofmann et al., 1999). Interestingly, HIV-1 replication was blocked in most OWM-derived cell lines and one species of NWM, the Owl monkey. Because of its initial definition as a genetic barrier to lentiviral infection, the restriction factor was originally named lentivirus susceptibility factor-1 (Lv-1) (Cowan et al., 2002).

TRIM5α was not identified as the protein responsible for the OWM block until it was isolated during a cDNA screen of HIV-1 resistant rhesus macaque cells. In the study, a cDNA library was created from HIV-1 resistant rhesus macaque cells and the cDNA clones from this library were transduced into an HIV-1 sensitive human cell line (HeLa) (Stremlau et al., 2004). The human cells were then challenged with HIV-1 and screened for resistant clones, which identified the rhesus orthologue of TRIM5α (rhTRIM5α). Excitingly, around the same time, TRIM5α was also linked to the HIV-1 block in Owl monkey cells; however, the Owl monkey version of *TRIM5α* encoded a TRIM5α-cyclophilin A fusion protein (Sayah et al., 2004). Cyclophilin A (CypA) was previously shown to bind to the HIV-1 capsid protein and promote HIV-1 replication in human cells; however, in Owl monkey cells CypA seemed to restrict HIV-1 (Luban, 2007, Sokolskaja & Luban, 2006). The discovery of a TRIM5α-CypA fusion protein explained these results and indicated that CypA may target TRIM5α-CypA to incoming HIV-1 capsid proteins.

Fig. 3. The HIV-1 lifecycle. The HIV-1 envelope protein (Env) binds to the CD4+ receptor and CXCR4/CCR5 co-receptor on the host cell. The viral core is released into the cytoplasm, where the RNA genome is reverse-transcribed into double-stranded cDNA. The cDNA is imported into the nucleus, where it integrates into the host genome. The viral genes are transcribed and the RNA is exported to the cytoplasm, where it is translated into protein. The viral proteins traffic to the membrane where they assemble and bud out of the host cell.

## **2.1.2 TRIM5α: Structure and function**

146 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

transcriptase enzyme, and precedes the formation of the multimeric pre-integration complex. This complex, which consists of both host and viral proteins, is transported along microtubules to the nucleus, where the HIV-1 integrase enzyme facilitates integration of the viral cDNA into the host genome. Following integration, HIV-1 transcription occurs from the 5' long terminal repeat (LTR) promoter, and leads to the synthesis of spliced HIV-1 RNA and unspliced HIV-1 genomic RNA. The RNA is exported into the cytoplasm where the main structural protein of HIV-1, the Gag polyprotein, is translated along with other viral proteins. The Gag protein oligomerizes and traffics to the cell membrane, where it forms higher-order structures and assembles into virions with other viral proteins. Cellular proteins are also involved in assembly, particularly Tsg101 and AIP1/ALIX, which participate in the budding and release of immature, non-infectious virions from the cell membrane (Garrus et al., 2001, Strack et al., 2003). As budding occurs, the Gag protein is cleaved into its four structural domains (matrix, capsid, nucleocapsid and p6) by the viral protease, generating mature infectious viral particles that are released from the cell membrane (Ganser-Pornillos et al., 2008, Ono, 2009). The following sections review the main HIV-1 cellular restriction factors in the order of their point of attack in the lifecycle,

For years, researchers have been aware of a barrier to HIV-1 infection in Old world monkey (OWM) cells, however they have only recently begun to understand the nature of it. Early in the AIDS epidemic, the discovery that the host-range of HIV-1 was limited to humans and apes suggested that other primates may have an internal mechanism to combat HIV-1 infection (Alter et al., 1984, Gajdusek et al., 1985, Lusso et al., 1988). A large number of mammalian cell lines were tested for susceptibility to HIV-1 infection, including cells derived from humans, OWMs (monkeys of African and Asian origin) and New world monkeys (NWM) (monkeys of Central and South American origin) (Hofmann et al., 1999). Interestingly, HIV-1 replication was blocked in most OWM-derived cell lines and one species of NWM, the Owl monkey. Because of its initial definition as a genetic barrier to lentiviral infection, the restriction factor was originally named lentivirus susceptibility

TRIM5α was not identified as the protein responsible for the OWM block until it was isolated during a cDNA screen of HIV-1 resistant rhesus macaque cells. In the study, a cDNA library was created from HIV-1 resistant rhesus macaque cells and the cDNA clones from this library were transduced into an HIV-1 sensitive human cell line (HeLa) (Stremlau et al., 2004). The human cells were then challenged with HIV-1 and screened for resistant clones, which identified the rhesus orthologue of TRIM5α (rhTRIM5α). Excitingly, around the same time, TRIM5α was also linked to the HIV-1 block in Owl monkey cells; however, the Owl monkey version of *TRIM5α* encoded a TRIM5α-cyclophilin A fusion protein (Sayah et al., 2004). Cyclophilin A (CypA) was previously shown to bind to the HIV-1 capsid protein and promote HIV-1 replication in human cells; however, in Owl monkey cells CypA seemed to restrict HIV-1 (Luban, 2007, Sokolskaja & Luban, 2006). The discovery of a TRIM5α-CypA fusion protein explained these results and indicated that CypA may target

beginning with capsid uncoating and ending with viral release.

**2. Lifecycle target: HIV-1 capsid uncoating** 

**2.1.1 TRIM5α: History and background** 

factor-1 (Lv-1) (Cowan et al., 2002).

TRIM5α-CypA to incoming HIV-1 capsid proteins.

**2.1 TRIM5α**

TRIM5α belongs to the tripartite motif-containing (TRIM) family of proteins, of which there are currently 77 identified members (Nisole et al., 2005). Although the *TRIM5* gene gives rise to several isoforms through differential splicing, TRIM5α is the only isoform with potent anti-HIV-1 activity. All TRIM proteins have a conserved RBCC motif, which consists of a RING domain, one or two B-box domains and a predicted coiled-coil region. The majority of TRIM proteins, including TRIM5α, have a C-terminal B30.2 domain. The **R**eally **I**nteresting **N**ew **G**ene (RING) domain has intrinsic E3 ligase activity, and together with an E1 activating enzyme and an E2 conjugating enzyme it can transfer ubiquitin or ubiquitin-like molecules to target proteins (Ozato et al., 2008). This modification can alter a protein's halflife, subcellular localization or interaction with other proteins. Importantly, two RING domain cysteine residues (C15 and C18) are essential for the E3 ligase activity of the RING domain (C15 and C18). These two residues are also required by rhTRIM5α for restricting HIV-1 replication (Diaz-Griffero et al., 2006).

Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 149

on the HIV-1 capsid by the B30.2 domain of rhTRIM5α and subsequent block at the level of reverse transcription. Interestingly, human TRIM5α (huTRIM5α) only modestly inhibits HIV-1 replication and substitution of a single amino acid (R332) in its B30.2 domain enables it to

The human apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) family is part of a larger family of APOBEC cytidine deaminases, capable of converting cytosine to uracil in RNA or DNA. The APOBEC3 family is found on chromosome 22 and contains seven members (A-H), which are believed to be the result of multiple duplication events (Conticello et al., 2005). Interestingly, APOBEC3 proteins appear to be under positive selective pressure, possibly as a defence against endogenous retroelements, and have been shown to have antiviral activity against a variety of viruses, including murine leukemia virus, human T-lymphotropic virus, simian immunodeficiency virus and the recently discovered Xenotropic Murine leukemia virus-Related Virus (XMRV) (Sawyer et al., 2004, Sawyer et al., 2004, Groom et al., 2010, Groom et al., 2010, Aguiar & Peterlin, 2008, Niewiadomska & Yu, 2009). In addition, all seven members have been implicated in HIV-1 restriction, however APOBEC3F/G are the best studied and appear to be the most potent

Before APOBEC3 proteins were specifically identified, it was discovered that HIV-1 clones with the accessory protein Vif deleted were capable of replicating in certain cell lines. These cells were termed "permissive cells", and included HeLa, HEK 293T, SupT1 and CEM-SS lines. In other cells, such as primary human T-lymphocytes or macrophages, or the H9 and CEM T cell lines, virions produced from Vif-deficient strains were up to 1,000 times less infectious compared to virions from wild-type strains (Gabuzda et al., 1992). Cell fusion experiments revealed that this "non-permissive" phenotype was dominant, and comparison of the related CEM T and CEM-SS cell lines revealed a 1.5 kb cDNA segment expressed in CEM T cells that was not produced in CEM-SS cells (Madani & Kabat, 1998, Sheehy et al., 2002, Simon et al., 1998). This protein, termed CEM15 and later renamed APOBEC3G (A3G), was shown to be suppressed by Vif, thus resulting in productive infection of non-permissive

In the absence of Vif, A3G is packaged into newly formed virions and blocks HIV-1 replication after infection of a new cell. Two mechanisms of HIV-1 inhibition have been reported for A3G: 1) the production of hyper-mutated viral DNA and 2) decreased accumulation of viral DNA (Figure 5) (Anderson & Hope, 2008, Bishop et al., 2008, Lecossier et al., 2003, Mangeat et al., 2003, Simon & Malim, 1996). During reverse transcription, A3G induces cytidine deamination (CU mutations) in the negative strand of newly synthesized viral cDNA. The latter results in GA hyper-mutated viral DNA and increases the probability of producing premature stop codons or mutated, non-functional viral proteins. Interestingly, cytidine deamination also recruits cellular uracil-DNA glycosylases that cleave off the uracil side chain as part of the base-excision repair pathway. The resulting abasic site may then prevent plus-strand DNA synthesis or lead to degradation of viral DNA by endonucleases (Klarmann et al., 2003, Yang et al., 2007). However, there is some controversy over the degree to which APOBEC3-mediated cytidine deamination contributes to reduced

restrict HIV-1 as potently as rhTRIM5α (Yap et al., 2005).

**3. Lifecycle target: HIV-1 reverse transcription** 

**3.1.1 APOBEC3: Structure and function** 

restrictors (Hultquist & Harris, 2009).

cells with wild-type HIV-1 (Sheehy et al., 2002).

**3.1.2 APOBEC3-mediated HIV-1 restriction** 

**3.1 APOBEC3** 

The function of the B-box domain remains largely uncharacterized; however, it is an interesting domain because it is unique to TRIM proteins. Deletion of the B-box domain of TRIM5α eliminates the ability of TRIM5α to restrict HIV-1, suggesting that this domain is critical for TRIM5α-mediated restriction (Stremlau et al., 2004, Javanbakht et al., 2005, Li & Sodroski, 2008, Perez-Caballero et al., 2005). In addition, it was recently shown that the Bbox domain promotes HIV-1 capsid binding by mediating higher-order self-association (Li & Sodroski, 2008). The coiled-coil region is involved in protein-protein interactions and more specifically, it is thought to mediate TRIM5α oligomer formation. It has been proposed that oligomer formation is important for positioning the B30.2 domain of TRIM5α for optimal HIV-1 capsid binding and accordingly, TRIM5α coiled-coil mutants fail to restrict HIV-1 (Perez-Caballero et al., 2005, Javanbakht et al., 2006). Finally, the specificity and interspecies variability of TRIM5α is found in the B30.2 domain. Sequence analysis has shown a significant amount of interspecies variability within the B30.2 domain of both NWM and OWM, especially on several variable loops that are thought to form the binding surface for HIV-1 capsid recognition (Ohkura et al., 2006, Woo et al., 2006, Yao et al., 2006).

#### **2.1.3 TRIM5α-mediated HIV-1 restriction**

To date, rhTRIM5α is the earliest-acting HIV-1 restriction factor and targets virus replication immediately after HIV-1 entry into target cells. Several studies have shown that rhTRIM5α blocks reverse transcription and nuclear import of viral cDNA. The mechanisms underlying this restriction are thought to include: sequestration of the virus core in the cytoplasm, modification of the virus core leading to degradation, or interference in the trafficking of the preintegration complex (Bieniasz, 2004, Chatterji et al., 2006, Stremlau et al., 2006). The most favoured mechanism involves rhTRIM5α binding to the viral core and disrupting the normal uncoating process of the core (Figure 4). This binding involves the recognition of specific sequences

Fig. 4. TRIM5α-mediated HIV-1 restriction. RhTRIM5α binds to incoming HIV-1 capsid proteins via its B30.2 domain, causing them to rapidly dissociate and prematurely disassemble. This leads to a block in HIV-1 reverse transcription and inhibits nuclear import of viral cDNA, restricting further propagation of the virus.

on the HIV-1 capsid by the B30.2 domain of rhTRIM5α and subsequent block at the level of reverse transcription. Interestingly, human TRIM5α (huTRIM5α) only modestly inhibits HIV-1 replication and substitution of a single amino acid (R332) in its B30.2 domain enables it to restrict HIV-1 as potently as rhTRIM5α (Yap et al., 2005).

## **3. Lifecycle target: HIV-1 reverse transcription**

## **3.1 APOBEC3**

148 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

The function of the B-box domain remains largely uncharacterized; however, it is an interesting domain because it is unique to TRIM proteins. Deletion of the B-box domain of TRIM5α eliminates the ability of TRIM5α to restrict HIV-1, suggesting that this domain is critical for TRIM5α-mediated restriction (Stremlau et al., 2004, Javanbakht et al., 2005, Li & Sodroski, 2008, Perez-Caballero et al., 2005). In addition, it was recently shown that the Bbox domain promotes HIV-1 capsid binding by mediating higher-order self-association (Li & Sodroski, 2008). The coiled-coil region is involved in protein-protein interactions and more specifically, it is thought to mediate TRIM5α oligomer formation. It has been proposed that oligomer formation is important for positioning the B30.2 domain of TRIM5α for optimal HIV-1 capsid binding and accordingly, TRIM5α coiled-coil mutants fail to restrict HIV-1 (Perez-Caballero et al., 2005, Javanbakht et al., 2006). Finally, the specificity and interspecies variability of TRIM5α is found in the B30.2 domain. Sequence analysis has shown a significant amount of interspecies variability within the B30.2 domain of both NWM and OWM, especially on several variable loops that are thought to form the binding surface for HIV-1 capsid recognition (Ohkura et al., 2006, Woo et al., 2006, Yao et al., 2006).

To date, rhTRIM5α is the earliest-acting HIV-1 restriction factor and targets virus replication immediately after HIV-1 entry into target cells. Several studies have shown that rhTRIM5α blocks reverse transcription and nuclear import of viral cDNA. The mechanisms underlying this restriction are thought to include: sequestration of the virus core in the cytoplasm, modification of the virus core leading to degradation, or interference in the trafficking of the preintegration complex (Bieniasz, 2004, Chatterji et al., 2006, Stremlau et al., 2006). The most favoured mechanism involves rhTRIM5α binding to the viral core and disrupting the normal uncoating process of the core (Figure 4). This binding involves the recognition of specific sequences

Fig. 4. TRIM5α-mediated HIV-1 restriction. RhTRIM5α binds to incoming HIV-1 capsid proteins via its B30.2 domain, causing them to rapidly dissociate and prematurely

of viral cDNA, restricting further propagation of the virus.

disassemble. This leads to a block in HIV-1 reverse transcription and inhibits nuclear import

**2.1.3 TRIM5α-mediated HIV-1 restriction** 

## **3.1.1 APOBEC3: Structure and function**

The human apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) family is part of a larger family of APOBEC cytidine deaminases, capable of converting cytosine to uracil in RNA or DNA. The APOBEC3 family is found on chromosome 22 and contains seven members (A-H), which are believed to be the result of multiple duplication events (Conticello et al., 2005). Interestingly, APOBEC3 proteins appear to be under positive selective pressure, possibly as a defence against endogenous retroelements, and have been shown to have antiviral activity against a variety of viruses, including murine leukemia virus, human T-lymphotropic virus, simian immunodeficiency virus and the recently discovered Xenotropic Murine leukemia virus-Related Virus (XMRV) (Sawyer et al., 2004, Sawyer et al., 2004, Groom et al., 2010, Groom et al., 2010, Aguiar & Peterlin, 2008, Niewiadomska & Yu, 2009). In addition, all seven members have been implicated in HIV-1 restriction, however APOBEC3F/G are the best studied and appear to be the most potent restrictors (Hultquist & Harris, 2009).

Before APOBEC3 proteins were specifically identified, it was discovered that HIV-1 clones with the accessory protein Vif deleted were capable of replicating in certain cell lines. These cells were termed "permissive cells", and included HeLa, HEK 293T, SupT1 and CEM-SS lines. In other cells, such as primary human T-lymphocytes or macrophages, or the H9 and CEM T cell lines, virions produced from Vif-deficient strains were up to 1,000 times less infectious compared to virions from wild-type strains (Gabuzda et al., 1992). Cell fusion experiments revealed that this "non-permissive" phenotype was dominant, and comparison of the related CEM T and CEM-SS cell lines revealed a 1.5 kb cDNA segment expressed in CEM T cells that was not produced in CEM-SS cells (Madani & Kabat, 1998, Sheehy et al., 2002, Simon et al., 1998). This protein, termed CEM15 and later renamed APOBEC3G (A3G), was shown to be suppressed by Vif, thus resulting in productive infection of non-permissive cells with wild-type HIV-1 (Sheehy et al., 2002).

## **3.1.2 APOBEC3-mediated HIV-1 restriction**

In the absence of Vif, A3G is packaged into newly formed virions and blocks HIV-1 replication after infection of a new cell. Two mechanisms of HIV-1 inhibition have been reported for A3G: 1) the production of hyper-mutated viral DNA and 2) decreased accumulation of viral DNA (Figure 5) (Anderson & Hope, 2008, Bishop et al., 2008, Lecossier et al., 2003, Mangeat et al., 2003, Simon & Malim, 1996). During reverse transcription, A3G induces cytidine deamination (CU mutations) in the negative strand of newly synthesized viral cDNA. The latter results in GA hyper-mutated viral DNA and increases the probability of producing premature stop codons or mutated, non-functional viral proteins. Interestingly, cytidine deamination also recruits cellular uracil-DNA glycosylases that cleave off the uracil side chain as part of the base-excision repair pathway. The resulting abasic site may then prevent plus-strand DNA synthesis or lead to degradation of viral DNA by endonucleases (Klarmann et al., 2003, Yang et al., 2007). However, there is some controversy over the degree to which APOBEC3-mediated cytidine deamination contributes to reduced

Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 151

In recent years, the resting T cell theory has received criticism, mainly because several results in the original work were not repeatable. However, it should be noted that many results were still repeatable including the presence of LMM/HMM in resting/activated T cells and the enzymatically active nature of LMM forms (Chiu et al., 2005). Furthermore, it was observed that factors such as IL-2 and IL-15 both increase susceptibility of resting T cells to HIV-1 infection and induce a shift of A3G organization from LMM to HMM forms, suggesting that these results may warrant further investigation. In addition, several APOBEC3 proteins appear to have cytidine deaminase-independent antiviral activity against Hepatitis B virus, Adeno-associated virus, and a number of retroelements, further supporting the existence of a cytidine deaminase-independent antiviral mechanism (Bogerd et al., 2006, Chen et al., 2006, Stenglein & Harris, 2006, Turelli et al., 2004). As such, the extent to which cytidine deaminase-dependent or –independent functions contribute to the

antiviral activity of APOBEC3 proteins against HIV-1 requires further elucidation.

Vif restores HIV-1 infectivity by inducing the degradation of APOBEC3 proteins (Conticello et al., 2003, Marin et al., 2003, Sheehy et al., 2003). Vif and APOBEC3 proteins physically interact with each other, and these interactions are specific, however they differ depending on the APOBEC3 member being targeted (Niewiadomska & Yu, 2009). Vif also contains several conserved elements that allow it to form a complex with certain cellular proteins. Specifically, the SLQxLA motif in Vif interacts with ElonginC, allowing the recruitment of ElonginB and Cul5, which bind Vif through another conserved motif. Rbc1 is also recruited to the complex, creating an E3 ligase capable of polyubiquitinating APOBEC3 proteins and targeting them for 26S proteasomal degradation (Figure 6) (Kobayashi et al., 2005, Mehle et al., 2004,

Fig. 6. Vif-mediated degradation of APOBEC3. Vif interacts with cellular factors to create a Skp1-cullin-F-box (SCF)-like complex that polyubiquitinates and degrades APOBEC3 molecules. Vif binds Cullin5 (Cul5) through two conserved motifs and other cellular factors,

forming a scaffold for other E3 ligase components.

**3.1.3 Countermeasures to APOBEC3-mediated HIV-1 restriction** 

accumulation of HIV-1 DNA, and it has been reported that the HIV-1 accessory protein Vpu induces the degradation of certain cellular uracil-DNA glycosylases (Schrofelbauer et al., 2005). Furthermore, loss of certain uracil-DNA glycosylases does not appear to affect APOBEC3 restriction of HIV-1, and similar results have been obtained for A3G restriction of other viruses (Kaiser & Emerman, 2006, Nguyen et al., 2007). Nevertheless, this does not rule out the possible involvement of other glycosylases, and additional studies will be required to fully elucidate the effects of APOBEC3 cytidine deaminase activity on HIV-1 replication.

Cytidine deaminase mutants of both A3F and A3G still retain a degree of anti-HIV-1 activity, and both proteins have been shown to affect the initiation of reverse transcription by interfering with tRNA3Lys binding to viral RNA. In addition, A3G has been shown to inhibit both plus and minus strand transfer RNA integration and DNA elongation during reverse transcription (Hultquist & Harris, 2009). Interestingly, endogenous A3G in resting naive and memory CD4+ T cells may inhibit newly infecting virus, contributing to the known resistance of quiescent T cells to HIV-1 infection (Chiu et al., 2005, Muckenfuss et al., 2006). In this model, A3G can exist in two forms: an inactive high molecular mass complex (HMM) in activated T cells or an enzymatically active low molecular mass form (LMM) in resting T cells. Since A3G can interfere with multiple steps of reverse transcription, this hypothesis is consistent with infection of resting T cells, in which cDNA synthesis is initiated, but the viral genome is not completely reverse transcribed.

Fig. 5. APOBEC3G-mediated restriction of Vif-deficient HIV-1. APOBEC3G (A3G) is packaged into newly formed virions and interferes with viral replication upon infection of new cells. A3G directly interferes with reverse transcription, resulting in decreased amounts of viral cDNA. In addition, A3G acts as a cytidine deaminase, inducing CU mutations in minus-strand viral cDNA. This leads to base excision by cellular uracil-DNA glycosylases or uracil bases are replaced with thymine, resulting in extensiveGA hypermutations in the viral genome.

accumulation of HIV-1 DNA, and it has been reported that the HIV-1 accessory protein Vpu induces the degradation of certain cellular uracil-DNA glycosylases (Schrofelbauer et al., 2005). Furthermore, loss of certain uracil-DNA glycosylases does not appear to affect APOBEC3 restriction of HIV-1, and similar results have been obtained for A3G restriction of other viruses (Kaiser & Emerman, 2006, Nguyen et al., 2007). Nevertheless, this does not rule out the possible involvement of other glycosylases, and additional studies will be required to fully elucidate the effects of APOBEC3 cytidine deaminase activity on HIV-1

Cytidine deaminase mutants of both A3F and A3G still retain a degree of anti-HIV-1 activity, and both proteins have been shown to affect the initiation of reverse transcription by interfering with tRNA3Lys binding to viral RNA. In addition, A3G has been shown to inhibit both plus and minus strand transfer RNA integration and DNA elongation during reverse transcription (Hultquist & Harris, 2009). Interestingly, endogenous A3G in resting naive and memory CD4+ T cells may inhibit newly infecting virus, contributing to the known resistance of quiescent T cells to HIV-1 infection (Chiu et al., 2005, Muckenfuss et al., 2006). In this model, A3G can exist in two forms: an inactive high molecular mass complex (HMM) in activated T cells or an enzymatically active low molecular mass form (LMM) in resting T cells. Since A3G can interfere with multiple steps of reverse transcription, this hypothesis is consistent with infection of resting T cells, in which cDNA synthesis is

initiated, but the viral genome is not completely reverse transcribed.

Fig. 5. APOBEC3G-mediated restriction of Vif-deficient HIV-1. APOBEC3G (A3G) is packaged into newly formed virions and interferes with viral replication upon infection of new cells. A3G directly interferes with reverse transcription, resulting in decreased amounts of viral cDNA. In addition, A3G acts as a cytidine deaminase, inducing CU mutations in minus-strand viral cDNA. This leads to base excision by cellular uracil-DNA glycosylases or uracil bases are replaced with thymine, resulting in extensiveGA hypermutations in the

replication.

viral genome.

In recent years, the resting T cell theory has received criticism, mainly because several results in the original work were not repeatable. However, it should be noted that many results were still repeatable including the presence of LMM/HMM in resting/activated T cells and the enzymatically active nature of LMM forms (Chiu et al., 2005). Furthermore, it was observed that factors such as IL-2 and IL-15 both increase susceptibility of resting T cells to HIV-1 infection and induce a shift of A3G organization from LMM to HMM forms, suggesting that these results may warrant further investigation. In addition, several APOBEC3 proteins appear to have cytidine deaminase-independent antiviral activity against Hepatitis B virus, Adeno-associated virus, and a number of retroelements, further supporting the existence of a cytidine deaminase-independent antiviral mechanism (Bogerd et al., 2006, Chen et al., 2006, Stenglein & Harris, 2006, Turelli et al., 2004). As such, the extent to which cytidine deaminase-dependent or –independent functions contribute to the antiviral activity of APOBEC3 proteins against HIV-1 requires further elucidation.

## **3.1.3 Countermeasures to APOBEC3-mediated HIV-1 restriction**

Vif restores HIV-1 infectivity by inducing the degradation of APOBEC3 proteins (Conticello et al., 2003, Marin et al., 2003, Sheehy et al., 2003). Vif and APOBEC3 proteins physically interact with each other, and these interactions are specific, however they differ depending on the APOBEC3 member being targeted (Niewiadomska & Yu, 2009). Vif also contains several conserved elements that allow it to form a complex with certain cellular proteins. Specifically, the SLQxLA motif in Vif interacts with ElonginC, allowing the recruitment of ElonginB and Cul5, which bind Vif through another conserved motif. Rbc1 is also recruited to the complex, creating an E3 ligase capable of polyubiquitinating APOBEC3 proteins and targeting them for 26S proteasomal degradation (Figure 6) (Kobayashi et al., 2005, Mehle et al., 2004,

Fig. 6. Vif-mediated degradation of APOBEC3. Vif interacts with cellular factors to create a Skp1-cullin-F-box (SCF)-like complex that polyubiquitinates and degrades APOBEC3 molecules. Vif binds Cullin5 (Cul5) through two conserved motifs and other cellular factors, forming a scaffold for other E3 ligase components.

Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 153

Upon activation, PKR inhibits HIV-1 replication through a number of different mechanisms. One mechanism involves phosphorylation of the enzyme RNA Helicase A (RHA), which has been shown to enhance HIV-1 transcriptional activity and binds to the HIV-1 transactivation response (TAR) element (Fujii et al., 2001, Jeang & Yedavalli, 2006). The HIV-1 TAR element is required for trans-activation of the viral promoter and binds to the viral trans-activator of transcription (Tat) protein. This interaction greatly increases transcription of viral genes from the HIV-1 promoter, and results in the production of many full-length HIV-1 transcripts. The TAR element is an unusual stem-loop RNA structure that is located at the 5' end of all HIV-1 mRNAs (Adelson et al., 1999, Nagai et al., 1997). PKR recognizes TAR RNA as dsRNA, and it binds to the upper bulge of the lower stem-loop structure using

Like PKR, RHA contains a dsRBD with two dsRNA binding motifs, which are required for TAR recognition and binding (Fujii et al., 2001). A lysine residue at position 236 of RHA is essential for TAR binding (Fujii et al., 2001, Jeang & Yedavalli, 2006). Recently, PKR has been shown to phosphorylate the dsRBD of RHA; a modification that depends on lysine 296 of the PKR protein (Sadler et al., 2009). Phosphorylation of RHA by PKR seems to inhibit RHA-TAR binding, which in turn decreases the levels of HIV-1 mRNA transcripts (Sadler et

Upon activation, PKR inhibits HIV-1 replication through a number of different mechanisms. The most widely researched pathway occurs through PKR-mediated phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2α), a key regulator of protein synthesis (Figure 7) (Dey et al., 2005, Rojas et al., 2010). In eukaryotic cells, eIF2α bound to GTP mediates the formation of a trimeric complex with methionine transfer RNA (mettRNA). This complex leads to met-tRNA binding to the 40S ribosomal subunit, and allows for the initiation of translation (Rojas et al., 2010, Nallagatla et al., 2011). However, phosphorylation of eIF2α by PKR prevents the eIF2α-GTP interaction and eliminates the formation of the trimeric complex, thus inhibiting translation of all mRNA, including viral

The HIV-1 trans-activation response (TAR) element is required for trans-activation of the viral promoter and binds to the viral trans-activator of transcription (Tat) protein. This interaction greatly increases viral gene expression from the HIV-1 promoter by inducing chromatin remodelling and by recruiting elongation-competent transcriptional complexes onto the viral LTR. The TAR element is an unusual stem-loop RNA structure that is located at the 5' end of all HIV-1 mRNAs (Adelson et al., 1999, Nagai et al., 1997). PKR recognizes TAR RNA as dsRNA, and it binds to the upper bulge of the lower stem-loop structure using both of its dsRNA binding motifs (Carpick et al., 1997, Kim et al., 2006). This leads to activation of PKR and phosphorylation of eIF2α, which subsequently inhibits protein

Although *in vitro* studies have shown that PKR can inhibit HIV-1 replication, *in vivo* models of infection fail to exhibit viral restriction. Interestingly, low levels of dsRNA have been shown to have beneficial effects on PKR activation; however, higher levels of dsRNA

**4.1.2 PKR-mediated inhibition of HIV-1 transcription** 

**4.1.3 PKR-mediated inhibition of HIV-1 translation** 

translation (Nallagatla et al., 2011, Roy et al., 1991)

**4.1.4 Countermeasures to PKR-mediated HIV-1 restriction** 

mRNA (Nallagatla et al., 2011).

al., 2009).

both of its dsRNA binding motifs (Carpick et al., 1997, Kim et al., 2006).

Yu et al., 2003). Interestingly, one report suggests that Vif instead of A3G is actually polyubiquitinated, possibly to serve as a way to transport A3G to the 26S proteasome for degradation (Dang et al., 2008). However, mutated and modified forms of Vif that are still capable of degrading A3G are unable to restore viral infectivity (Mehle et al., 2004, Kao et al., 2007). Thus, Vif may also inhibit A3G through degradation-independent mechanisms. Possible theories include a competitive inhibition model, where Vif binds to a common target, preventing A3G packaging, or that Vif inactivates A3G by inducing the formation of high molecular mass complexes (Goila-Gaur & Strebel, 2008, Goila-Gaur et al., 2008).

#### **3.1.4 APOBEC3: Effects on HIV-1 replication** *in vivo*

The extent to which APOBEC3 proteins are functional during HIV-1 infection *in vivo* is a highly contested topic. One report has observed the presence of extensive GA hypermutation in virus samples collected from one HIV-1 long-term non-progressor (Wang et al., 2003)(Kao et al., 2007)(Kao et al., 2007)(Kao et al., 2007)(Kao et al., 2007)(Kao et al., 2007)(Kao et al., 2007)(Kao et al., 2007). Although this may suggest a role for APOBEC3 in controlling infection, it appears to be the only reported case, and thus the effects of APOBEC3 may have been secondary to some other mechanism of control. Still, it is possible that APOBEC3 proteins are more functional in certain patients/infections than in others. There are reports of both significant correlations and lack of correlation, between hypermutation and reduced viral load/higher CD4+ cell counts (Land et al., 2008, Piantadosi et al., 2009, Ulenga et al., 2008). Though hypermutation-independent mechanisms may exist, other groups have shown a negative correlation between A3G mRNA expression levels and HIV-1 viremia, and a positive correlation between A3G mRNA levels and CD4+ cell counts (Ulenga et al., 2008, Vazquez-Perez et al., 2009). Furthermore, it was shown that A3F/G mRNA levels post-infection are higher in patients with low viral set points than in patients with high viral set points, and higher in seronegative patients compared to healthy controls. Nevertheless, another group observed no correlation between A3F/G mRNA levels and viremia or CD4+ cell counts (Cho et al., 2006). Although conflicting reports exist, these may, in part, be explained by varying levels of APOBEC3 mRNA between donors (Koning et al., 2009). More detailed studies into both APOBEC3 expression and activity at the host level, in addition to correlation with disease progression, will be required to further elucidate its relationship to HIV-1 infection.

#### **4. Lifecycle target: HIV-1 RNA**

#### **4.1 PKR**

#### **4.1.1 PKR: Structure and function**

Protein kinase R (PKR) is constitutively expressed in human cells as an inactive monomer. In the presence of double-stranded RNA (dsRNA), which forms the genetic material of some viruses, PKR forms a dimer and phosphorylates itself to become active (Dey et al., 2005, Garcia et al., 2007). Activation of PKR is also possible through type I IFN signalling, and PKR is upregulated in the presence of IFNs. PKR contains a N-terminal dsRNA binding domain (dsRBD), which is made up of two dsRBD motifs and can bind to dsRNA as short as 30bp (Figure 1) (Lemaire et al., 2008). Dimerization relies strongly on the dsRBD, and studies deleting this domain show impaired dimerization as well as lack of PKR activation (Cosentino et al., 1995). In addition to its dsRBD, PKR contains a C-terminal kinase domain that has intrinsic phosphotransferase activity. This function hinges on a key lysine residue at position 296, without which the kinase domain is inactive (Sadler et al., 2009).

## **4.1.2 PKR-mediated inhibition of HIV-1 transcription**

152 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Yu et al., 2003). Interestingly, one report suggests that Vif instead of A3G is actually polyubiquitinated, possibly to serve as a way to transport A3G to the 26S proteasome for degradation (Dang et al., 2008). However, mutated and modified forms of Vif that are still capable of degrading A3G are unable to restore viral infectivity (Mehle et al., 2004, Kao et al., 2007). Thus, Vif may also inhibit A3G through degradation-independent mechanisms. Possible theories include a competitive inhibition model, where Vif binds to a common target, preventing A3G packaging, or that Vif inactivates A3G by inducing the formation of high

The extent to which APOBEC3 proteins are functional during HIV-1 infection *in vivo* is a highly contested topic. One report has observed the presence of extensive GA hypermutation in virus samples collected from one HIV-1 long-term non-progressor (Wang et al., 2003)(Kao et al., 2007)(Kao et al., 2007)(Kao et al., 2007)(Kao et al., 2007)(Kao et al., 2007)(Kao et al., 2007)(Kao et al., 2007). Although this may suggest a role for APOBEC3 in controlling infection, it appears to be the only reported case, and thus the effects of APOBEC3 may have been secondary to some other mechanism of control. Still, it is possible that APOBEC3 proteins are more functional in certain patients/infections than in others. There are reports of both significant correlations and lack of correlation, between hypermutation and reduced viral load/higher CD4+ cell counts (Land et al., 2008, Piantadosi et al., 2009, Ulenga et al., 2008). Though hypermutation-independent mechanisms may exist, other groups have shown a negative correlation between A3G mRNA expression levels and HIV-1 viremia, and a positive correlation between A3G mRNA levels and CD4+ cell counts (Ulenga et al., 2008, Vazquez-Perez et al., 2009). Furthermore, it was shown that A3F/G mRNA levels post-infection are higher in patients with low viral set points than in patients with high viral set points, and higher in seronegative patients compared to healthy controls. Nevertheless, another group observed no correlation between A3F/G mRNA levels and viremia or CD4+ cell counts (Cho et al., 2006). Although conflicting reports exist, these may, in part, be explained by varying levels of APOBEC3 mRNA between donors (Koning et al., 2009). More detailed studies into both APOBEC3 expression and activity at the host level, in addition to correlation with disease progression,

molecular mass complexes (Goila-Gaur & Strebel, 2008, Goila-Gaur et al., 2008).

will be required to further elucidate its relationship to HIV-1 infection.

position 296, without which the kinase domain is inactive (Sadler et al., 2009).

Protein kinase R (PKR) is constitutively expressed in human cells as an inactive monomer. In the presence of double-stranded RNA (dsRNA), which forms the genetic material of some viruses, PKR forms a dimer and phosphorylates itself to become active (Dey et al., 2005, Garcia et al., 2007). Activation of PKR is also possible through type I IFN signalling, and PKR is upregulated in the presence of IFNs. PKR contains a N-terminal dsRNA binding domain (dsRBD), which is made up of two dsRBD motifs and can bind to dsRNA as short as 30bp (Figure 1) (Lemaire et al., 2008). Dimerization relies strongly on the dsRBD, and studies deleting this domain show impaired dimerization as well as lack of PKR activation (Cosentino et al., 1995). In addition to its dsRBD, PKR contains a C-terminal kinase domain that has intrinsic phosphotransferase activity. This function hinges on a key lysine residue at

**4. Lifecycle target: HIV-1 RNA** 

**4.1.1 PKR: Structure and function** 

**4.1 PKR** 

**3.1.4 APOBEC3: Effects on HIV-1 replication** *in vivo*

Upon activation, PKR inhibits HIV-1 replication through a number of different mechanisms. One mechanism involves phosphorylation of the enzyme RNA Helicase A (RHA), which has been shown to enhance HIV-1 transcriptional activity and binds to the HIV-1 transactivation response (TAR) element (Fujii et al., 2001, Jeang & Yedavalli, 2006). The HIV-1 TAR element is required for trans-activation of the viral promoter and binds to the viral trans-activator of transcription (Tat) protein. This interaction greatly increases transcription of viral genes from the HIV-1 promoter, and results in the production of many full-length HIV-1 transcripts. The TAR element is an unusual stem-loop RNA structure that is located at the 5' end of all HIV-1 mRNAs (Adelson et al., 1999, Nagai et al., 1997). PKR recognizes TAR RNA as dsRNA, and it binds to the upper bulge of the lower stem-loop structure using both of its dsRNA binding motifs (Carpick et al., 1997, Kim et al., 2006).

Like PKR, RHA contains a dsRBD with two dsRNA binding motifs, which are required for TAR recognition and binding (Fujii et al., 2001). A lysine residue at position 236 of RHA is essential for TAR binding (Fujii et al., 2001, Jeang & Yedavalli, 2006). Recently, PKR has been shown to phosphorylate the dsRBD of RHA; a modification that depends on lysine 296 of the PKR protein (Sadler et al., 2009). Phosphorylation of RHA by PKR seems to inhibit RHA-TAR binding, which in turn decreases the levels of HIV-1 mRNA transcripts (Sadler et al., 2009).

## **4.1.3 PKR-mediated inhibition of HIV-1 translation**

Upon activation, PKR inhibits HIV-1 replication through a number of different mechanisms. The most widely researched pathway occurs through PKR-mediated phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2α), a key regulator of protein synthesis (Figure 7) (Dey et al., 2005, Rojas et al., 2010). In eukaryotic cells, eIF2α bound to GTP mediates the formation of a trimeric complex with methionine transfer RNA (mettRNA). This complex leads to met-tRNA binding to the 40S ribosomal subunit, and allows for the initiation of translation (Rojas et al., 2010, Nallagatla et al., 2011). However, phosphorylation of eIF2α by PKR prevents the eIF2α-GTP interaction and eliminates the formation of the trimeric complex, thus inhibiting translation of all mRNA, including viral mRNA (Nallagatla et al., 2011).

The HIV-1 trans-activation response (TAR) element is required for trans-activation of the viral promoter and binds to the viral trans-activator of transcription (Tat) protein. This interaction greatly increases viral gene expression from the HIV-1 promoter by inducing chromatin remodelling and by recruiting elongation-competent transcriptional complexes onto the viral LTR. The TAR element is an unusual stem-loop RNA structure that is located at the 5' end of all HIV-1 mRNAs (Adelson et al., 1999, Nagai et al., 1997). PKR recognizes TAR RNA as dsRNA, and it binds to the upper bulge of the lower stem-loop structure using both of its dsRNA binding motifs (Carpick et al., 1997, Kim et al., 2006). This leads to activation of PKR and phosphorylation of eIF2α, which subsequently inhibits protein translation (Nallagatla et al., 2011, Roy et al., 1991)

### **4.1.4 Countermeasures to PKR-mediated HIV-1 restriction**

Although *in vitro* studies have shown that PKR can inhibit HIV-1 replication, *in vivo* models of infection fail to exhibit viral restriction. Interestingly, low levels of dsRNA have been shown to have beneficial effects on PKR activation; however, higher levels of dsRNA

Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 155

Tripartite motif-containing protein 22 (TRIM22) was originally isolated during a search for IFN-induced genes in Daudi cells, and is located at chromosomal position 11p15, immediately adjacent to the TRIM5α gene (Tissot & Mechti, 1995). Similar to TRIM5α, TRIM22 belongs to the TRIM family of proteins and is upregulated in response to Type I and Type II IFNs (Bouazzaoui et al., 2006). TRIM22 expression is altered by multiple cytokines and viral antigens/infections, including Hepatitis B virus, encephalomyocarditis virus, and HIV-1 (Gao et al., 2009, Gao et al., 2009, Eldin et al., 2009). TRIM22 may also play a role in cellular processes such as cell differentiation/proliferation, as it is a known p53 target gene and has been suggested to have potential anti-proliferative functions (Obad et

To date, several studies have addressed the effect of TRIM22 on HIV-1 replication. TRIM22 expression was first shown to reduce HIV-1 transcription from a luciferase reporter construct under control of the HIV-1 long terminal repeat promoter (LTR) (Tissot & Mechti, 1995). Although this report did not follow up these observations in the context of full-length, replication competent HIV-1, it was fundamental in identifying TRIM22 as a potential HIV-1 restriction factor. Eleven years later, TRIM22 expression was shown to be increased in response to *ex vivo* HIV-1 infection of primary monocyte-derived macrophages, a biological target of HIV-1 (Bouazzaoui et al., 2006). In addition, overexpression of TRIM22 restricted HIV-1 infection by 70-90% and prevented the formation of syncytia. In 2008, TRIM22 was confirmed to be a potent effector of the IFN response against HIV-1 infection, and two different methods of TRIM22-mediated latestage HIV-1 restriction were observed: one dependent on, and a second independent of,

It has since been confirmed that TRIM22 is capable of restricting HIV-1 mediated transcription (Kajaste-Rudnitski et al., 2011). Different clones of the U937 promonocytic cell line have previously been identified to be either permissive or nonpermissive to HIV-1 replication (Franzoso et al., 1994). Examination of multiple IFN-induced restriction factors revealed that only TRIM22 was present in nonpermissive clones and absent in permissive clones. LTR-driven transcription between permissive and nonpermissive clones was examined using a reporter construct expressing luciferase under control of the HIV-1 LTR. Basal transcription levels were decreased 7-10 fold in nonpermissive clones, but recovered to levels observed in permissive cells when shRNA was used to knockdown TRIM22 expression. Furthermore, LTR-driven transcription was decreased in permissive cells transduced with TRIM22, suggesting that the constitutive expression of TRIM22 is responsible for the restrictive phenotype observed in nonpermissive clones (Kajaste-Rudnitski et al., 2011). Reduced LTR-driven luciferase expression and HIV-1 replication were also observed in A3.01 cells (T cell line) expressing TRIM22, further supporting the effects of TRIM22 on HIV-1 infection in critical cell targets (Kajaste-Rudnitski et al., 2011). TRIM22 appears to strongly target basal transcription from the HIV-1 LTR. In further experiments using LTR-driven luciferase constructs, TRIM22 had no effect on transcription when cells were transfected with a plasmid encoding the HIV-1 Tat protein (Kajaste-Rudnitski et al., 2011). Although statistical significance was not reached, this may be due to the effects of exogenously provided Tat masking the efficacy of TRIM22. It should also be

**4.2 TRIM22** 

**4.2.1 TRIM22: Structure and function** 

the HIV-1 Gag polyprotein (Barr et al., 2008).

**4.2.2 TRIM22-mediated inhibition of HIV-1 transcription** 

al., 2004, Obad et al., 2007).

(greater than a 1:1 ratio of dsRNA:PKR) may actually inhibit PKR activity (Chu et al., 1998, Hunter et al., 1975). During the initial stages of HIV-1 infection, low levels of TAR RNA (dsRNA) are produced and this leads to PKR activation (Lemaire et al., 2008). Conversely, in later stages of viral replication (when Tat-TAR binding enhances transcription), much more TAR RNA (dsRNA) is generated, which seems to inhibit PKR activity (Lemaire et al., 2008, Hunter et al., 1975, Clerzius et al., 2011, Manche et al., 1992). It has been proposed that high concentrations of dsRNA cause PKR to bind dsRNA as a monomer, which inhibits PKR dimerization and subsequent activation (Manche et al., 1992, Cole, 2007).

PKR activation is also interrupted by the HIV-1 Tat protein. Tat binds to the HIV-1 TAR element and in doing so, masks TAR recognition by PKR and inhibits PKR activation (Cai et al., 2000). Furthermore, Tat binds to PKR directly and therefore competes for PKR binding with eIF2α (Brand et al., 1997). There is a high degree of sequence homology between the Tat and eIF2α binding sites in PKR, which leads to a decrease in eIF2α phosphorylation (Cai et al., 2000, Brand et al., 1997). In addition, Tat binding to PKR inhibits autophosphorylation, possibly by inhibiting PKR dimerization, which is necessary for its antiviral activity (Cai et al., 2000, Brand et al., 1997). There is also evidence that phosphorylation of Tat by PKR at several key amino acids (S62, T64, S68) actually enhances Tat's ability to initiate transcription; however, the precise mechanism of transcriptional enhancement is not yet clear (Endo-Munoz et al., 2005).

Fig. 7. PKR-mediated inhibition of HIV-1 protein translation. PKR recognizes the HIV-1 TAR element (dsRNA) and dimerizes and phosphorylates itself to become active. Active PKR phosphorylates eIF2α, preventing guanine nucleotide exchange and eIF2α activation. Inactive eIF2α is unable to transfer methionine-tRNA to the 40S ribosome for mRNA translation, and protein translation is inhibited.

#### **4.2 TRIM22**

154 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

(greater than a 1:1 ratio of dsRNA:PKR) may actually inhibit PKR activity (Chu et al., 1998, Hunter et al., 1975). During the initial stages of HIV-1 infection, low levels of TAR RNA (dsRNA) are produced and this leads to PKR activation (Lemaire et al., 2008). Conversely, in later stages of viral replication (when Tat-TAR binding enhances transcription), much more TAR RNA (dsRNA) is generated, which seems to inhibit PKR activity (Lemaire et al., 2008, Hunter et al., 1975, Clerzius et al., 2011, Manche et al., 1992). It has been proposed that high concentrations of dsRNA cause PKR to bind dsRNA as a monomer, which inhibits PKR

PKR activation is also interrupted by the HIV-1 Tat protein. Tat binds to the HIV-1 TAR element and in doing so, masks TAR recognition by PKR and inhibits PKR activation (Cai et al., 2000). Furthermore, Tat binds to PKR directly and therefore competes for PKR binding with eIF2α (Brand et al., 1997). There is a high degree of sequence homology between the Tat and eIF2α binding sites in PKR, which leads to a decrease in eIF2α phosphorylation (Cai et al., 2000, Brand et al., 1997). In addition, Tat binding to PKR inhibits autophosphorylation, possibly by inhibiting PKR dimerization, which is necessary for its antiviral activity (Cai et al., 2000, Brand et al., 1997). There is also evidence that phosphorylation of Tat by PKR at several key amino acids (S62, T64, S68) actually enhances Tat's ability to initiate transcription; however, the precise mechanism of transcriptional enhancement is not yet

Fig. 7. PKR-mediated inhibition of HIV-1 protein translation. PKR recognizes the HIV-1 TAR element (dsRNA) and dimerizes and phosphorylates itself to become active. Active PKR phosphorylates eIF2α, preventing guanine nucleotide exchange and eIF2α activation. Inactive eIF2α is unable to transfer methionine-tRNA to the 40S ribosome for mRNA

dimerization and subsequent activation (Manche et al., 1992, Cole, 2007).

clear (Endo-Munoz et al., 2005).

translation, and protein translation is inhibited.

#### **4.2.1 TRIM22: Structure and function**

Tripartite motif-containing protein 22 (TRIM22) was originally isolated during a search for IFN-induced genes in Daudi cells, and is located at chromosomal position 11p15, immediately adjacent to the TRIM5α gene (Tissot & Mechti, 1995). Similar to TRIM5α, TRIM22 belongs to the TRIM family of proteins and is upregulated in response to Type I and Type II IFNs (Bouazzaoui et al., 2006). TRIM22 expression is altered by multiple cytokines and viral antigens/infections, including Hepatitis B virus, encephalomyocarditis virus, and HIV-1 (Gao et al., 2009, Gao et al., 2009, Eldin et al., 2009). TRIM22 may also play a role in cellular processes such as cell differentiation/proliferation, as it is a known p53 target gene and has been suggested to have potential anti-proliferative functions (Obad et al., 2004, Obad et al., 2007).

To date, several studies have addressed the effect of TRIM22 on HIV-1 replication. TRIM22 expression was first shown to reduce HIV-1 transcription from a luciferase reporter construct under control of the HIV-1 long terminal repeat promoter (LTR) (Tissot & Mechti, 1995). Although this report did not follow up these observations in the context of full-length, replication competent HIV-1, it was fundamental in identifying TRIM22 as a potential HIV-1 restriction factor. Eleven years later, TRIM22 expression was shown to be increased in response to *ex vivo* HIV-1 infection of primary monocyte-derived macrophages, a biological target of HIV-1 (Bouazzaoui et al., 2006). In addition, overexpression of TRIM22 restricted HIV-1 infection by 70-90% and prevented the formation of syncytia. In 2008, TRIM22 was confirmed to be a potent effector of the IFN response against HIV-1 infection, and two different methods of TRIM22-mediated latestage HIV-1 restriction were observed: one dependent on, and a second independent of, the HIV-1 Gag polyprotein (Barr et al., 2008).

#### **4.2.2 TRIM22-mediated inhibition of HIV-1 transcription**

It has since been confirmed that TRIM22 is capable of restricting HIV-1 mediated transcription (Kajaste-Rudnitski et al., 2011). Different clones of the U937 promonocytic cell line have previously been identified to be either permissive or nonpermissive to HIV-1 replication (Franzoso et al., 1994). Examination of multiple IFN-induced restriction factors revealed that only TRIM22 was present in nonpermissive clones and absent in permissive clones. LTR-driven transcription between permissive and nonpermissive clones was examined using a reporter construct expressing luciferase under control of the HIV-1 LTR. Basal transcription levels were decreased 7-10 fold in nonpermissive clones, but recovered to levels observed in permissive cells when shRNA was used to knockdown TRIM22 expression. Furthermore, LTR-driven transcription was decreased in permissive cells transduced with TRIM22, suggesting that the constitutive expression of TRIM22 is responsible for the restrictive phenotype observed in nonpermissive clones (Kajaste-Rudnitski et al., 2011). Reduced LTR-driven luciferase expression and HIV-1 replication were also observed in A3.01 cells (T cell line) expressing TRIM22, further supporting the effects of TRIM22 on HIV-1 infection in critical cell targets (Kajaste-Rudnitski et al., 2011).

TRIM22 appears to strongly target basal transcription from the HIV-1 LTR. In further experiments using LTR-driven luciferase constructs, TRIM22 had no effect on transcription when cells were transfected with a plasmid encoding the HIV-1 Tat protein (Kajaste-Rudnitski et al., 2011). Although statistical significance was not reached, this may be due to the effects of exogenously provided Tat masking the efficacy of TRIM22. It should also be

Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 157

Overexpression studies have shown that OAS1/RNaseL-mediated HIV-1 restriction is possible; however, when RNaseL is expressed at biologically relevant levels, HIV-1 can inhibit this pathway. The HIV-1 Tat protein sequesters the TAR element *in vivo*, thus preventing OAS1-TAR binding and RNaseL activation (Schroder et al., 1990b). It is possible that this pathway is responsible for the low levels of mRNA production during the initial stages of HIV-1 infection; however, as Tat expression increases, activation of the OAS1/RNaseL pathway decreases and the production of HIV-1 mRNA rises significantly. In addition, cells that contain latent virus may be kept under control by the OAS1/RNaseL pathway. Nevertheless, the ability of cells to endocytose Tat from other apoptotic cells may lead to trans-activation of the TAR element and HIV-1 mRNA production in these latently

Although PKR and OAS1/RNaseL are potent HIV-1 restriction factors *in vitro*, the HIV-1 Tat protein is an effective viral countermeasure *in vivo*. Research on these restriction factors however, does provide insight into future therapeutics that may target the HIV-1 Tat protein, or overpower Tat for TAR binding. *In vitro* studies have already shown that shRNA directed against Tat or a TAR RNA decoy can provide long-term inhibition of HIV-1

Interestingly, TRIM22 appears to restrict HIV-1 replication by multiple mechanisms. TRIM22 has been shown to be an integral part of IFNβ-mediated HIV-1 restriction, and its expression can restrict HIV-1 replication in several transformed cell lines. In cell lines such as human osteosarcoma (HOS) and HeLa, TRIM22 restricts the release of virus, but has little to no effect on intracellular levels of the HIV-1 structural protein Gag. Conversely, in the osteosarcoma cell lines U2OS and 143B, TRIM22 expression not only restricts the release of virus, but also prevents the intracellular accumulation of Gag protein (Barr et al., 2008). The presence of different mechanisms in different cell lines, and the fact that multiple localizations for TRIM22 have been observed, suggests that there are many complex details

Further investigation of the mechanism of TRIM22-induced restriction in HOS cells revealed that TRIM22 likely interferes with intracellular trafficking of the HIV-1 Gag protein (Barr et al., 2008). Of note, Gag is both necessary and sufficient for budding and release of virus particles. This property allows Gag, in the absence of other viral proteins, to assemble and bud from the cell membrane, resulting in the production of non-infectious, virus-like particles (VLP). Importantly, TRIM22 expression was shown to inhibit the release of VLPs and prevent accumulation of Gag at the cell membrane, a step that is critical for virus assembly. These effects were dependent on the E3 ubiquitin ligase activity of TRIM22.

RhTRIM5α was originally shown to target incoming HIV-1 capsid proteins, thus inhibiting early stages of HIV-1 replication (see Section 2.1). For this reason, it was initially assumed that rhTRIM5α didn't affect late stages of HIV-1 replication. However, subsequent research showed that it could also restrict HIV-1 through rapid degradation of the Gag polyprotein, the main structural component of HIV-1 (Sakuma et al., 2007). Treatment of cells

replication; however, these studies must still be confirmed *in vivo* (Li et al., 2005).

**5.1.2 Countermeasures to OAS1/RNaseL-mediated HIV-1 restriction** 

infected cells (Schroder et al., 1990b, Frankel & Pabo, 1988).

of TRIM22 function that remain to be discovered (Figure 9).

**5.2.1 TRIM22-mediated HIV-1 restriction** 

**5.3.1 TRIM5α-mediated HIV-1 restriction** 

**5.2 TRIM22** 

**5.3 TRIM5α**

noted that all direct evidence to date of TRIM22 inhibiting HIV-1 transcription has been through the use of LTR-driven reporter constructs. It will be important to test the effects of TRIM22 on replication-competent HIV-1, which will provide a more natural scenario of virus transcription, Tat-induction, and possible effects of other HIV-1 accessory proteins.

## **4.2.3 TRIM22-mediated effects on HIV-1 replication** *in vivo*

Interestingly, there is evidence to support a role for TRIM22 as an anti-HIV effector *in vivo.*  A study monitoring gene expression in high-risk HIV-1 negative individuals detected a positive correlation between TRIM22 expression and increased control of HIV-1 infection (Singh et al., 2011). It was observed that IFNβ and TRIM22 levels in peripheral blood mononuclear cells (PBMCs) were increased in patients after HIV-1 infection. In addition, infected patients expressing higher TRIM22 levels exhibited significantly lower viral loads and significantly higher CD4+ T cell counts, suggesting that TRIM22 may play a role in controlling HIV-1 infection. Surprisingly, a significant inverse correlation was observed between the closely related, IFN-inducible TRIM5α protein and IFNβ expression (Singh et al., 2011). TRIM22 and TRIM5α have been under positive selection episodically for approximately 23 million years; however, these two genes have evolved in a mutually exclusive manner, with only one being selected for in a given primate lineage (Sawyer et al., 2007). Since human TRIM5α has little to no inhibitory effect on HIV-1 replication compared to the potent inhibitory effects of rhesus TRIM5α, it is possible that human TRIM22 has evolved to compensate for the loss of antiretroviral activity of human TRIM5α.

## **5. Lifecycle target: HIV-1 protein**

## **5.1 OAS1/RNaseL**

#### **5.1.1 OAS1/RNaseL-mediated inhibition of HIV-1 translation**

Similar to PKR, 2'5' oligoadenylate synthetase 1 (OAS1) senses viral infection by recognizing dsRNA, and is constitutively expressed in an inactive monomeric form (Sadler et al., 2009). However, unlike PKR, OAS1 recognizes dsRNA in the absence of a dsRBD. Exactly how OAS1 recognizes dsRNA without this domain remains unclear (Kodym et al., 2009, Marie et al., 1990, Sadler & Williams, 2008). Once OAS1 is activated by dsRNA, it forms a tetramer, which converts ATP molecules into 2'5' oligoadenylates (2-5A) (Marie et al., 1990, Hovanessian, 2007). These 2-5As are strong inducers of an enzyme called RNaseL. By binding to the N-terminus of RNaseL, 2-5As activate the ribonuclease activity of RNaseL, which then degrades single-stranded RNA (ssRNA) by cleaving the phosphodiester bonds of uracil rich sequences to produce products with 3' monophosphate and 5' hydroxyl termini (Chakrabarti et al., 2011, Malathi et al., 2007). This leads to a reduction in mRNA translation and induces the RIG-I and/or MDA5 pathways, which are positive regulators of interferon signalling and the antiviral response (Figure 8) (Malathi et al., 2007).

The HIV-1 TAR element (dsRNA) is sufficient for OAS1 recognition and activation, and OAS1-TAR binding leads to 2-5A production, RNaseL recognition, cleavage of HIV-1 transcripts and inhibition of protein translation (Maitra et al., 1994). Interestingly, Jurkat T cells that overexpress RNaseL show a substantial decrease in HIV-1 mRNA production, as well as a 1000-fold decrease in HIV-1 replication two weeks post-infection (Maitra & Silverman, 1998). In addition, overexpression of RNaseL leads to accelerated HIV-induced apoptotic cell death, possibly through Fas-Fas ligand-mediated signalling (Maitra & Silverman, 1998). In contrast, cells devoid of RNaseL are unable to restrict HIV-1 replication, highlighting the importance of this pathway in cellular restriction of virus replication.

## **5.1.2 Countermeasures to OAS1/RNaseL-mediated HIV-1 restriction**

Overexpression studies have shown that OAS1/RNaseL-mediated HIV-1 restriction is possible; however, when RNaseL is expressed at biologically relevant levels, HIV-1 can inhibit this pathway. The HIV-1 Tat protein sequesters the TAR element *in vivo*, thus preventing OAS1-TAR binding and RNaseL activation (Schroder et al., 1990b). It is possible that this pathway is responsible for the low levels of mRNA production during the initial stages of HIV-1 infection; however, as Tat expression increases, activation of the OAS1/RNaseL pathway decreases and the production of HIV-1 mRNA rises significantly. In addition, cells that contain latent virus may be kept under control by the OAS1/RNaseL pathway. Nevertheless, the ability of cells to endocytose Tat from other apoptotic cells may lead to trans-activation of the TAR element and HIV-1 mRNA production in these latently infected cells (Schroder et al., 1990b, Frankel & Pabo, 1988).

Although PKR and OAS1/RNaseL are potent HIV-1 restriction factors *in vitro*, the HIV-1 Tat protein is an effective viral countermeasure *in vivo*. Research on these restriction factors however, does provide insight into future therapeutics that may target the HIV-1 Tat protein, or overpower Tat for TAR binding. *In vitro* studies have already shown that shRNA directed against Tat or a TAR RNA decoy can provide long-term inhibition of HIV-1 replication; however, these studies must still be confirmed *in vivo* (Li et al., 2005).

## **5.2 TRIM22**

156 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

noted that all direct evidence to date of TRIM22 inhibiting HIV-1 transcription has been through the use of LTR-driven reporter constructs. It will be important to test the effects of TRIM22 on replication-competent HIV-1, which will provide a more natural scenario of virus transcription, Tat-induction, and possible effects of other HIV-1 accessory proteins.

Interestingly, there is evidence to support a role for TRIM22 as an anti-HIV effector *in vivo.*  A study monitoring gene expression in high-risk HIV-1 negative individuals detected a positive correlation between TRIM22 expression and increased control of HIV-1 infection (Singh et al., 2011). It was observed that IFNβ and TRIM22 levels in peripheral blood mononuclear cells (PBMCs) were increased in patients after HIV-1 infection. In addition, infected patients expressing higher TRIM22 levels exhibited significantly lower viral loads and significantly higher CD4+ T cell counts, suggesting that TRIM22 may play a role in controlling HIV-1 infection. Surprisingly, a significant inverse correlation was observed between the closely related, IFN-inducible TRIM5α protein and IFNβ expression (Singh et al., 2011). TRIM22 and TRIM5α have been under positive selection episodically for approximately 23 million years; however, these two genes have evolved in a mutually exclusive manner, with only one being selected for in a given primate lineage (Sawyer et al., 2007). Since human TRIM5α has little to no inhibitory effect on HIV-1 replication compared to the potent inhibitory effects of rhesus TRIM5α, it is possible that human TRIM22 has

evolved to compensate for the loss of antiretroviral activity of human TRIM5α.

interferon signalling and the antiviral response (Figure 8) (Malathi et al., 2007).

Similar to PKR, 2'5' oligoadenylate synthetase 1 (OAS1) senses viral infection by recognizing dsRNA, and is constitutively expressed in an inactive monomeric form (Sadler et al., 2009). However, unlike PKR, OAS1 recognizes dsRNA in the absence of a dsRBD. Exactly how OAS1 recognizes dsRNA without this domain remains unclear (Kodym et al., 2009, Marie et al., 1990, Sadler & Williams, 2008). Once OAS1 is activated by dsRNA, it forms a tetramer, which converts ATP molecules into 2'5' oligoadenylates (2-5A) (Marie et al., 1990, Hovanessian, 2007). These 2-5As are strong inducers of an enzyme called RNaseL. By binding to the N-terminus of RNaseL, 2-5As activate the ribonuclease activity of RNaseL, which then degrades single-stranded RNA (ssRNA) by cleaving the phosphodiester bonds of uracil rich sequences to produce products with 3' monophosphate and 5' hydroxyl termini (Chakrabarti et al., 2011, Malathi et al., 2007). This leads to a reduction in mRNA translation and induces the RIG-I and/or MDA5 pathways, which are positive regulators of

The HIV-1 TAR element (dsRNA) is sufficient for OAS1 recognition and activation, and OAS1-TAR binding leads to 2-5A production, RNaseL recognition, cleavage of HIV-1 transcripts and inhibition of protein translation (Maitra et al., 1994). Interestingly, Jurkat T cells that overexpress RNaseL show a substantial decrease in HIV-1 mRNA production, as well as a 1000-fold decrease in HIV-1 replication two weeks post-infection (Maitra & Silverman, 1998). In addition, overexpression of RNaseL leads to accelerated HIV-induced apoptotic cell death, possibly through Fas-Fas ligand-mediated signalling (Maitra & Silverman, 1998). In contrast, cells devoid of RNaseL are unable to restrict HIV-1 replication, highlighting the importance of this pathway in cellular restriction of virus replication.

**5.1.1 OAS1/RNaseL-mediated inhibition of HIV-1 translation** 

**4.2.3 TRIM22-mediated effects on HIV-1 replication** *in vivo*

**5. Lifecycle target: HIV-1 protein** 

**5.1 OAS1/RNaseL** 

#### **5.2.1 TRIM22-mediated HIV-1 restriction**

Interestingly, TRIM22 appears to restrict HIV-1 replication by multiple mechanisms. TRIM22 has been shown to be an integral part of IFNβ-mediated HIV-1 restriction, and its expression can restrict HIV-1 replication in several transformed cell lines. In cell lines such as human osteosarcoma (HOS) and HeLa, TRIM22 restricts the release of virus, but has little to no effect on intracellular levels of the HIV-1 structural protein Gag. Conversely, in the osteosarcoma cell lines U2OS and 143B, TRIM22 expression not only restricts the release of virus, but also prevents the intracellular accumulation of Gag protein (Barr et al., 2008). The presence of different mechanisms in different cell lines, and the fact that multiple localizations for TRIM22 have been observed, suggests that there are many complex details of TRIM22 function that remain to be discovered (Figure 9).

Further investigation of the mechanism of TRIM22-induced restriction in HOS cells revealed that TRIM22 likely interferes with intracellular trafficking of the HIV-1 Gag protein (Barr et al., 2008). Of note, Gag is both necessary and sufficient for budding and release of virus particles. This property allows Gag, in the absence of other viral proteins, to assemble and bud from the cell membrane, resulting in the production of non-infectious, virus-like particles (VLP). Importantly, TRIM22 expression was shown to inhibit the release of VLPs and prevent accumulation of Gag at the cell membrane, a step that is critical for virus assembly. These effects were dependent on the E3 ubiquitin ligase activity of TRIM22.

#### **5.3 TRIM5α**

#### **5.3.1 TRIM5α-mediated HIV-1 restriction**

RhTRIM5α was originally shown to target incoming HIV-1 capsid proteins, thus inhibiting early stages of HIV-1 replication (see Section 2.1). For this reason, it was initially assumed that rhTRIM5α didn't affect late stages of HIV-1 replication. However, subsequent research showed that it could also restrict HIV-1 through rapid degradation of the Gag polyprotein, the main structural component of HIV-1 (Sakuma et al., 2007). Treatment of cells

Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 159

Fig. 9. Possible mechanisms of TRIM22-mediated HIV-1 restriction. TRIM22 prevents accumulation of the Gag polyprotein at the plasma membrane, and as such may bind directly to Gag. Alternatively, TRIM22 may mono-ubiquitinate or polyubiquitinate Gag. Experiments using LTR-luciferase reporter constructs have also shown that TRIM22 restricts

Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein (Ubl) that was first discovered in 1979, and is highly induced in the presence of IFN-/ (Herrmann et al., 2007, Farrell et al., 1979). The ISG15 protein is composed of two ubiquitin-like domains that can modify substrate proteins similarly to ubiquitin (Jeon et al., 2010). In addition, the Cterminus of ISG15 contains the Gly-Gly motif which is required for ISG15 conjugation to target proteins. ISG15ylation requires the aid of an E1 activating protein, an E2 conjugating protein, and an E3 ligase protein. First, the ISG15-specific E1 activating protein, Ube1L, uses ATP to adenylate the Gly-Gly motif of ISG15. Ube1L then forms a thioester bond between its catalytic cysteine residue and the C-terminal Gly residue of ISG15. With the help of the E2 conjugating protein, UbcH8, and a substrate-specific E3 ligase, ISG15 forms a covalent bond with the -NH2 of a substrate lysine residue (reviewed in (Kerscher et al., 2006)). Importantly, ISG15 is conjugated to both viral and host proteins, and can have an antiviral effect by altering the activity of substrate proteins required for viral propagation (Harty et

ISG15ylation has been implicated in restriction of HIV-1 replication at the budding stage of the HIV-1 lifecycle (Okumura et al., 2006, Pincetic et al., 2010). The HIV-1 Gag protein contains a late-budding or L domain that has a PTAP motif, and can interact with endosomal sorting complex required for transport (ESCRT)-I. Specifically, tumour susceptibility gene 101 (TSG101), a component of ESCRT-I, interacts with the PTAP motif on

transcription from the 5' HIV-1 LTR.

**6.1 ISG15** 

al., 2009, Shi et al., 2010).

**6. Lifecycle target: HIV-1 budding** 

Fig. 8. OAS1/RNaseL-mediated inhibition of HIV-1 protein translation. Following recognition of the HIV-1 TAR element (dsRNA), OAS1 forms a tetramer whose catalytic activity turns ATP molecules into 2'5'oligoadenylates (2-5As). The 2-5As activate the RNaseL enzyme, which leads to its dimerization and stimulates it to cleave ssRNA (such as mRNA). Cleavage of mRNA by RNaseL results in the inhibition of protein translation, including the translation of viral proteins.

with the proteasome inhibitors MG132 and MG115 did not restore HIV-1 Gag protein stability, suggesting that late restriction by rhTRIM5α occurs independently of the ubiquitin/proteasome system. Interestingly, similar to early-stage restriction, the human orthologue of rhTRIM5α did not restrict late stages of HIV-1 replication (Sakuma et al., 2007, Sakuma et al., 2007, Sakuma et al., 2010).

Unlike early-stage rhTRIM5α-mediated restriction, the B30.2 domain was dispensable for Gag degradation. However, two amino acids in the coiled-coil domain (M133 and T146) and the E3 ligase activity of rhTRIM5α were required for late-stage restriction (Sakuma et al., 2010). It is possible that rhTRIM5α acts synergistically with other TRIM proteins or cell proteases to degrade the Gag polyprotein. For example, TRIM22 has been shown to affect late stages of HIV-1 replication and thus may be involved in rhTRIM5α-mediated restriction (Barr et al., 2008). Additional research will determine if other TRIM proteins are involved and help define the exact mechanism of late-stage rhTRIM5α-mediated restriction.

Fig. 9. Possible mechanisms of TRIM22-mediated HIV-1 restriction. TRIM22 prevents accumulation of the Gag polyprotein at the plasma membrane, and as such may bind directly to Gag. Alternatively, TRIM22 may mono-ubiquitinate or polyubiquitinate Gag. Experiments using LTR-luciferase reporter constructs have also shown that TRIM22 restricts transcription from the 5' HIV-1 LTR.

## **6. Lifecycle target: HIV-1 budding**

## **6.1 ISG15**

158 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Fig. 8. OAS1/RNaseL-mediated inhibition of HIV-1 protein translation. Following recognition of the HIV-1 TAR element (dsRNA), OAS1 forms a tetramer whose catalytic activity turns ATP molecules into 2'5'oligoadenylates (2-5As). The 2-5As activate the RNaseL enzyme, which leads to its dimerization and stimulates it to cleave ssRNA (such as mRNA). Cleavage of mRNA by RNaseL results in the inhibition of protein translation, including the

with the proteasome inhibitors MG132 and MG115 did not restore HIV-1 Gag protein stability, suggesting that late restriction by rhTRIM5α occurs independently of the ubiquitin/proteasome system. Interestingly, similar to early-stage restriction, the human orthologue of rhTRIM5α did not restrict late stages of HIV-1 replication (Sakuma et al., 2007,

Unlike early-stage rhTRIM5α-mediated restriction, the B30.2 domain was dispensable for Gag degradation. However, two amino acids in the coiled-coil domain (M133 and T146) and the E3 ligase activity of rhTRIM5α were required for late-stage restriction (Sakuma et al., 2010). It is possible that rhTRIM5α acts synergistically with other TRIM proteins or cell proteases to degrade the Gag polyprotein. For example, TRIM22 has been shown to affect late stages of HIV-1 replication and thus may be involved in rhTRIM5α-mediated restriction (Barr et al., 2008). Additional research will determine if other TRIM proteins are involved

and help define the exact mechanism of late-stage rhTRIM5α-mediated restriction.

translation of viral proteins.

Sakuma et al., 2007, Sakuma et al., 2010).

Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein (Ubl) that was first discovered in 1979, and is highly induced in the presence of IFN-/ (Herrmann et al., 2007, Farrell et al., 1979). The ISG15 protein is composed of two ubiquitin-like domains that can modify substrate proteins similarly to ubiquitin (Jeon et al., 2010). In addition, the Cterminus of ISG15 contains the Gly-Gly motif which is required for ISG15 conjugation to target proteins. ISG15ylation requires the aid of an E1 activating protein, an E2 conjugating protein, and an E3 ligase protein. First, the ISG15-specific E1 activating protein, Ube1L, uses ATP to adenylate the Gly-Gly motif of ISG15. Ube1L then forms a thioester bond between its catalytic cysteine residue and the C-terminal Gly residue of ISG15. With the help of the E2 conjugating protein, UbcH8, and a substrate-specific E3 ligase, ISG15 forms a covalent bond with the -NH2 of a substrate lysine residue (reviewed in (Kerscher et al., 2006)). Importantly, ISG15 is conjugated to both viral and host proteins, and can have an antiviral effect by altering the activity of substrate proteins required for viral propagation (Harty et al., 2009, Shi et al., 2010).

ISG15ylation has been implicated in restriction of HIV-1 replication at the budding stage of the HIV-1 lifecycle (Okumura et al., 2006, Pincetic et al., 2010). The HIV-1 Gag protein contains a late-budding or L domain that has a PTAP motif, and can interact with endosomal sorting complex required for transport (ESCRT)-I. Specifically, tumour susceptibility gene 101 (TSG101), a component of ESCRT-I, interacts with the PTAP motif on

Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 161

2009). Conversely, the N-terminal transmembrane region of tetherin may anchor to the cell membrane whereas the GPI terminus may associate with the budding virus. Another plausible hypothesis is that one tetherin molecule binds to the cell membrane and another tetherin molecule binds to the budding virus. In this case, HIV-1 release would be inhibited by the interaction between coiled-coil regions in the tetherin dimer (Perez-Caballero et al.,

In spite of tetherin-induced restriction of HIVΔVpu virus, trapping virus at the membrane is not sufficient to prevent cell-to-cell transmission of HIV-1 (Casartelli et al., 2010, Kuhl et al., 2010). This binding leads to efficient cell-to-cell transmission of the virus. Interestingly, tetherin has also been shown to prevent HIV-1 transmission through this route. Specifically, tetherin appears to link budding HIV-1 particles together in a chain-like fashion, tethering them to the cell membrane in viral aggregates (Hammonds et al., 2010). The formation of these aggregates prevents HIV-1 transmission through the virological synapse, possibly because the aggregates cannot fuse properly to the target cells (Casartelli et al., 2010). It is possible that tetherin is incorporated into the budding virions and that this causes abnormal virus fusion to the target cell; however, more studies are needed to confirm the presence of tetherin in HIV-1 particles and further define its role in HIV-1 restriction at the virological synapse. Taken as a whole, current research suggests that tetherin may have two roles in HIV-1 restriction: tethering virus particles to the cell membrane and preventing cell-to-cell

Fig. 10. Tetherin-induced inhibition of HIV-1 particle release. Each model centres on the dimerization of two tetherin molecules. In model 1, the N-terminal transmembrane regions of the tetherin dimer anchor to the cell surface and the C-terminal GPI domains associate with the budding virus. Model 2 is the opposite of model 1. In model 3, each tetherin molecule of the dimer associates with either only the budding virus or only the cell

membrane, and HIV-1 restriction depends on the interaction between the coiled-coil regions.

2009) (Figure 10).

transmission of HIV-1 to uninfected target cells.

the HIV-1 Gag protein, and subsequently recruits ESCRT-II and ESCRT-III (VerPlank et al., 2001). ESCRT-III promotes viral budding and the recruitment of vacuolar protein sorting (Vps4), an ATPase that releases ESCRT factors from the membrane (reviewed in (Usami et al., 2009)(Williams & Urbe, 2007)). Interestingly, ISG15 has been shown to interrupt the interaction between TSG101 and the HIV-1 Gag protein; however, neither TSG101 nor HIV-1 Gag are directly modified with ISG15 (Okumura et al., 2006). ISG15 was also shown to interfere with the recruitment of Vps4 to the HIV-1 budding complex; however, the mechanism of this interruption has not yet been characterized. It is possible that charged multi-vesicular body protein CHMP-5, a component of ESCRT-III, prevents the recruitment of Vps4 as it was shown to be ISG15ylated (Pincetic et al., 2010). Further characterization of ISG15-mediated HIV-1 restriction is required to understand the antiviral effects of ISG15 on HIV-1 budding.

## **7. Lifecycle target: HIV-1 release**

#### **7.1 Tetherin**

#### **7.1.1 Tetherin: History and structure**

For the past two decades, scientists have known that the HIV-1 Vpu protein is required for efficient release of virus particles (Gottlinger et al., 1993). HIV-1 particles lacking Vpu (HIV∆Vpu) cannot release properly from certain cells; however, until recently the cause of this phenotype was unknown (Varthakavi et al., 2003). Tetherin (also known as BST-2 and CD317) was first suggested to be an antiviral protein in 2006, when it was shown to target the K5 protein of Kaposi's sarcoma-associated herpes virus (Bartee et al., 2006). A few years later, tetherin was identified as the causative agent of the HIVΔVpu phenotype when it was shown to inhibit the release of HIVΔVpu particles at the cell membrane of certain restrictive cells such as the HeLa cell line (Neil et al., 2008).

Tetherin is an interferon-induced, transmembrane protein that contains a short cytoplasmic N-terminus, a transmembrane region, an ectodomain, and a C-terminal glycosylphosphatidylinositol (GPI) anchor (Kupzig et al., 2003). Both the transmembrane region and the ectodomain are made from a single alpha helix, and the ectodomain contains an additional coiled-coil region. Tetherin exists as a homodimer, which is formed by disulphide bridges between the coiled-coil ectodomain regions of two tetherin proteins. Importantly, tetherin dimerization has been shown to be crucial for HIV∆Vpu restriction (Andrew et al., 2009).

#### **7.1.2 Tetherin-mediated restriction of HIV-1 release**

Currently, the precise mechanism of tetherin-induced HIV∆Vpu restriction is uncertain. Among the proposed models, two aspects seem to be consistent: 1) tetherin proteins form homodimers via the coiled-coil regions in their ectodomains and 2) the N- and C-terminus of tetherin are incorporated into the cell and/or viral membrane (Perez-Caballero et al., 2009). Tetherin homodimers localize to the cell membrane where they associate with HIV-1 Gag oligomers on lipid rafts (where budding of the virus occurs) (Neil et al., 2008, Nguyen & Hildreth, 2000). Details of the tethering mechanism underlying restriction are poorly understood. One favourable hypothesis involves the C-terminal GPI being anchored to the cell membrane and the N-terminal transmembrane region being associated with the Gag oliogomers of the budding virus. As budding occurs, the cell membrane-bound C-terminus tethers the budding virus to the cell via the virion-bound N-terminus (Perez-Caballero et al.,

the HIV-1 Gag protein, and subsequently recruits ESCRT-II and ESCRT-III (VerPlank et al., 2001). ESCRT-III promotes viral budding and the recruitment of vacuolar protein sorting (Vps4), an ATPase that releases ESCRT factors from the membrane (reviewed in (Usami et al., 2009)(Williams & Urbe, 2007)). Interestingly, ISG15 has been shown to interrupt the interaction between TSG101 and the HIV-1 Gag protein; however, neither TSG101 nor HIV-1 Gag are directly modified with ISG15 (Okumura et al., 2006). ISG15 was also shown to interfere with the recruitment of Vps4 to the HIV-1 budding complex; however, the mechanism of this interruption has not yet been characterized. It is possible that charged multi-vesicular body protein CHMP-5, a component of ESCRT-III, prevents the recruitment of Vps4 as it was shown to be ISG15ylated (Pincetic et al., 2010). Further characterization of ISG15-mediated HIV-1 restriction is required to understand the antiviral effects of ISG15 on

For the past two decades, scientists have known that the HIV-1 Vpu protein is required for efficient release of virus particles (Gottlinger et al., 1993). HIV-1 particles lacking Vpu (HIV∆Vpu) cannot release properly from certain cells; however, until recently the cause of this phenotype was unknown (Varthakavi et al., 2003). Tetherin (also known as BST-2 and CD317) was first suggested to be an antiviral protein in 2006, when it was shown to target the K5 protein of Kaposi's sarcoma-associated herpes virus (Bartee et al., 2006). A few years later, tetherin was identified as the causative agent of the HIVΔVpu phenotype when it was shown to inhibit the release of HIVΔVpu particles at the cell membrane of certain restrictive

Tetherin is an interferon-induced, transmembrane protein that contains a short cytoplasmic N-terminus, a transmembrane region, an ectodomain, and a C-terminal glycosylphosphatidylinositol (GPI) anchor (Kupzig et al., 2003). Both the transmembrane region and the ectodomain are made from a single alpha helix, and the ectodomain contains an additional coiled-coil region. Tetherin exists as a homodimer, which is formed by disulphide bridges between the coiled-coil ectodomain regions of two tetherin proteins. Importantly, tetherin dimerization has been shown to be crucial for HIV∆Vpu restriction

Currently, the precise mechanism of tetherin-induced HIV∆Vpu restriction is uncertain. Among the proposed models, two aspects seem to be consistent: 1) tetherin proteins form homodimers via the coiled-coil regions in their ectodomains and 2) the N- and C-terminus of tetherin are incorporated into the cell and/or viral membrane (Perez-Caballero et al., 2009). Tetherin homodimers localize to the cell membrane where they associate with HIV-1 Gag oligomers on lipid rafts (where budding of the virus occurs) (Neil et al., 2008, Nguyen & Hildreth, 2000). Details of the tethering mechanism underlying restriction are poorly understood. One favourable hypothesis involves the C-terminal GPI being anchored to the cell membrane and the N-terminal transmembrane region being associated with the Gag oliogomers of the budding virus. As budding occurs, the cell membrane-bound C-terminus tethers the budding virus to the cell via the virion-bound N-terminus (Perez-Caballero et al.,

HIV-1 budding.

**7.1 Tetherin** 

(Andrew et al., 2009).

**7. Lifecycle target: HIV-1 release** 

**7.1.1 Tetherin: History and structure** 

cells such as the HeLa cell line (Neil et al., 2008).

**7.1.2 Tetherin-mediated restriction of HIV-1 release** 

2009). Conversely, the N-terminal transmembrane region of tetherin may anchor to the cell membrane whereas the GPI terminus may associate with the budding virus. Another plausible hypothesis is that one tetherin molecule binds to the cell membrane and another tetherin molecule binds to the budding virus. In this case, HIV-1 release would be inhibited by the interaction between coiled-coil regions in the tetherin dimer (Perez-Caballero et al., 2009) (Figure 10).

In spite of tetherin-induced restriction of HIVΔVpu virus, trapping virus at the membrane is not sufficient to prevent cell-to-cell transmission of HIV-1 (Casartelli et al., 2010, Kuhl et al., 2010). This binding leads to efficient cell-to-cell transmission of the virus. Interestingly, tetherin has also been shown to prevent HIV-1 transmission through this route. Specifically, tetherin appears to link budding HIV-1 particles together in a chain-like fashion, tethering them to the cell membrane in viral aggregates (Hammonds et al., 2010). The formation of these aggregates prevents HIV-1 transmission through the virological synapse, possibly because the aggregates cannot fuse properly to the target cells (Casartelli et al., 2010). It is possible that tetherin is incorporated into the budding virions and that this causes abnormal virus fusion to the target cell; however, more studies are needed to confirm the presence of tetherin in HIV-1 particles and further define its role in HIV-1 restriction at the virological synapse. Taken as a whole, current research suggests that tetherin may have two roles in HIV-1 restriction: tethering virus particles to the cell membrane and preventing cell-to-cell transmission of HIV-1 to uninfected target cells.

Fig. 10. Tetherin-induced inhibition of HIV-1 particle release. Each model centres on the dimerization of two tetherin molecules. In model 1, the N-terminal transmembrane regions of the tetherin dimer anchor to the cell surface and the C-terminal GPI domains associate with the budding virus. Model 2 is the opposite of model 1. In model 3, each tetherin molecule of the dimer associates with either only the budding virus or only the cell membrane, and HIV-1 restriction depends on the interaction between the coiled-coil regions.

Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 163

create effective therapies. In the short-term, drug-based therapies are the most likely to be successful, and due to the constant development of HIV-1 resistance, new drugs are always needed. To date, there are 32 antiretroviral drugs approved by the FDA, and none of these drugs target the same steps in the HIV-1 lifecycle as cellular restriction factors. This makes restriction factors excellent candidates for drug design, specifically proteins such as TRIM22 or ISG15, which do not appear to be directly targeted by any HIV-1 proteins. With the development of any new HIV-1 drug, resistance is always a concern; however, identifying new stages of the HIV-1 lifecycle to antagonize may reduce viral replication enough to

Alternatively, drugs targeting cellular restriction factor antagonists could be developed. For example, the HIV-1 Vif protein antagonizes APOBEC3 by marking it for proteasomal degradation (Yu et al., 2003). Inhibiting the interaction between APOBEC3 and Vif may prevent this degradation, and many studies have focused on identifying important interacting regions on both proteins (Chen et al., 2009, Huthoff & Malim, 2007, Yamashita et al., 2008). Unfortunately, many of the interacting regions on Vif differ depending on the specific APOBEC3 protein it is interacting with, and it is likely that three-dimensional structures of APOBEC3 proteins bound to Vif will be needed to effectively target this interaction (Russell et al., 2009, Tian et al., 2006). Despite these challenges, one small molecule antagonist of Vif was recently identified (RN-18) and shown to decrease levels of Vif protein *in vitro* (Nathans et al., 2008)*.* It will be interesting to follow-up this research *in vivo*, and learn whether RN-18 has an effect on HIV-1 replication in infected individuals. Finally, another option to shield APOBEC3 from Vif involves designing a molecule that binds to APOBEC3 and prevents this interaction; however, this molecule would also have to

The HIV-1 Tat protein is another attractive drug target, since it is essential for HIV-1 replication and antagonizes two HIV-1 restriction factors (PKR and OAS1). For unknown reasons, Tat hasn't received much attention as a potential drug target, possibly because its actions are hard to re-create *in vitro*. However, inhibition of Tat potently inhibits HIV-1 replication, and further research in this area is certainly warranted. Drugs that target the HIV-1 Vpu protein could also be considered; however, because Vpu is not critical for HIV-1 replication *in vivo* it may not be an ideal candidate (Friborg et al., 1995, Terwilliger et al., 1989). Conversely, drugs that mimic the effects of tetherin, but are resistant to Vpu, may successfully reduce HIV-1 replication. In fact, an artificial Vpu-resistant tetherin protein was recently engineered; however, it has not yet been tested in clinical trials (Perez-Caballero et al., 2009). Inhibiting the action of host proteins that assist HIV-1 replication is another possibility. For example, cyclophilin A (CypA) is required for HIV-1 replication, and without Cyp A HIV-1 virions are not infectious (Sokolskaja & Luban, 2006, Thali et al., 1994). Small molecule inhibitors targeting Cyp A may block HIV-1 replication at the uncoating stage, and targeting a host protein avoids the problem of viral resistance. However, HIV-1 propagation in the absence of Cyp A may allow HIV-1 variants to evolve

An alternative approach to HIV-1 therapy involves using cellular restriction factors in conjunction with gene therapy. In this approach, DNA encoding one or more cellular restriction factors is inserted into target cells to interfere with HIV-1 infection or replication.

preserve APOBEC3's antiviral function (Albin & Harris, 2010).

that no longer require Cyp A for replication.

**8.2 Gene therapy approach** 

prevent escape mutants.

#### **7.1.3 Countermeasures to tetherin-mediated HIV-1 restriction**

The HIV-1 Vpu protein has been shown to degrade tetherin, thus abolishing its anti-HIV-1 effects. Specifically, the transmembrane domain of Vpu can bind to tetherin, and this domain is necessary for Vpu localization to the cell membrane and subsequent association with tetherin (Kobayashi et al., 2011, Skasko et al., 2011, Vigan & Neil, 2010). Interestingly, mutating a single amino acid in the Vpu transmembrane domain (A18H) traps Vpu in the endoplasmic reticulum, where it is unable to translocate to the cell membrane or degrade tetherin (Skasko et al., 2011). Furthermore, it has recently been shown that four amino acids in the Vpu transmembrane domain (I34, L37, L41, and T45) are necessary for Vpu interaction with and antagonism of tetherin (Kobayashi et al., 2011). Mutational experiments with tetherin show that its transmembrane domain is also important for Vpu-tetherin interactions. Given this data, it is likely that Vpu and tetherin interact through their respective transmembrane domains and thus, that these domains are critical for Vpumediated tetherin degradation.

There are currently two major hypotheses for the mechanism of Vpu-mediated tetherin degradation. The first hypothesis involves tetherin degradation at a post-translational step, as there is no decrease in tetherin transcript levels in the presence of Vpu, but there is a decrease in protein expression (Douglas et al., 2009, Mangeat et al., 2009). It is possible that this degradation is mediated by Vpu binding to β-transducin repeat-containing protein (β-TrCP), which is a substrate adaptor for a multi-subunit E3 ligase complex and is able to interact with Vpu through its C-terminus. The consequence of Vpu binding to the β-TrCP-E3 ligase complex is the ubiquitination of cell surface proteins, including tetherin, on lysine residues at positions 18 and/or 21 (Mangeat et al., 2009, Guatelli, 2009, Iwabu et al., 2009, Pardieu et al., 2010). Tetherin ubiquitination leads to its endocytosis from the cell membrane and degradation through either the proteasomal or lysosomal degradation pathways (Douglas et al., 2009, Mitchell et al., 2009, Van Damme et al., 2008).

Tetherin degradation by Vpu and the β-TrCP-E3 ligase complex however, is insufficient to explain one interesting finding: Vpu constructs that contain mutations in the motif that recognizes β-TrCP can still partially, or in some cases totally, overcome tetherin-mediated HIV-1 restriction (Douglas et al., 2009, Mangeat et al., 2009, Mangeat et al., 2009, Mitchell et al., 2009, Miyagi et al., 2009). Thus, a second hypothesis has been proposed that involves tetherin degradation in late endosomal compartments. It has previously been shown that Vpu is distributed throughout the trans-golgi network, and that it can modulate tetherin cell surface expression by sequestering it intracellularly. Sequestration of tetherin prevents its anterograde trafficking to the cell membrane and subsequently delivers it to late endosomal compartments (Dube et al., 2010, Dube et al., 2010, Hauser et al., 2010). Of note, the specifics of this mechanism of Vpu-mediated degradation are still largely uncharacterized and further studies are needed to elucidate the details of this mechanism. However, taken together, current research suggests that there may be two mechanisms by which Vpu counteracts the antiviral activity of tetherin.

#### **8. Conclusion**

#### **8.1 Pharmaceutical approach**

In the future, it is probable that new HIV-1 therapies will be developed based on the actions of cellular restriction factors. Currently, many studies are focused on defining the molecular mechanisms of these factors; however, it is still unclear how this information will be used to

The HIV-1 Vpu protein has been shown to degrade tetherin, thus abolishing its anti-HIV-1 effects. Specifically, the transmembrane domain of Vpu can bind to tetherin, and this domain is necessary for Vpu localization to the cell membrane and subsequent association with tetherin (Kobayashi et al., 2011, Skasko et al., 2011, Vigan & Neil, 2010). Interestingly, mutating a single amino acid in the Vpu transmembrane domain (A18H) traps Vpu in the endoplasmic reticulum, where it is unable to translocate to the cell membrane or degrade tetherin (Skasko et al., 2011). Furthermore, it has recently been shown that four amino acids in the Vpu transmembrane domain (I34, L37, L41, and T45) are necessary for Vpu interaction with and antagonism of tetherin (Kobayashi et al., 2011). Mutational experiments with tetherin show that its transmembrane domain is also important for Vpu-tetherin interactions. Given this data, it is likely that Vpu and tetherin interact through their respective transmembrane domains and thus, that these domains are critical for Vpu-

There are currently two major hypotheses for the mechanism of Vpu-mediated tetherin degradation. The first hypothesis involves tetherin degradation at a post-translational step, as there is no decrease in tetherin transcript levels in the presence of Vpu, but there is a decrease in protein expression (Douglas et al., 2009, Mangeat et al., 2009). It is possible that this degradation is mediated by Vpu binding to β-transducin repeat-containing protein (β-TrCP), which is a substrate adaptor for a multi-subunit E3 ligase complex and is able to interact with Vpu through its C-terminus. The consequence of Vpu binding to the β-TrCP-E3 ligase complex is the ubiquitination of cell surface proteins, including tetherin, on lysine residues at positions 18 and/or 21 (Mangeat et al., 2009, Guatelli, 2009, Iwabu et al., 2009, Pardieu et al., 2010). Tetherin ubiquitination leads to its endocytosis from the cell membrane and degradation through either the proteasomal or lysosomal degradation pathways

Tetherin degradation by Vpu and the β-TrCP-E3 ligase complex however, is insufficient to explain one interesting finding: Vpu constructs that contain mutations in the motif that recognizes β-TrCP can still partially, or in some cases totally, overcome tetherin-mediated HIV-1 restriction (Douglas et al., 2009, Mangeat et al., 2009, Mangeat et al., 2009, Mitchell et al., 2009, Miyagi et al., 2009). Thus, a second hypothesis has been proposed that involves tetherin degradation in late endosomal compartments. It has previously been shown that Vpu is distributed throughout the trans-golgi network, and that it can modulate tetherin cell surface expression by sequestering it intracellularly. Sequestration of tetherin prevents its anterograde trafficking to the cell membrane and subsequently delivers it to late endosomal compartments (Dube et al., 2010, Dube et al., 2010, Hauser et al., 2010). Of note, the specifics of this mechanism of Vpu-mediated degradation are still largely uncharacterized and further studies are needed to elucidate the details of this mechanism. However, taken together, current research suggests that there may be two mechanisms by which Vpu

In the future, it is probable that new HIV-1 therapies will be developed based on the actions of cellular restriction factors. Currently, many studies are focused on defining the molecular mechanisms of these factors; however, it is still unclear how this information will be used to

**7.1.3 Countermeasures to tetherin-mediated HIV-1 restriction** 

(Douglas et al., 2009, Mitchell et al., 2009, Van Damme et al., 2008).

counteracts the antiviral activity of tetherin.

**8. Conclusion** 

**8.1 Pharmaceutical approach** 

mediated tetherin degradation.

create effective therapies. In the short-term, drug-based therapies are the most likely to be successful, and due to the constant development of HIV-1 resistance, new drugs are always needed. To date, there are 32 antiretroviral drugs approved by the FDA, and none of these drugs target the same steps in the HIV-1 lifecycle as cellular restriction factors. This makes restriction factors excellent candidates for drug design, specifically proteins such as TRIM22 or ISG15, which do not appear to be directly targeted by any HIV-1 proteins. With the development of any new HIV-1 drug, resistance is always a concern; however, identifying new stages of the HIV-1 lifecycle to antagonize may reduce viral replication enough to prevent escape mutants.

Alternatively, drugs targeting cellular restriction factor antagonists could be developed. For example, the HIV-1 Vif protein antagonizes APOBEC3 by marking it for proteasomal degradation (Yu et al., 2003). Inhibiting the interaction between APOBEC3 and Vif may prevent this degradation, and many studies have focused on identifying important interacting regions on both proteins (Chen et al., 2009, Huthoff & Malim, 2007, Yamashita et al., 2008). Unfortunately, many of the interacting regions on Vif differ depending on the specific APOBEC3 protein it is interacting with, and it is likely that three-dimensional structures of APOBEC3 proteins bound to Vif will be needed to effectively target this interaction (Russell et al., 2009, Tian et al., 2006). Despite these challenges, one small molecule antagonist of Vif was recently identified (RN-18) and shown to decrease levels of Vif protein *in vitro* (Nathans et al., 2008)*.* It will be interesting to follow-up this research *in vivo*, and learn whether RN-18 has an effect on HIV-1 replication in infected individuals. Finally, another option to shield APOBEC3 from Vif involves designing a molecule that binds to APOBEC3 and prevents this interaction; however, this molecule would also have to preserve APOBEC3's antiviral function (Albin & Harris, 2010).

The HIV-1 Tat protein is another attractive drug target, since it is essential for HIV-1 replication and antagonizes two HIV-1 restriction factors (PKR and OAS1). For unknown reasons, Tat hasn't received much attention as a potential drug target, possibly because its actions are hard to re-create *in vitro*. However, inhibition of Tat potently inhibits HIV-1 replication, and further research in this area is certainly warranted. Drugs that target the HIV-1 Vpu protein could also be considered; however, because Vpu is not critical for HIV-1 replication *in vivo* it may not be an ideal candidate (Friborg et al., 1995, Terwilliger et al., 1989). Conversely, drugs that mimic the effects of tetherin, but are resistant to Vpu, may successfully reduce HIV-1 replication. In fact, an artificial Vpu-resistant tetherin protein was recently engineered; however, it has not yet been tested in clinical trials (Perez-Caballero et al., 2009). Inhibiting the action of host proteins that assist HIV-1 replication is another possibility. For example, cyclophilin A (CypA) is required for HIV-1 replication, and without Cyp A HIV-1 virions are not infectious (Sokolskaja & Luban, 2006, Thali et al., 1994). Small molecule inhibitors targeting Cyp A may block HIV-1 replication at the uncoating stage, and targeting a host protein avoids the problem of viral resistance. However, HIV-1 propagation in the absence of Cyp A may allow HIV-1 variants to evolve that no longer require Cyp A for replication.

#### **8.2 Gene therapy approach**

An alternative approach to HIV-1 therapy involves using cellular restriction factors in conjunction with gene therapy. In this approach, DNA encoding one or more cellular restriction factors is inserted into target cells to interfere with HIV-1 infection or replication.

Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 165

& Rossi, 2006, Morris & Rossi, 2006, Lee et al., 2005, Lee et al., 2005). However, the use of viral vectors for gene delivery poses several potential problems such as toxicity, immunogenicity, and insertion mutagenesis. As such, the use of non-viral vectors for gene delivery must be further explored, and nanotechnology is one promising option (Lundin et al., 2009, Mintzer & Simanek, 2009). One example is the use of RNA interference (RNAi) for HIV/AIDS therapies. RNAi may have therapeutic potential in the treatment of HIV/AIDS; however, delivery of siRNA to specific cells continues to be a problem (Haasnoot et al., 2007, Haasnoot et al., 2007, Whitehead et al., 2009, Whitehead et al., 2009, Berkhout & ter Brake, 2009). Nanosuspensions of siRNA are currently being tested in humans for cancer treatment, and have recently entered Phase I clinical trials (Davis, 2009). If this technique is successful, it could be applied to cellular restriction factor-based HIV/AIDS gene therapy. For example, nanosuspensions of siRNA could be targeted to HIV-1-infected cells to knockdown the viral mRNA of restriction factor antagonists, such as Vif, Tat and Vpu. This would increase the antiviral activity of restriction factors, specifically reducing HIV-1 replication in infected cells. Alternatively, DNA from one or more cellular restriction factors could be delivered to HIV-1 infected cells using nanotechnology platforms. This may provide a safe and effective way to deliver cellular restriction factor genes to HIV-1 infected

Zinc finger nucleases (ZFN) have recently emerged as an important technology for gene modification, and there are several potential applications for ZFNs in HIV/AIDS therapy. ZFNs function by inducing a double-stranded break in a specific DNA sequence and generate the desired gene modification during DNA repair (Urnov et al., 2010). One of the main advantages of ZFNs is that the changes they make are both permanent and heritable, eliminating the need for persistent therapeutic intervention. For HIV-1, most ZFN research has focused on the manipulation of the human CCR5 gene, which encodes one of HIV-1's co-receptors and is required for viral entry into the host cell. Deletion of a 32-bp region from this gene (CCR5Δ32) results in a non-functional receptor, and people with this mutation are resistant to HIV-1 infection (Huang et al., 1996). Thus far, ZFN researchers have succeeded in deleting the 32-bp region from the human CCR5 gene, both in primary CD4+ T-cells and hematopoietic stem cells (Bobis-Wozowicz et al., 2011, Lei et al., 2011, Perez et al., 2008). Furthermore, there are two Phase I clinical trials in progress testing the efficacy of *ex vivo* expansion and infusion of these modified cells in HIV-1 infected individuals (Urnov et al.,

In addition to gene deletion, ZFNs have also been used successfully for gene correction (allele editing) and gene addition (Urnov et al., 2005, Urnov et al., 2005, Moehle et al., 2007). Both gene correction and addition may be useful for cellular restriction factor-based HIV-1 therapies; however, to date this has never been experimentally tested. For example, the addition of one or more cellular restriction factor genes to HIV-1 target cells may produce a 'super-restrictive' phenotype, whereby cells with multiple genes express higher levels of restriction factor proteins, thus increasing their capacity to fight HIV-1 infection. Several HIV-1 restriction factors have been shown to be more effective restrictors when expressed at higher levels. For example, higher expression of TRIM22 was recently shown to be correlated with lower levels of viremia and higher CD4+ T-cell counts in HIV-1-infected individuals (Singh et al., 2011). Another possibility involves adding cellular restriction factor

cells.

2010).

**8.3.2 Zinc finger nucleases** 

One advantage of this approach is that cellular restriction factors are naturally expressed in human cells, and as such may be less toxic or immunogenic *in vivo* (Barr, 2010). Since there are no known viral countermeasures to TRIM22, Rhesus TRIM5α or ISG15, these proteins are currently the best candidates for gene therapy. Another possibility involves using molecular engineering to create modified restriction factors that are resistant to viral antagonists, making them more suitable candidates for gene therapy. For example, a human protein modeled after the TRIM5α-CypA fusion protein in Owl monkeys was recently engineered, and shown to block HIV-1 replication in primary CD4+ T-cells and macrophages (Neagu et al., 2009). In addition, mice engrafted with inhibitor-expressing CD4+ T-cells had decreased viremia and increased levels of CD4+ T-cells. It is possible that this human TRIM5α-CypA protein could be used for gene therapy, and it is likely that it will be tested clinically in the near future.

Since many cellular restriction factors are IFN-inducible, they are not constitutively expressed in cells. As such, it is desirable to employ a gene therapy approach that mimics this pattern of expression. One interesting strategy involves creating a construct that contains restriction factor genes under the control of the HIV-1 LTR promoter. In this strategy, target cells are preloaded with the construct, and when HIV-1 infects these cells, Tat expression activates transcription of the LTR-fused restriction factor genes. Restriction factor expression reduces HIV-1 replication in infected cells, limiting further propagation of the virus. Notably, this approach has been successfully tested *in vitro* using the restriction factors PKR, OAS1 and ISG15; however, more experiments are needed to validate this strategy *in vivo*, and to test various construct delivery methods (Muto et al., 1999, Schroder et al., 1990a, Su et al., 1995). Gene therapy continues to be a promising approach for the treatment of HIV/AIDS; however, several problems need to be addressed before this technology can be fully realized. Some of these issues include, but are not limited to, increasing the stability of DNA and longevity of target cells, avoiding adverse immune responses, and targeting specific cells or tissues.

#### **8.3 Additional approaches**

#### **8.3.1 Nanotechnology**

Nanotechnology is revolutionizing many areas of medicine, particularly in the realm of drug delivery. With nanotechnology, it is now possible to target drugs to specific cells or tissues, a method that could be used to direct antiretroviral drugs to CD4+ T-cells and macrophages (Farokhzad, 2008, Farokhzad & Langer, 2009). In addition, targeted antiviral delivery to the brain or other organs could ensure that drugs reach latent HIV-1 reservoirs (Vyas et al., 2006, Vyas et al., 2006, Amiji et al., 2006). The development of controlled-release delivery systems could also allow antiretroviral drugs to be released over longer times, and enhance their half-lives. For example, a new anti-HIV-1 drug called Rilpivirine was recently administered to dogs and mice in nanosuspensions (Baert et al., 2009). This resulted in the sustained release of the drug over 3 months in dogs and 3 weeks in mice, compared to a half-life of 38 hours for free drug. Importantly, this type of drug delivery system could have major implications in reducing antiretroviral toxicity and improving drug adherence. Thus, nanotechnology should be considered in the development of new antiretroviral drugs, including drugs that mimic the effects of cellular restriction factors.

In addition to improving antiretroviral therapies, there are ongoing efforts to apply nanotechnology to gene therapy. Early attempts in gene therapy for HIV/AIDS have used viral vectors as gene delivery systems, with some encouraging results (Li et al., 2005, Morris & Rossi, 2006, Morris & Rossi, 2006, Lee et al., 2005, Lee et al., 2005). However, the use of viral vectors for gene delivery poses several potential problems such as toxicity, immunogenicity, and insertion mutagenesis. As such, the use of non-viral vectors for gene delivery must be further explored, and nanotechnology is one promising option (Lundin et al., 2009, Mintzer & Simanek, 2009). One example is the use of RNA interference (RNAi) for HIV/AIDS therapies. RNAi may have therapeutic potential in the treatment of HIV/AIDS; however, delivery of siRNA to specific cells continues to be a problem (Haasnoot et al., 2007, Haasnoot et al., 2007, Whitehead et al., 2009, Whitehead et al., 2009, Berkhout & ter Brake, 2009). Nanosuspensions of siRNA are currently being tested in humans for cancer treatment, and have recently entered Phase I clinical trials (Davis, 2009). If this technique is successful, it could be applied to cellular restriction factor-based HIV/AIDS gene therapy. For example, nanosuspensions of siRNA could be targeted to HIV-1-infected cells to knockdown the viral mRNA of restriction factor antagonists, such as Vif, Tat and Vpu. This would increase the antiviral activity of restriction factors, specifically reducing HIV-1 replication in infected cells. Alternatively, DNA from one or more cellular restriction factors could be delivered to HIV-1 infected cells using nanotechnology platforms. This may provide a safe and effective way to deliver cellular restriction factor genes to HIV-1 infected cells.

#### **8.3.2 Zinc finger nucleases**

164 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

One advantage of this approach is that cellular restriction factors are naturally expressed in human cells, and as such may be less toxic or immunogenic *in vivo* (Barr, 2010). Since there are no known viral countermeasures to TRIM22, Rhesus TRIM5α or ISG15, these proteins are currently the best candidates for gene therapy. Another possibility involves using molecular engineering to create modified restriction factors that are resistant to viral antagonists, making them more suitable candidates for gene therapy. For example, a human protein modeled after the TRIM5α-CypA fusion protein in Owl monkeys was recently engineered, and shown to block HIV-1 replication in primary CD4+ T-cells and macrophages (Neagu et al., 2009). In addition, mice engrafted with inhibitor-expressing CD4+ T-cells had decreased viremia and increased levels of CD4+ T-cells. It is possible that this human TRIM5α-CypA protein could be used for gene therapy, and it is likely that it will be tested

Since many cellular restriction factors are IFN-inducible, they are not constitutively expressed in cells. As such, it is desirable to employ a gene therapy approach that mimics this pattern of expression. One interesting strategy involves creating a construct that contains restriction factor genes under the control of the HIV-1 LTR promoter. In this strategy, target cells are preloaded with the construct, and when HIV-1 infects these cells, Tat expression activates transcription of the LTR-fused restriction factor genes. Restriction factor expression reduces HIV-1 replication in infected cells, limiting further propagation of the virus. Notably, this approach has been successfully tested *in vitro* using the restriction factors PKR, OAS1 and ISG15; however, more experiments are needed to validate this strategy *in vivo*, and to test various construct delivery methods (Muto et al., 1999, Schroder et al., 1990a, Su et al., 1995). Gene therapy continues to be a promising approach for the treatment of HIV/AIDS; however, several problems need to be addressed before this technology can be fully realized. Some of these issues include, but are not limited to, increasing the stability of DNA and longevity of target cells, avoiding adverse immune

Nanotechnology is revolutionizing many areas of medicine, particularly in the realm of drug delivery. With nanotechnology, it is now possible to target drugs to specific cells or tissues, a method that could be used to direct antiretroviral drugs to CD4+ T-cells and macrophages (Farokhzad, 2008, Farokhzad & Langer, 2009). In addition, targeted antiviral delivery to the brain or other organs could ensure that drugs reach latent HIV-1 reservoirs (Vyas et al., 2006, Vyas et al., 2006, Amiji et al., 2006). The development of controlled-release delivery systems could also allow antiretroviral drugs to be released over longer times, and enhance their half-lives. For example, a new anti-HIV-1 drug called Rilpivirine was recently administered to dogs and mice in nanosuspensions (Baert et al., 2009). This resulted in the sustained release of the drug over 3 months in dogs and 3 weeks in mice, compared to a half-life of 38 hours for free drug. Importantly, this type of drug delivery system could have major implications in reducing antiretroviral toxicity and improving drug adherence. Thus, nanotechnology should be considered in the development of new antiretroviral drugs,

In addition to improving antiretroviral therapies, there are ongoing efforts to apply nanotechnology to gene therapy. Early attempts in gene therapy for HIV/AIDS have used viral vectors as gene delivery systems, with some encouraging results (Li et al., 2005, Morris

clinically in the near future.

**8.3 Additional approaches 8.3.1 Nanotechnology** 

responses, and targeting specific cells or tissues.

including drugs that mimic the effects of cellular restriction factors.

Zinc finger nucleases (ZFN) have recently emerged as an important technology for gene modification, and there are several potential applications for ZFNs in HIV/AIDS therapy. ZFNs function by inducing a double-stranded break in a specific DNA sequence and generate the desired gene modification during DNA repair (Urnov et al., 2010). One of the main advantages of ZFNs is that the changes they make are both permanent and heritable, eliminating the need for persistent therapeutic intervention. For HIV-1, most ZFN research has focused on the manipulation of the human CCR5 gene, which encodes one of HIV-1's co-receptors and is required for viral entry into the host cell. Deletion of a 32-bp region from this gene (CCR5Δ32) results in a non-functional receptor, and people with this mutation are resistant to HIV-1 infection (Huang et al., 1996). Thus far, ZFN researchers have succeeded in deleting the 32-bp region from the human CCR5 gene, both in primary CD4+ T-cells and hematopoietic stem cells (Bobis-Wozowicz et al., 2011, Lei et al., 2011, Perez et al., 2008). Furthermore, there are two Phase I clinical trials in progress testing the efficacy of *ex vivo* expansion and infusion of these modified cells in HIV-1 infected individuals (Urnov et al., 2010).

In addition to gene deletion, ZFNs have also been used successfully for gene correction (allele editing) and gene addition (Urnov et al., 2005, Urnov et al., 2005, Moehle et al., 2007). Both gene correction and addition may be useful for cellular restriction factor-based HIV-1 therapies; however, to date this has never been experimentally tested. For example, the addition of one or more cellular restriction factor genes to HIV-1 target cells may produce a 'super-restrictive' phenotype, whereby cells with multiple genes express higher levels of restriction factor proteins, thus increasing their capacity to fight HIV-1 infection. Several HIV-1 restriction factors have been shown to be more effective restrictors when expressed at higher levels. For example, higher expression of TRIM22 was recently shown to be correlated with lower levels of viremia and higher CD4+ T-cell counts in HIV-1-infected individuals (Singh et al., 2011). Another possibility involves adding cellular restriction factor

Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 167

An, P., Johnson, R., Phair, J., Kirk, G.D., Yu, X.F., Donfield, S., Buchbinder, S., Goedert, J.J. &

Anderson, J.L. & Hope, T.J. 2008, APOBEC3G restricts early HIV-1 replication in the

Andrew, A.J., Miyagi, E., Kao, S. & Strebel, K. 2009, The formation of cysteine-linked dimers

Baert, L., van 't Klooster, G., Dries, W., Francois, M., Wouters, A., Basstanie, E., Iterbeke, K.,

Ball, T.B., Ji, H., Kimani, J., McLaren, P., Marlin, C., Hill, A.V. & Plummer, F.A. 2007,

Barr, S.D. 2010, Cellular HIV-1 restriction factors: a new avenue for AIDS therapy?, *Future* 

Barr, S.D., Smiley, J.R. & Bushman, F.D. 2008, The interferon response inhibits HIV particle production by induction of TRIM22, *PLoS pathogens,* vol. 4, no. 2, pp. e1000007. Barre-Sinoussi, F., Chermann, J.C., Rey, F., Nugeyre, M.T., Chamaret, S., Gruest, J., Dauguet,

Bartee, E., McCormack, A. & Fruh, K. 2006, Quantitative membrane proteomics reveals new

Baum, A. & Garcia-Sastre, A. 2010, Induction of type I interferon by RNA viruses: cellular receptors and their substrates, *Amino acids,* vol. 38, no. 5, pp. 1283-1299. Berkhout, B. & ter Brake, O. 2009, Towards a durable RNAi gene therapy for HIV-AIDS,

Beyrer, C., Artenstein, A.W., Rugpao, S., Stephens, H., VanCott, T.C., Robb, M.L., Rinkaew,

Bieniasz, P.D. 2004, Intrinsic immunity: a front-line defense against viral attack, *Nature* 

Bishop, K.N., Verma, M., Kim, E.Y., Wolinsky, S.M. & Malim, M.H. 2008, APOBEC3G

*Expert opinion on biological therapy,* vol. 9, no. 2, pp. 161-170.

*infectious diseases,* vol. 179, no. 1, pp. 59-67.

*immunology,* vol. 5, no. 11, pp. 1109-1115.

*of infectious diseases,* vol. 200, no. 7, pp. 1054-1058.

sensitivity to Vpu, *Retrovirology,* vol. 6, pp. 80.

1091-1101.

pp. 868-871.

e1000231.

*Virology,* vol. 5, no. 4, pp. 417-433.

cytoplasm of target cells, *Virology,* vol. 375, no. 1, pp. 1-12.

*Pharmazeutische Verfahrenstechnik e.V,* vol. 72, no. 3, pp. 502-508.

Winkler, C.A. 2009, APOBEC3B deletion and risk of HIV-1 acquisition, *The Journal* 

of BST-2/tetherin is important for inhibition of HIV-1 virus release but not for

Stappers, F., Stevens, P., Schueller, L., Van Remoortere, P., Kraus, G., Wigerinck, P. & Rosier, J. 2009, Development of a long-acting injectable formulation with nanoparticles of rilpivirine (TMC278) for HIV treatment, *European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur* 

Polymorphisms in IRF-1 associated with resistance to HIV-1 infection in highly exposed uninfected Kenyan sex workers, *AIDS (London, England),* vol. 21, no. 9, pp.

C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W. & Montagnier, L. 1983, Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS), *Science (New York, N.Y.),* vol. 220, no. 4599,

cellular targets of viral immune modulators, *PLoS pathogens,* vol. 2, no. 10, pp. e107.

M., Birx, D.L., Khamboonruang, C., Zimmerman, P.A., Nelson, K.E. & Natpratan, C. 1999, Epidemiologic and biologic characterization of a cohort of human immunodeficiency virus type 1 highly exposed, persistently seronegative female sex workers in northern Thailand. Chiang Mai HEPS Working Group, *The Journal of* 

inhibits elongation of HIV-1 reverse transcripts, *PLoS pathogens,* vol. 4, no. 12, pp.

genes to hematopoietic stem cells, allowing the generation of 'super-restrictive' cells in all blood lineages (including macrophages and dendritic cells, which are often the first cells to encounter HIV-1 *in vivo*). However, many more studies need to be performed, particularly to identify any deleterious effects caused by amplified expression of cellular restriction factors.

#### **8.3.3 Next-generation sequencing**

Next-generation sequencing is another new and exciting technology that has potential applications in HIV/AIDS therapy. With this calibre of sequencing, it is now possible to read hundreds of DNA samples simultaneously, an approach that has helped researchers identify polymorphisms in different human genes. It is well known that people differ significantly in their susceptibility to HIV-1 infection and disease progression to AIDS, and polymorphisms in cellular restriction factors may contribute to these differences (Ball et al., 2007, Beyrer et al., 1999, Cao et al., 1995). For example, there is research suggesting that various polymorphisms in the TRIM5α and APOBEC3 genes contribute to HIV-1 disease progression; however, due to confounding reports further research is needed in this area (van Manen et al., 2008, van Manen et al., 2008, An et al., 2009, Goldschmidt et al., 2006, Harari et al., 2009, Valcke et al., 2006). In addition, polymorphisms in other cellular restriction factors may influence the clinical course of HIV-1 infection, but many of these factors have never been tested. In the future, it may be possible to generate an individual's cellular restriction factor polymorphism "blueprint"(Barr, 2010). This blueprint could help predict a person's susceptibility to HIV-1 infection and progression to AIDS, and may potentially lead to a more personalized HIV-1 treatment regime. Alternatively, with the advent of ZFN technology it may also be possible to "edit" multiple restriction factor genes (change disadvantageous polymorphisms to advantageous polymorphisms) to create an optimal cellular restriction factor blueprint *in vivo*, and better equip individuals to fight HIV-1 infection.

## **9. References**


genes to hematopoietic stem cells, allowing the generation of 'super-restrictive' cells in all blood lineages (including macrophages and dendritic cells, which are often the first cells to encounter HIV-1 *in vivo*). However, many more studies need to be performed, particularly to identify any deleterious effects caused by amplified expression of cellular restriction

Next-generation sequencing is another new and exciting technology that has potential applications in HIV/AIDS therapy. With this calibre of sequencing, it is now possible to read hundreds of DNA samples simultaneously, an approach that has helped researchers identify polymorphisms in different human genes. It is well known that people differ significantly in their susceptibility to HIV-1 infection and disease progression to AIDS, and polymorphisms in cellular restriction factors may contribute to these differences (Ball et al., 2007, Beyrer et al., 1999, Cao et al., 1995). For example, there is research suggesting that various polymorphisms in the TRIM5α and APOBEC3 genes contribute to HIV-1 disease progression; however, due to confounding reports further research is needed in this area (van Manen et al., 2008, van Manen et al., 2008, An et al., 2009, Goldschmidt et al., 2006, Harari et al., 2009, Valcke et al., 2006). In addition, polymorphisms in other cellular restriction factors may influence the clinical course of HIV-1 infection, but many of these factors have never been tested. In the future, it may be possible to generate an individual's cellular restriction factor polymorphism "blueprint"(Barr, 2010). This blueprint could help predict a person's susceptibility to HIV-1 infection and progression to AIDS, and may potentially lead to a more personalized HIV-1 treatment regime. Alternatively, with the advent of ZFN technology it may also be possible to "edit" multiple restriction factor genes (change disadvantageous polymorphisms to advantageous polymorphisms) to create an optimal cellular restriction factor blueprint *in vivo*, and better equip individuals to fight

Adelson, M.E., Martinand-Mari, C., Iacono, K.T., Muto, N.F. & Suhadolnik, R.J. 1999,

Aguiar, R.S. & Peterlin, B.M. 2008, APOBEC3 proteins and reverse transcription, *Virus* 

Albin, J.S. & Harris, R.S. 2010, Interactions of host APOBEC3 restriction factors with HIV-1

Alter, H.J., Eichberg, J.W., Masur, H., Saxinger, W.C., Gallo, R., Macher, A.M., Lane, H.C. &

Amiji, M.M., Vyas, T.K. & Shah, L.K. 2006, Role of nanotechnology in HIV/AIDS treatment:

*biochemistry / FEBS,* vol. 264, no. 3, pp. 806-815.

*research,* vol. 134, no. 1-2, pp. 74-85.

Inhibition of human immunodeficiency virus (HIV-1) replication in SupT1 cells transduced with an HIV-1 LTR-driven PKR cDNA construct, *European journal of* 

in vivo: implications for therapeutics, *Expert reviews in molecular medicine,* vol. 12,

Fauci, A.S. 1984, Transmission of HTLV-III infection from human plasma to chimpanzees: an animal model for AIDS, *Science (New York, N.Y.),* vol. 226, no.

potential to overcome the viral reservoir challenge, *Discovery medicine,* vol. 6, no. 34,

factors.

HIV-1 infection.

**9. References** 

pp. e4.

4674, pp. 549-552.

pp. 157-162.

**8.3.3 Next-generation sequencing** 


Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 169

Chen, G., He, Z., Wang, T., Xu, R. & Yu, X.F. 2009, A patch of positively charged amino acids

Chen, H., Lilley, C.E., Yu, Q., Lee, D.V., Chou, J., Narvaiza, I., Landau, N.R. & Weitzman,

Chiu, Y.L., Soros, V.B., Kreisberg, J.F., Stopak, K., Yonemoto, W. & Greene, W.C. 2005,

Cho, S.J., Drechsler, H., Burke, R.C., Arens, M.Q., Powderly, W. & Davidson, N.O. 2006,

Chu, W.M., Ballard, R., Carpick, B.W., Williams, B.R. & Schmid, C.W. 1998, Potential Alu

Clerzius, G., Gelinas, J.F. & Gatignol, A. 2011, Multiple levels of PKR inhibition during HIV-

Cole, J.L. 2007, Activation of PKR: an open and shut case?, *Trends in biochemical sciences,* vol.

Conticello, S.G., Thomas, C.J., Petersen-Mahrt, S.K. & Neuberger, M.S. 2005, Evolution of the

Conticello, S.G., Harris, R.S. & Neuberger, M.S. 2003, The Vif protein of HIV triggers

Cosentino, G.P., Venkatesan, S., Serluca, F.C., Green, S.R., Mathews, M.B. & Sonenberg, N.

Cowan, S., Hatziioannou, T., Cunningham, T., Muesing, M.A., Gottlinger, H.G. & Bieniasz,

Dang, Y., Siew, L.M. & Zheng, Y.H. 2008, APOBEC3G is degraded by the proteasomal

Davis, M.E. 2009, The first targeted delivery of siRNA in humans via a self-assembling,

Deng, H., Liu, R., Ellmeier, W., Choe, S., Unutmaz, D., Burkhart, M., Di Marzio, P., Marmon,

*Sciences of the United States of America,* vol. 92, no. 21, pp. 9445-9449.

*the United States of America,* vol. 99, no. 18, pp. 11914-11919.

*of biological chemistry,* vol. 283, no. 19, pp. 13124-13131.

*pharmaceutics,* vol. 6, no. 3, pp. 659-668.

HIV-1, *Nature,* vol. 381, no. 6584, pp. 661-666.

1 replication, *Reviews in medical virology,* vol. 21, no. 1, pp. 42-53.

retrotransposons, *Current biology : CB,* vol. 16, no. 5, pp. 480-485.

8674-8682.

435, no. 7038, pp. 108-114.

32, no. 2, pp. 57-62.

*virology,* vol. 80, no. 4, pp. 2069-2072.

*Molecular and cellular biology,* vol. 18, no. 1, pp. 58-68.

*biology and evolution,* vol. 22, no. 2, pp. 367-377.

*biology : CB,* vol. 13, no. 22, pp. 2009-2013.

surrounding the human immunodeficiency virus type 1 Vif SLVx4Yx9Y motif influences its interaction with APOBEC3G, *Journal of virology,* vol. 83, no. 17, pp.

M.D. 2006, APOBEC3A is a potent inhibitor of adeno-associated virus and

Cellular APOBEC3G restricts HIV-1 infection in resting CD4+ T cells, *Nature,* vol.

APOBEC3F and APOBEC3G mRNA levels do not correlate with human immunodeficiency virus type 1 plasma viremia or CD4+ T-cell count, *Journal of* 

function: regulation of the activity of double-stranded RNA-activated kinase PKR,

AID/APOBEC family of polynucleotide (deoxy)cytidine deaminases, *Molecular* 

degradation of the human antiretroviral DNA deaminase APOBEC3G, *Current* 

1995, Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo, *Proceedings of the National Academy of* 

P.D. 2002, Cellular inhibitors with Fv1-like activity restrict human and simian immunodeficiency virus tropism, *Proceedings of the National Academy of Sciences of* 

pathway in a Vif-dependent manner without being polyubiquitylated, *The Journal* 

cyclodextrin polymer-based nanoparticle: from concept to clinic, *Molecular* 

S., Sutton, R.E., Hill, C.M., Davis, C.B., Peiper, S.C., Schall, T.J., Littman, D.R. & Landau, N.R. 1996, Identification of a major co-receptor for primary isolates of


Bobis-Wozowicz, S., Osiak, A., Rahman, S.H. & Cathomen, T. 2011, Targeted genome

Bogerd, H.P., Wiegand, H.L., Hulme, A.E., Garcia-Perez, J.L., O'Shea, K.S., Moran, J.V. &

Borrow, P. & Bhardwaj, N. 2008, Innate immune responses in primary HIV-1 infection,

Bouazzaoui, A., Kreutz, M., Eisert, V., Dinauer, N., Heinzelmann, A., Hallenberger, S.,

Broder, S. & Gallo, R.C. 1984, A pathogenic retrovirus (HTLV-III) linked to AIDS, *The New* 

Buchbinder, S.P., Mehrotra, D.V., Duerr, A., Fitzgerald, D.W., Mogg, R., Li, D., Gilbert, P.B.,

Cai, R., Carpick, B., Chun, R.F., Jeang, K.T. & Williams, B.R. 2000, HIV-I TAT inhibits PKR

Cao, Y., Qin, L., Zhang, L., Safrit, J. & Ho, D.D. 1995, Virologic and immunologic

Casartelli, N., Sourisseau, M., Feldmann, J., Guivel-Benhassine, F., Mallet, A., Marcelin,

Chakrabarti, A., Jha, B.K. & Silverman, R.H. 2011, New insights into the role of RNase L in

Chatterji, U., Bobardt, M.D., Gaskill, P., Sheeter, D., Fox, H. & Gallay, P.A. 2006, Trim5alpha

*The Journal of biological chemistry,* vol. 281, no. 48, pp. 37025-37033.

infection, *The New England journal of medicine,* vol. 332, no. 4, pp. 201-208. Carpick, B.W., Graziano, V., Schneider, D., Maitra, R.K., Lee, X. & Williams, B.R. 1997,

*Calif.),* vol. 53, no. 4, pp. 339-346.

272, no. 13, pp. 8388-8395.

vol. 372, no. 9653, pp. 1881-1893.

*immunology,* vol. 22, no. 4, pp. 488-496.

*of America,* vol. 103, no. 23, pp. 8780-8785.

*Current opinion in HIV and AIDS,* vol. 3, no. 1, pp. 36-44.

*England journal of medicine,* vol. 311, no. 20, pp. 1292-1297.

*Biochemistry and Biophysics,* vol. 373, no. 2, pp. 361-367.

*Journal of biological chemistry,* vol. 272, no. 14, pp. 9510-9516.

cell transmission, *PLoS pathogens,* vol. 6, no. 6, pp. e1000955.

editing in pluripotent stem cells using zinc-finger nucleases, *Methods (San Diego,* 

Cullen, B.R. 2006, Cellular inhibitors of long interspersed element 1 and Alu retrotransposition, *Proceedings of the National Academy of Sciences of the United States* 

Strayle, J., Walker, R., Rubsamen-Waigmann, H., Andreesen, R. & von Briesen, H. 2006, Stimulated trans-acting factor of 50 kDa (Staf50) inhibits HIV-1 replication in human monocyte-derived macrophages, *Virology,* vol. 356, no. 1-2, pp. 79-94. Brand, S.R., Kobayashi, R. & Mathews, M.B. 1997, The Tat protein of human

immunodeficiency virus type 1 is a substrate and inhibitor of the interferoninduced, virally activated protein kinase, PKR, *The Journal of biological chemistry,* vol.

Lama, J.R., Marmor, M., Del Rio, C., McElrath, M.J., Casimiro, D.R., Gottesdiener, K.M., Chodakewitz, J.A., Corey, L., Robertson, M.N. & Step Study Protocol Team 2008, Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-concept trial, *Lancet,* 

activity by both RNA-dependent and RNA-independent mechanisms, *Archives of* 

characterization of long-term survivors of human immunodeficiency virus type 1

Characterization of the solution complex between the interferon-induced, doublestranded RNA-activated protein kinase and HIV-I trans-activating region RNA, *The* 

A.G., Guatelli, J. & Schwartz, O. 2010, Tetherin restricts productive HIV-1 cell-to-

innate immunity, *Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research,* vol. 31, no. 1, pp. 49-57. Chakrabarti, L.A. & Simon, V. 2010, Immune mechanisms of HIV control, *Current opinion in* 

accelerates degradation of cytosolic capsid associated with productive HIV-1 entry,


Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 171

Fujii, R., Okamoto, M., Aratani, S., Oishi, T., Ohshima, T., Taira, K., Baba, M., Fukamizu, A.

Gabuzda, D.H., Lawrence, K., Langhoff, E., Terwilliger, E., Dorfman, T., Haseltine, W.A. &

Ganser-Pornillos, B.K., Yeager, M. & Sundquist, W.I. 2008, The structural biology of HIV assembly, *Current opinion in structural biology,* vol. 18, no. 2, pp. 203-217. Gao, B., Duan, Z., Xu, W. & Xiong, S. 2009, Tripartite motif-containing 22 inhibits the activity

Garcia, M.A., Meurs, E.F. & Esteban, M. 2007, The dsRNA protein kinase PKR: virus and cell

Garrus, J.E., von Schwedler, U.K., Pornillos, O.W., Morham, S.G., Zavitz, K.H., Wang, H.E.,

Goila-Gaur, R., Khan, M.A., Miyagi, E., Kao, S., Opi, S., Takeuchi, H. & Strebel, K. 2008, HIV-

Goila-Gaur, R. & Strebel, K. 2008, HIV-1 Vif, APOBEC, and intrinsic immunity, *Retrovirology,* 

Goldschmidt, V., Bleiber, G., May, M., Martinez, R., Ortiz, M., Telenti, A. & Swiss HIV

Gottlinger, H.G., Dorfman, T., Cohen, E.A. & Haseltine, W.A. 1993, Vpu protein of human

Guatelli, J.C. 2009, Interactions of viral protein U (Vpu) with cellular factors, *Current topics in* 

Haasnoot, J., Westerhout, E.M. & Berkhout, B. 2007, RNA interference against viruses: strike and counterstrike, *Nature biotechnology,* vol. 25, no. 12, pp. 1435-1443.

*of Sciences of the United States of America,* vol. 90, no. 15, pp. 7381-7385. Groom, H.C., Yap, M.W., Galao, R.P., Neil, S.J. & Bishop, K.N. 2010, Susceptibility of

domain, *Hepatology (Baltimore, Md.),* vol. 50, no. 2, pp. 424-433.

in CD4+ T lymphocytes, *Journal of virology,* vol. 66, no. 11, pp. 6489-6495. Gajdusek, D.C., Amyx, H.L., Gibbs, C.J.,Jr, Asher, D.M., Rodgers-Johnson, P., Epstein, L.G.,

patients, *Lancet,* vol. 1, no. 8419, pp. 55-56.

control, *Biochimie,* vol. 89, no. 6-7, pp. 799-811.

budding, *Cell,* vol. 107, no. 1, pp. 55-65.

*Virology,* vol. 372, no. 1, pp. 136-146.

progression, *Retrovirology,* vol. 3, pp. 54.

*microbiology and immunology,* vol. 339, pp. 27-45.

vol. 107, no. 11, pp. 5166-5171.

vol. 5, pp. 51.

pp. 5445-5451.

Vpu protein, *Journal of acquired immune deficiency syndromes and human retrovirology : official publication of the International Retrovirology Association,* vol. 8, no. 1, pp. 10-22.

& Nakajima, T. 2001, A Role of RNA Helicase A in cis-Acting Transactivation Response Element-mediated Transcriptional Regulation of Human Immunodeficiency Virus Type 1, *The Journal of biological chemistry,* vol. 276, no. 8,

Sodroski, J. 1992, Role of vif in replication of human immunodeficiency virus type 1

Sarin, P.S., Gallo, R.C., Maluish, A. & Arthur, L.O. 1985, Infection of chimpanzees by human T-lymphotropic retroviruses in brain and other tissues from AIDS

of hepatitis B virus core promoter, which is dependent on nuclear-located RING

Wettstein, D.A., Stray, K.M., Cote, M., Rich, R.L., Myszka, D.G. & Sundquist, W.I. 2001, Tsg101 and the vacuolar protein sorting pathway are essential for hiv-1

1 Vif promotes the formation of high molecular mass APOBEC3G complexes,

Cohort Study 2006, Role of common human TRIM5alpha variants in HIV-1 disease

immunodeficiency virus type 1 enhances the release of capsids produced by gag gene constructs of widely divergent retroviruses, *Proceedings of the National Academy* 

xenotropic murine leukemia virus-related virus (XMRV) to retroviral restriction factors, *Proceedings of the National Academy of Sciences of the United States of America,* 


Dey, M., Cao, C., Dar, A.C., Tamura, T., Ozato, K., Sicheri, F. & Dever, T.E. 2005, Mechanistic

Diaz-Griffero, F., Li, X., Javanbakht, H., Song, B., Welikala, S., Stremlau, M. & Sodroski, J.

Douglas, J.L., Viswanathan, K., McCarroll, M.N., Gustin, J.K., Fruh, K. & Moses, A.V. 2009,

Dube, M., Roy, B.B., Guiot-Guillain, P., Binette, J., Mercier, J., Chiasson, A. & Cohen, E.A.

Eldin, P., Papon, L., Oteiza, A., Brocchi, E., Lawson, T.G. & Mechti, N. 2009, TRIM22 E3

Endo-Munoz, L., Warby, T., Harrich, D. & McMillan, N.A. 2005, Phosphorylation of HIV Tat

Farokhzad, O.C. & Langer, R. 2009, Impact of nanotechnology on drug delivery, *ACS nano,* 

Farokhzad, O.C. 2008, Nanotechnology for drug delivery: the perfect partnership, *Expert* 

Farrell, P.J., Broeze, R.J. & Lengyel, P. 1979, Accumulation of an mRNA and protein in

Fischl, M.A., Richman, D.D., Grieco, M.H., Gottlieb, M.S., Volberding, P.A., Laskin, O.L.,

Flynn, N.M., Forthal, D.N., Harro, C.D., Judson, F.N., Mayer, K.H., Para, M.F. & rgp120 HIV

Frankel, A.D. & Pabo, C.O. 1988, Cellular uptake of the tat protein from human

Franzoso, G., Biswas, P., Poli, G., Carlson, L.M., Brown, K.D., Tomita-Yamaguchi, M., Fauci,

Friborg, J., Ladha, A., Gottlinger, H., Haseltine, W.A. & Cohen, E.A. 1995, Functional

immunodeficiency virus, *Cell,* vol. 55, no. 6, pp. 1189-1193.

*The Journal of experimental medicine,* vol. 180, no. 4, pp. 1445-1456.

recognition, *Cell,* vol. 122, no. 6, pp. 901-913.

TRIM5, *Virology,* vol. 349, no. 2, pp. 300-315.

vol. 83, no. 16, pp. 7931-7947.

545.

525.

pp. 185-191.

vol. 191, no. 5, pp. 654-665.

*journal,* vol. 2, pp. 17.

vol. 3, no. 1, pp. 16-20.

*pathogens,* vol. 6, no. 4, pp. e1000856.

*opinion on drug delivery,* vol. 5, no. 9, pp. 927-929.

link between PKR dimerization, autophosphorylation, and eIF2alpha substrate

2006, Rapid turnover and polyubiquitylation of the retroviral restriction factor

Vpu directs the degradation of the human immunodeficiency virus restriction factor BST-2/Tetherin via a {beta}TrCP-dependent mechanism, *Journal of virology,* 

2010, Antagonism of tetherin restriction of HIV-1 release by Vpu involves binding and sequestration of the restriction factor in a perinuclear compartment, *PLoS* 

ubiquitin ligase activity is required to mediate antiviral activity against encephalomyocarditis virus, *The Journal of general virology,* vol. 90, no. Pt 3, pp. 536-

by PKR increases interaction with TAR RNA and enhances transcription, *Virology* 

interferon-treated Ehrlich ascites tumour cells, *Nature,* vol. 279, no. 5713, pp. 523-

Leedom, J.M., Groopman, J.E., Moldvan, D., Schooley, R.T. & al., e. 1987, The efficacy of azidothymidine (AZT) in the treatment of patients with AIDS and AIDSrelated complex. A double-blind, placebo-controlled trial, *N.Engl.J.Med.,* vol. 317,

Vaccine Study Group 2005, Placebo-controlled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection, *The Journal of infectious diseases,* 

A.S. & Siebenlist, U.K. 1994, A family of serine proteases expressed exclusively in myelo-monocytic cells specifically processes the nuclear factor-kappa B subunit p65 in vitro and may impair human immunodeficiency virus replication in these cells,

analysis of the phosphorylation sites on the human immunodeficiency virus type 1

Vpu protein, *Journal of acquired immune deficiency syndromes and human retrovirology : official publication of the International Retrovirology Association,* vol. 8, no. 1, pp. 10-22.


Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 173

Jeang, K.T. & Yedavalli, V. 2006, Role of RNA helicases in HIV-1 replication, *Nucleic acids* 

Jeon, Y.J., Yoo, H.M. & Chung, C.H. 2010, ISG15 and immune diseases, *Biochimica et* 

Kaiser, S.M. & Emerman, M. 2006, Uracil DNA glycosylase is dispensable for human

Kajaste-Rudnitski, A., Marelli, S.S., Pultrone, C., Pertel, T., Uchil, P.D., Mechti, N., Mothes,

Kao, S., Goila-Gaur, R., Miyagi, E., Khan, M.A., Opi, S., Takeuchi, H. & Strebel, K. 2007,

Kerscher, O., Felberbaum, R. & Hochstrasser, M. 2006, Modification of proteins by ubiquitin and ubiquitin-like proteins, *Annu Rev Cell Dev Biol,* vol. 22, pp. 159-180. Kim, I., Liu, C.W. & Puglisi, J.D. 2006, Specific recognition of HIV TAR RNA by the dsRNA

Klarmann, G.J., Chen, X., North, T.W. & Preston, B.D. 2003, Incorporation of uracil into

Kobayashi, M., Takaori-Kondo, A., Miyauchi, Y., Iwai, K. & Uchiyama, T. 2005,

Kobayashi, T., Ode, H., Yoshida, T., Sato, K., Gee, P., Yamamoto, S.P., Ebina, H., Strebel, K.,

Kodym, R., Kodym, E. & Story, M.D. 2009, 2'-5'-Oligoadenylate synthetase is activated by a

Koning, F.A., Newman, E.N., Kim, E.Y., Kunstman, K.J., Wolinsky, S.M. & Malim, M.H.

Kuhl, B.D., Sloan, R.D., Donahue, D.A., Bar-Magen, T., Liang, C. & Wainberg, M.A. 2010, Tetherin restricts direct cell-to-cell infection of HIV-1, *Retrovirology,* vol. 7, pp. 115. Kumagai, Y., Takeuchi, O. & Akira, S. 2008, Pathogen recognition by innate receptors,

cell subsets, *Journal of virology,* vol. 83, no. 18, pp. 9474-9485.

immunodeficiency virus type 1 replication and does not contribute to the antiviral effects of the cytidine deaminase Apobec3G, *Journal of virology,* vol. 80, no. 2, pp.

W., Poli, G., Luban, J. & Vicenzi, E. 2011, TRIM22 Inhibits HIV-1 Transcription Independently of Its E3-Ubiquitin Ligase Activity, Tat and NF-{kappa}B

Production of infectious virus and degradation of APOBEC3G are separable functional properties of human immunodeficiency virus type 1 Vif, *Virology,* vol.

binding domains (dsRBD1-dsRBD2) of PKR, *Journal of Molecular Biology,* vol. 358,

minus strand DNA affects the specificity of plus strand synthesis initiation during lentiviral reverse transcription, *The Journal of biological chemistry,* vol. 278, no. 10, pp.

Ubiquitination of APOBEC3G by an HIV-1 Vif-Cullin5-Elongin B-Elongin C complex is essential for Vif function, *The Journal of biological chemistry,* vol. 280, no.

Sato, H. & Koyanagi, Y. 2011, Identification of amino acids in the human tetherin transmembrane domain responsible for HIV-1 Vpu interaction and susceptibility,

specific RNA sequence motif, *Biochemical and biophysical research communications,* 

2009, Defining APOBEC3 expression patterns in human tissues and hematopoietic

*Journal of infection and chemotherapy : official journal of the Japan Society of* 

*research,* vol. 34, no. 15, pp. 4198-4205.

875-882.

369, no. 2, pp. 329-339.

no. 2, pp. 430-442.

19, pp. 18573-18578.

vol. 388, no. 2, pp. 317-322.

7902-7909.

*biophysica acta,* vol. 1802, no. 5, pp. 485-496.

Responsive LTR Elements, *Journal of virology,* .

*Journal of virology,* vol. 85, no. 2, pp. 932-945.

*Chemotherapy,* vol. 14, no. 2, pp. 86-92.


Hammonds, J., Wang, J.J., Yi, H. & Spearman, P. 2010, Immunoelectron microscopic

Harari, A., Ooms, M., Mulder, L.C. & Simon, V. 2009, Polymorphisms and splice variants

Harty, R.N., Pitha, P.M. & Okumura, A. 2009, Antiviral activity of innate immune protein

Hauser, H., Lopez, L.A., Yang, S.J., Oldenburg, J.E., Exline, C.M., Guatelli, J.C. & Cannon,

Herrmann, J., Lerman, L.O. & Lerman, A. 2007, Ubiquitin and ubiquitin-like proteins in protein regulation, *Circulation research,* vol. 100, no. 9, pp. 1276-1291. Hofmann, W., Schubert, D., LaBonte, J., Munson, L., Gibson, S., Scammell, J., Ferrigno, P. &

Hovanessian, A.G. 2007, On the discovery of interferon-inducible, double-stranded RNA

Huang, Y., Paxton, W.A., Wolinsky, S.M., Neumann, A.U., Zhang, L., He, T., Kang, S.,

Hultquist, J.F. & Harris, R.S. 2009, Leveraging APOBEC3 proteins to alter the HIV mutation

Hunter, T., Hunt, T., Jackson, R.J. & Robertson, H.D. 1975, The characteristics of inhibition of

Huthoff, H. & Malim, M.H. 2007, Identification of amino acid residues in APOBEC3G

Ikeda, H., Laigret, F., Martin, M.A. & Repaske, R. 1985, Characterization of a molecularly

Iwabu, Y., Fujita, H., Kinomoto, M., Kaneko, K., Ishizaka, Y., Tanaka, Y., Sata, T. &

Javanbakht, H., Yuan, W., Yeung, D.F., Song, B., Diaz-Griffero, F., Li, Y., Li, X., Stremlau, M.

Javanbakht, H., Diaz-Griffero, F., Stremlau, M., Si, Z. & Sodroski, J. 2005, The contribution of

plasma membrane, *PLoS pathogens,* vol. 6, no. 2, pp. e1000749.

ISG15, *Journal of innate immunity,* vol. 1, no. 5, pp. 397-404.

in a perinuclear compartment, *Retrovirology,* vol. 7, pp. 51.

virus infection, *Journal of virology,* vol. 73, no. 12, pp. 10020-10028.

*Cytokine & growth factor reviews,* vol. 18, no. 5-6, pp. 351-361.

rate and combat AIDS, *Future virology,* vol. 4, no. 6, pp. 605.

encapsidation, *Journal of virology,* vol. 81, no. 8, pp. 3807-3815.

*Journal of biological chemistry,* vol. 250, no. 2, pp. 409-417.

*biological chemistry,* vol. 284, no. 50, pp. 35060-35072.

83, no. 1, pp. 295-303.

pp. 1240-1243.

55, no. 3, pp. 768-777.

no. 1, pp. 234-246.

evidence for Tetherin/BST2 as the physical bridge between HIV-1 virions and the

influence the antiretroviral activity of human APOBEC3H, *Journal of virology,* vol.

P.M. 2010, HIV-1 Vpu and HIV-2 Env counteract BST-2/tetherin by sequestration

Sodroski, J. 1999, Species-specific, postentry barriers to primate immunodeficiency

activated enzymes: the 2'-5'oligoadenylate synthetases and the protein kinase PKR,

Ceradini, D., Jin, Z., Yazdanbakhsh, K., Kunstman, K., Erickson, D., Dragon, E., Landau, N.R., Phair, J., Ho, D.D. & Koup, R.A. 1996, The role of a mutant CCR5 allele in HIV-1 transmission and disease progression, *Nature medicine,* vol. 2, no. 11,

protein synthesis by double-stranded ribonucleic acid in reticulocyte lysates, *The* 

required for regulation by human immunodeficiency virus type 1 Vif and Virion

cloned retroviral sequence associated with Fv-4 resistance, *Journal of virology,* vol.

Tokunaga, K. 2009, HIV-1 accessory protein Vpu internalizes cell-surface BST-2/tetherin through transmembrane interactions leading to lysosomes, *The Journal of* 

& Sodroski, J. 2006, Characterization of TRIM5alpha trimerization and its contribution to human immunodeficiency virus capsid binding, *Virology,* vol. 353,

RING and B-box 2 domains to retroviral restriction mediated by monkey TRIM5alpha, *The Journal of biological chemistry,* vol. 280, no. 29, pp. 26933-26940.


Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 175

Maitra, R.K., McMillan, N.A., Desai, S., McSwiggen, J., Hovanessian, A.G., Sen, G., Williams,

Malathi, K., Dong, B., Gale, M.,Jr & Silverman, R.H. 2007, Small self-RNA generated by

Manche, L., Green, S.R., Schmedt, C. & Mathews, M.B. 1992, Interactions between double-

Mangeat, B., Gers-Huber, G., Lehmann, M., Zufferey, M., Luban, J. & Piguet, V. 2009, HIV-1

Marie, I., Svab, J., Robert, N., Galabru, J. & Hovanessian, A.G. 1990, Differential expression

Marin, M., Rose, K.M., Kozak, S.L. & Kabat, D. 2003, HIV-1 Vif protein binds the editing

McElrath, M.J., De Rosa, S.C., Moodie, Z., Dubey, S., Kierstead, L., Janes, H., Defawe, O.D.,

Mehle, A., Goncalves, J., Santa-Marta, M., McPike, M. & Gabuzda, D. 2004, Phosphorylation

Merson, M.H. 2006, The HIV-AIDS pandemic at 25--the global response, *The New England* 

Meylan, P.R., Guatelli, J.C., Munis, J.R., Richman, D.D. & Kornbluth, R.S. 1993, Mechanisms

Mintzer, M.A. & Simanek, E.E. 2009, Nonviral vectors for gene delivery, *Chemical reviews,* 

Mitchell, R.S., Katsura, C., Skasko, M.A., Fitzpatrick, K., Lau, D., Ruiz, A., Stephens, E.B.,

Miyagi, E., Andrew, A.J., Kao, S. & Strebel, K. 2009, Vpu enhances HIV-1 virus release in the

primary human macrophages, *Virology,* vol. 193, no. 1, pp. 138-148.

interferon-inducible enzymes, *Virology,* vol. 204, no. 2, pp. 823-827.

reverse transcripts, *Nature,* vol. 424, no. 6944, pp. 99-103.

cohort analysis, *Lancet,* vol. 372, no. 9653, pp. 1894-1905.

*journal of medicine,* vol. 354, no. 23, pp. 2414-2417.

*PLoS pathogens,* vol. 5, no. 5, pp. e1000450.

vol. 109, no. 2, pp. 259-302.

no. 8, pp. 2868-2873.

vol. 12, no. 11, pp. 5238-5248.

18607.

2866.

1398-1403.

B.R. & Silverman, R.H. 1994, HIV-1 TAR RNA has an intrinsic ability to activate

RNase L amplifies antiviral innate immunity, *Nature,* vol. 448, no. 7155, pp. 816-819.

stranded RNA regulators and the protein kinase DAI, *Molecular and cellular biology,* 

Vpu neutralizes the antiviral factor Tetherin/BST-2 by binding it and directing its beta-TrCP2-dependent degradation, *PLoS pathogens,* vol. 5, no. 9, pp. e1000574. Mangeat, B., Turelli, P., Caron, G., Friedli, M., Perrin, L. & Trono, D. 2003, Broad

antiretroviral defence by human APOBEC3G through lethal editing of nascent

and distinct structure of 69- and 100-kDa forms of 2-5A synthetase in human cells treated with interferon, *The Journal of biological chemistry,* vol. 265, no. 30, pp. 18601-

enzyme APOBEC3G and induces its degradation, *Nature medicine,* vol. 9, no. 11, pp.

Carter, D.K., Hural, J., Akondy, R., Buchbinder, S.P., Robertson, M.N., Mehrotra, D.V., Self, S.G., Corey, L., Shiver, J.W., Casimiro, D.R. & Step Study Protocol Team 2008, HIV-1 vaccine-induced immunity in the test-of-concept Step Study: a case-

of a novel SOCS-box regulates assembly of the HIV-1 Vif-Cul5 complex that promotes APOBEC3G degradation, *Genes & development,* vol. 18, no. 23, pp. 2861-

for the inhibition of HIV replication by interferons-alpha, -beta, and -gamma in

Margottin-Goguet, F., Benarous, R. & Guatelli, J.C. 2009, Vpu antagonizes BST-2 mediated restriction of HIV-1 release via beta-TrCP and endo-lysosomal trafficking,

absence of Bst-2 cell surface down-modulation and intracellular depletion, *Proceedings of the National Academy of Sciences of the United States of America,* vol. 106,


Kupzig, S., Korolchuk, V., Rollason, R., Sugden, A., Wilde, A. & Banting, G. 2003, Bst-

Land, A.M., Ball, T.B., Luo, M., Pilon, R., Sandstrom, P., Embree, J.E., Wachihi, C., Kimani, J.

Lecossier, D., Bouchonnet, F., Clavel, F. & Hance, A.J. 2003, Hypermutation of HIV-1 DNA

Lee, S.K., Dykxhoorn, D.M., Kumar, P., Ranjbar, S., Song, E., Maliszewski, L.E., Francois-

Lei, Y., Lee, C.L., Joo, K.I., Zarzar, J., Liu, Y., Dai, B., Fox, V. & Wang, P. 2011, Gene Editing

Lemaire, P.A., Anderson, E., Lary, J. & Cole, J.L. 2008, Mechanism of PKR Activation by

Li, M.J., Kim, J., Li, S., Zaia, J., Yee, J.K., Anderson, J., Akkina, R. & Rossi, J.J. 2005, Long-

Li, X. & Sodroski, J. 2008, The TRIM5alpha B-box 2 domain promotes cooperative binding to

Lilly, F. 1967, Susceptibility to two strains of Friend leukemia virus in mice, *Science (New* 

Luban, J. 2007, Cyclophilin A, TRIM5, and resistance to human immunodeficiency virus

Lundin, K.E., Simonson, O.E., Moreno, P.M., Zaghloul, E.M., Oprea, I.I., Svahn, M.G. &

Lusso, P., Markham, P.D., Ranki, A., Earl, P., Moss, B., Dorner, F., Gallo, R.C. & Krohn, K.J.

Maitra, R.K. & Silverman, R.H. 1998, Regulation of human immunodeficiency virus

type 1 infection, *Journal of virology,* vol. 81, no. 3, pp. 1054-1061.

and primary isolates of HIV, *Blood,* vol. 106, no. 3, pp. 818-826.

dsRNA, *Journal of Molecular Biology,* vol. 381, no. 2, pp. 351-360.

*Society of Gene Therapy,* vol. 12, no. 5, pp. 900-909.

vol. 82, no. 23, pp. 11495-11502.

no. 1, pp. 47-56.

72, no. 12, pp. 10251-10255.

no. 2, pp. 1146-1152.

*York, N.Y.),* vol. 155, no. 761, pp. 461-462.

*Traffic (Copenhagen, Denmark),* vol. 4, no. 10, pp. 694-709.

*Journal of virology,* vol. 82, no. 16, pp. 8172-8182.

1112.

*Therapy,* .

2/HM1.24 is a raft-associated apical membrane protein with an unusual topology,

& Plummer, F.A. 2008, Human immunodeficiency virus (HIV) type 1 proviral hypermutation correlates with CD4 count in HIV-infected women from Kenya,

in the absence of the Vif protein, *Science (New York, N.Y.),* vol. 300, no. 5622, pp.

Bongarcon, V., Goldfeld, A., Swamy, N.M., Lieberman, J. & Shankar, P. 2005, Lentiviral delivery of short hairpin RNAs protects CD4 T cells from multiple clades

of Human Embryonic Stem Cells via an Engineered Baculoviral Vector Carrying Zinc-finger Nucleases, *Molecular therapy : the journal of the American Society of Gene* 

term inhibition of HIV-1 infection in primary hematopoietic cells by lentiviral vector delivery of a triple combination of anti-HIV shRNA, anti-CCR5 ribozyme, and a nucleolar-localizing TAR decoy, *Molecular therapy : the journal of the American* 

the retroviral capsid by mediating higher-order self-association, *Journal of virology,* 

Smith, C.I. 2009, Nanotechnology approaches for gene transfer, *Genetica,* vol. 137,

1988, Cell-mediated immune response toward viral envelope and core antigens in gibbon apes (Hylobates lar) chronically infected with human immunodeficiency virus-1, *Journal of immunology (Baltimore, Md.: 1950),* vol. 141, no. 7, pp. 2467-2473. Madani, N. & Kabat, D. 1998, An endogenous inhibitor of human immunodeficiency virus

in human lymphocytes is overcome by the viral Vif protein, *Journal of virology,* vol.

replication by 2',5'-oligoadenylate-dependent RNase L, *Journal of virology,* vol. 72,


Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 177

Obad, S., Brunnstrom, H., Vallon-Christersson, J., Borg, A., Drott, K. & Gullberg, U. 2004,

Ohkura, S., Yap, M.W., Sheldon, T. & Stoye, J.P. 2006, All three variable regions of the

Okumura, A., Alce, T., Lubyova, B., Ezelle, H., Strebel, K. & Pitha, P.M. 2008, HIV-1

Okumura, A., Lu, G., Pitha-Rowe, I. & Pitha, P.M. 2006, Innate antiviral response targets

Ono, A. 2009, HIV-1 Assembly at the Plasma Membrane: Gag Trafficking and Localization,

Ozato, K., Shin, D.M., Chang, T.H. & Morse, H.C.,3rd 2008, TRIM family proteins and their

Pardieu, C., Vigan, R., Wilson, S.J., Calvi, A., Zang, T., Bieniasz, P., Kellam, P., Towers, G.J.

Perez, E.E., Wang, J., Miller, J.C., Jouvenot, Y., Kim, K.A., Liu, O., Wang, N., Lee, G.,

Perez-Caballero, D., Hatziioannou, T., Yang, A., Cowan, S. & Bieniasz, P.D. 2005, Human

Perez-Caballero, D., Zang, T., Ebrahimi, A., McNatt, M.W., Gregory, D.A., Johnson, M.C. &

Piantadosi, A., Humes, D., Chohan, B., McClelland, R.S. & Overbaugh, J. 2009, Analysis of

Pincetic, A., Kuang, Z., Seo, E.J. & Leis, J. 2010, The interferon-induced gene ISG15 blocks

Pincus, T., Hartley, J.W. & Rowe, W.P. 1971, A major genetic locus affecting resistance to

Pitisuttithum, P., Gilbert, P., Gurwith, M., Heyward, W., Martin, M., van Griensven, F., Hu,

U-937 cells, *Oncogene,* vol. 23, no. 23, pp. 4050-4059.

*Journal of virology,* vol. 80, no. 17, pp. 8554-8565.

degradation, *Virology,* vol. 373, no. 1, pp. 85-97.

of tetherin, *PLoS pathogens,* vol. 6, no. 4, pp. e1000843.

specificity, *J Virol,* vol. 79, no. 14, pp. 8969-8978.

cells, *Cell,* vol. 139, no. 3, pp. 499-511.

*S A,* vol. 103, no. 5, pp. 1440-1445.

849-860.

no. 7, pp. 808-816.

no. 16, pp. 7805-7814.

no. 9, pp. 4725-4736.

*Future virology,* vol. 4, no. 3, pp. 241-257.

Staf50 is a novel p53 target gene conferring reduced clonogenic growth of leukemic

TRIM5alpha B30.2 domain can contribute to the specificity of retrovirus restriction,

accessory proteins VPR and Vif modulate antiviral response by targeting IRF-3 for

HIV-1 release by the induction of ubiquitin-like protein ISG15, *Proc Natl Acad Sci U* 

emerging roles in innate immunity, *Nature reviews.Immunology,* vol. 8, no. 11, pp.

& Neil, S.J. 2010, The RING-CH ligase K5 antagonizes restriction of KSHV and HIV-1 particle release by mediating ubiquitin-dependent endosomal degradation

Bartsevich, V.V., Lee, Y.L., Guschin, D.Y., Rupniewski, I., Waite, A.J., Carpenito, C., Carroll, R.G., Orange, J.S., Urnov, F.D., Rebar, E.J., Ando, D., Gregory, P.D., Riley, J.L., Holmes, M.C. & June, C.H. 2008, Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases, *Nature biotechnology,* vol. 26,

tripartite motif 5alpha domains responsible for retrovirus restriction activity and

Bieniasz, P.D. 2009, Tetherin inhibits HIV-1 release by directly tethering virions to

the percentage of human immunodeficiency virus type 1 sequences that are hypermutated and markers of disease progression in a longitudinal cohort, including one individual with a partially defective Vif, *Journal of virology,* vol. 83,

retrovirus release from cells late in the budding process, *Journal of virology,* vol. 84,

infection with murine leukemia viruses. I. Tissue culture studies of naturally occurring viruses, *The Journal of experimental medicine,* vol. 133, no. 6, pp. 1219-1233.

D., Tappero, J.W., Choopanya, K. & Bangkok Vaccine Evaluation Group 2006,


Moehle, E.A., Rock, J.M., Lee, Y.L., Jouvenot, Y., DeKelver, R.C., Gregory, P.D., Urnov, F.D.

Muckenfuss, H., Hamdorf, M., Held, U., Perkovic, M., Lower, J., Cichutek, K., Flory, E.,

Muto, N.F., Martinand, C., Adelson, M.E. & Suhadolnik, R.J. 1999, Inhibition of Replication

Nagai, K., Wong, A.H., Li, S., Tam, W.N., Cuddihy, A.R., Sonenberg, N., Mathews, M.B.,

Nallagatla, S.R., Toroney, R. & Bevilacqua, P.C. 2011, Regulation of innate immunity

Nathans, R., Cao, H., Sharova, N., Ali, A., Sharkey, M., Stranska, R., Stevenson, M. & Rana,

Neagu, M.R., Ziegler, P., Pertel, T., Strambio-De-Castillia, C., Grutter, C., Martinetti, G.,

Neil, S.J., Zang, T. & Bieniasz, P.D. 2008, Tetherin inhibits retrovirus release and is

Nguyen, D.H., Gummuluru, S. & Hu, J. 2007, Deamination-independent inhibition of

Nguyen, D.H. & Hildreth, J.E. 2000, Evidence for budding of human immunodeficiency

Niewiadomska, A.M. & Yu, X.F. 2009, Host restriction of HIV-1 by APOBEC3 and viral

Nisole, S., Stoye, J.P. & Saib, A. 2005, TRIM family proteins: retroviral restriction and

Obad, S., Olofsson, T., Mechti, N., Gullberg, U. & Drott, K. 2007, Expression of the IFN-

antiviral defence, *Nat Rev Microbiol,* vol. 3, no. 10, pp. 799-808.

differentiation of human bone marrow, *Leuk Res,* .

*The Journal of clinical investigation,* vol. 119, no. 10, pp. 3035-3047.

antagonized by HIV-1 Vpu, *Nature,* vol. 451, no. 7177, pp. 425-430.

*Gene therapy,* vol. 13, no. 6, pp. 553-558.

Construct, *J.Virol.,* vol. 73, pp. 9021-9028.

*biology,* vol. 21, no. 1, pp. 119-127.

*virology,* vol. 74, no. 7, pp. 3264-3272.

10, pp. 1187-1192.

9, pp. 4465-4472.

22172.

1725.

& Holmes, M.C. 2007, Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases, *Proceedings of the National Academy of Sciences of the United States of America,* vol. 104, no. 9, pp. 3055-3060. Morris, K.V. & Rossi, J.J. 2006, Lentiviral-mediated delivery of siRNAs for antiviral therapy,

Schumann, G.G. & Munk, C. 2006, APOBEC3 proteins inhibit human LINE-1 retrotransposition, *The Journal of biological chemistry,* vol. 281, no. 31, pp. 22161-

of Reactivated Human Immunodeficiency Virus Type 1 (HIV-1) in Latently Infected U1 Cells Transduced with an HIV-1 Long Terminal Repeat-Driven PKR cDNA

Hiscott, J., Wainberg, M.A. & Koromilas, A.E. 1997, Induction of CD4 expression and human immunodeficiency virus type 1 replication by mutants of the interferon-inducible protein kinase PKR, *Journal of virology,* vol. 71, no. 2, pp. 1718-

through RNA structure and the protein kinase PKR, *Current opinion in structural* 

T.M. 2008, Small-molecule inhibition of HIV-1 Vif, *Nature biotechnology,* vol. 26, no.

Mazzucchelli, L., Grutter, M., Manz, M.G. & Luban, J. 2009, Potent inhibition of HIV-1 by TRIM5-cyclophilin fusion proteins engineered from human components,

hepatitis B virus reverse transcription by APOBEC3G, *Journal of virology,* vol. 81, no.

virus type 1 selectively from glycolipid-enriched membrane lipid rafts, *Journal of* 

evasion through Vif, *Current topics in microbiology and immunology,* vol. 339, pp. 1-25.

inducible p53-target gene TRIM22 is down-regulated during erythroid


Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 179

Sadler, A.J. & Williams, B.R. 2008, Interferon-inducible antiviral effectors, *Nature* 

Sakuma, R., Ohmine, S. & Ikeda, Y. 2010, Determinants for the rhesus monkey TRIM5alpha-

Sakuma, R., Noser, J.A., Ohmine, S. & Ikeda, Y. 2007, Rhesus monkey TRIM5alpha restricts

Sawyer, S.L., Emerman, M. & Malik, H.S. 2007, Discordant evolution of the adjacent

Sawyer, S.L., Emerman, M. & Malik, H.S. 2004, Ancient adaptive evolution of the primate antiviral DNA-editing enzyme APOBEC3G, *PLoS biology,* vol. 2, no. 9, pp. E275. Sayah, D.M., Sokolskaja, E., Berthoux, L. & Luban, J. 2004, Cyclophilin A retrotransposition

Schroder, H.C., Ugarkovic, D., Merz, H., Kuchino, Y., Okamoto, T. & Muller, W.E. 1990a,

Schroder, H.C., Ugarkovic, D., Wenger, R., Reuter, P., Okamoto, T. & Muller, W.E. 1990b,

Schrofelbauer, B., Yu, Q., Zeitlin, S.G. & Landau, N.R. 2005, Human immunodeficiency virus

Sheehy, A.M., Gaddis, N.C. & Malim, M.H. 2003, The antiretroviral enzyme APOBEC3G is

Sheehy, A.M., Gaddis, N.C., Choi, J.D. & Malim, M.H. 2002, Isolation of a human gene that

Shi, H.X., Yang, K., Liu, X., Liu, X.Y., Wei, B., Shan, Y.F., Zhu, L.H. & Wang, C. 2010, Positive

modification, *Molecular and cellular biology,* vol. 30, no. 10, pp. 2424-2436. Shiver, J.W., Fu, T.M., Chen, L., Casimiro, D.R., Davies, M.E., Evans, R.K., Zhang, Z.Q.,

*Societies for Experimental Biology,* vol. 4, no. 13, pp. 3124-3130.

glycosylases, *Journal of virology,* vol. 79, no. 17, pp. 10978-10987.

*and Human Retroviruses,* vol. 6, no. 5, pp. 659-672.

mediated block of the late phase of HIV-1 replication, *The Journal of biological* 

HIV-1 production through rapid degradation of viral Gag polyproteins, *Nat Med,* 

antiretroviral genes TRIM22 and TRIM5 in mammals, *PLoS pathogens,* vol. 3, no. 12,

into TRIM5 explains owl monkey resistance to HIV-1, *Nature,* vol. 430, no. 6999, pp.

Protection of HeLa-T4+ cells against human immunodeficiency virus (HIV) infection after stable transfection with HIV LTR-2',5'-oligoadenylate synthetase hybrid gene, *The FASEB journal : official publication of the Federation of American* 

Binding of Tat protein to TAR region of human immunodeficiency virus type 1 blocks TAR-mediated activation of (2'-5')oligoadenylate synthetase, *AIDS Research* 

type 1 Vpr induces the degradation of the UNG and SMUG uracil-DNA

degraded by the proteasome in response to HIV-1 Vif, *Nature medicine,* vol. 9, no.

inhibits HIV-1 infection and is suppressed by the viral Vif protein, *Nature,* vol. 418,

regulation of interferon regulatory factor 3 activation by Herc5 via ISG15

Simon, A.J., Trigona, W.L., Dubey, S.A., Huang, L., Harris, V.A., Long, R.S., Liang, X., Handt, L., Schleif, W.A., Zhu, L., Freed, D.C., Persaud, N.V., Guan, L., Punt, K.S., Tang, A., Chen, M., Wilson, K.A., Collins, K.B., Heidecker, G.J., Fernandez, V.R., Perry, H.C., Joyce, J.G., Grimm, K.M., Cook, J.C., Keller, P.M., Kresock, D.S., Mach, H., Troutman, R.D., Isopi, L.A., Williams, D.M., Xu, Z., Bohannon, K.E., Volkin, D.B., Montefiori, D.C., Miura, A., Krivulka, G.R., Lifton, M.A., Kuroda, M.J., Schmitz, J.E., Letvin, N.L., Caulfield, M.J., Bett, A.J., Youil, R., Kaslow, D.C. &

*reviews.Immunology,* vol. 8, no. 7, pp. 559-568.

*chemistry,* vol. 285, no. 6, pp. 3784-3793.

vol. 13, no. 5, pp. 631-635.

pp. e197.

569-573.

11, pp. 1404-1407.

no. 6898, pp. 646-50.

Randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 vaccine among injection drug users in Bangkok, Thailand, *The Journal of infectious diseases,* vol. 194, no. 12, pp. 1661-1671.


Priddy, F.H., Brown, D., Kublin, J., Monahan, K., Wright, D.P., Lalezari, J., Santiago, S.,

no. 4904, pp. 575-577.

*medicine,* vol. 361, no. 23, pp. 2209-2220.

323, no. 5919, pp. 1304-1307.

20, pp. 10457-10466.

vol. 5, no. 2, pp. e1000311.

Randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 vaccine among injection drug users in Bangkok, Thailand, *The Journal of infectious diseases,* vol. 194, no. 12, pp. 1661-1671. Poli, G., Orenstein, J.M., Kinter, A., Folks, T.M. & Fauci, A.S. 1989, Interferon-alpha but not

AZT suppresses HIV expression in chronically infected cell lines, *Science,* vol. 244,

Marmor, M., Lally, M., Novak, R.M., Brown, S.J., Kulkarni, P., Dubey, S.A., Kierstead, L.S., Casimiro, D.R., Mogg, R., DiNubile, M.J., Shiver, J.W., Leavitt, R.Y., Robertson, M.N., Mehrotra, D.V., Quirk, E. & Merck V520-016 Study Group 2008, Safety and immunogenicity of a replication-incompetent adenovirus type 5 HIV-1 clade B gag/pol/nef vaccine in healthy adults, *Clinical infectious diseases : an official publication of the Infectious Diseases Society of America,* vol. 46, no. 11, pp. 1769-1781. Pryciak, P.M. & Varmus, H.E. 1992, Fv-1 restriction and its effects on murine leukemia virus integration in vivo and in vitro, *Journal of virology,* vol. 66, no. 10, pp. 5959-5966. Rerks-Ngarm, S., Pitisuttithum, P., Nitayaphan, S., Kaewkungwal, J., Chiu, J., Paris, R.,

Premsri, N., Namwat, C., de Souza, M., Adams, E., Benenson, M., Gurunathan, S., Tartaglia, J., McNeil, J.G., Francis, D.P., Stablein, D., Birx, D.L., Chunsuttiwat, S., Khamboonruang, C., Thongcharoen, P., Robb, M.L., Michael, N.L., Kunasol, P., Kim, J.H. & MOPH-TAVEG Investigators 2009, Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand, *The New England journal of* 

2009, The challenge of finding a cure for HIV infection, *Science (New York, N.Y.),* vol.

to AZT and ddC during long-term combination therapy in patients with advanced

Thorner, A.R., Swanson, P.E., Gorgone, D.A., Lifton, M.A., Lemckert, A.A., Holterman, L., Chen, B., Dilraj, A., Carville, A., Mansfield, K.G., Goudsmit, J. & Barouch, D.H. 2006, Hexon-chimaeric adenovirus serotype 5 vectors circumvent

phosphorylation of eIF2alpha in rotavirus infection, *Journal of virology,* vol. 84, no.

stem structure of human immunodeficiency virus type 1 Tat-responsive sequence of RNA is required for interaction with the interferon-induced 68,000-Mr protein

within APOBEC3G and APOBEC3F interact with separate regions of human immunodeficiency virus type 1 Vif, *Journal of virology,* vol. 83, no. 4, pp. 1992-2003. Sadler, A.J., Latchoumanin, O., Hawkes, D., Mak, J. & Williams, B.R. 2009, An antiviral

response directed by PKR phosphorylation of the RNA helicase A, *PLoS pathogens,* 

Richman, D.D., Margolis, D.M., Delaney, M., Greene, W.C., Hazuda, D. & Pomerantz, R.J.

Richman, D.D., Meng, T.C., Spector, S.A., Fischl, M.A., Resnick, L. & Lai, S. 1994, Resistance

infection with human immunodeficiency virus, *JAIDS,* vol. 7, pp. 135-138. Roberts, D.M., Nanda, A., Havenga, M.J., Abbink, P., Lynch, D.M., Ewald, B.A., Liu, J.,

pre-existing anti-vector immunity, *Nature,* vol. 441, no. 7090, pp. 239-243. Rojas, M., Arias, C.F. & Lopez, S. 2010, Protein kinase R is responsible for the

Roy, S., Agy, M., Hovanessian, A.G., Sonenberg, N. & Katze, M.G. 1991, The integrity of the

Russell, R.A., Smith, J., Barr, R., Bhattacharyya, D. & Pathak, V.K. 2009, Distinct domains

kinase, *Journal of virology,* vol. 65, no. 2, pp. 632-640.


Cellular Restriction Factors: Exploiting the Body's Antiviral Proteins to Combat HIV-1/AIDS 181

Thali, M., Bukovsky, A., Kondo, E., Rosenwirth, B., Walsh, C.T., Sodroski, J. & Gottlinger,

Tian, C., Yu, X., Zhang, W., Wang, T., Xu, R. & Yu, X.F. 2006, Differential requirement for

Tissot, C. & Mechti, N. 1995, Molecular cloning of a new interferon-induced factor that

Turelli, P., Mangeat, B., Jost, S., Vianin, S. & Trono, D. 2004, Inhibition of hepatitis B virus replication by APOBEC3G, *Science (New York, N.Y.),* vol. 303, no. 5665, pp. 1829. Ulenga, N.K., Sarr, A.D., Hamel, D., Sankale, J.L., Mboup, S. & Kanki, P.J. 2008, The level of

Urnov, F.D., Rebar, E.J., Holmes, M.C., Zhang, H.S. & Gregory, P.D. 2010, Genome editing

Urnov, F.D., Miller, J.C., Lee, Y.L., Beausejour, C.M., Rock, J.M., Augustus, S., Jamieson,

Usami, Y., Popov, S., Popova, E., Inoue, M., Weissenhorn, W. & G Gottlinger, H. 2009, The

Valcke, H.S., Bernard, N.F., Bruneau, J., Alary, M., Tsoukas, C.M. & Roger, M. 2006,

van Manen, D., Rits, M.A., Beugeling, C., van Dort, K., Schuitemaker, H. & Kootstra, N.A.

Varthakavi, V., Smith, R.M., Bour, S.P., Strebel, K. & Spearman, P. 2003, Viral protein U

Vazquez-Perez, J.A., Ormsby, C.E., Hernandez-Juan, R., Torres, K.J. & Reyes-Teran, G. 2009,

VerPlank, L., Bouamr, F., LaGrassa, T.J., Agresta, B., Kikonyogo, A., Leis, J. & Carter, C.A.

372, pp. 363-365.

6, pp. 3112-3115.

10, pp. 1285-1290.

vol. 435, no. 7042, pp. 646-651.

*microbe,* vol. 3, no. 4, pp. 245-252.

*PLoS pathogens,* vol. 4, no. 2, pp. e18.

progression, *Retrovirology,* vol. 6, pp. 23.

no. 25, pp. 15154-15159.

636-646.

1, pp. 181-184.

*Biol Chem,* vol. 270, no. 25, pp. 14891-14898.

H.G. 1994, Functional association of cyclophilin A with HIV-1 virions, *Nature,* vol.

conserved tryptophans in human immunodeficiency virus type 1 Vif for the selective suppression of APOBEC3G and APOBEC3F, *Journal of virology,* vol. 80, no.

represses human immunodeficiency virus type 1 long terminal repeat expression, *J* 

APOBEC3G (hA3G)-related G-to-A mutations does not correlate with viral load in HIV type 1-infected individuals, *AIDS Research and Human Retroviruses,* vol. 24, no.

with engineered zinc finger nucleases, *Nature reviews.Genetics,* vol. 11, no. 9, pp.

A.C., Porteus, M.H., Gregory, P.D. & Holmes, M.C. 2005, Highly efficient endogenous human gene correction using designed zinc-finger nucleases, *Nature,* 

ESCRT pathway and HIV-1 budding, *Biochemical Society transactions,* vol. 37, no. Pt

APOBEC3G genetic variants and their association with risk of HIV infection in highly exposed Caucasians, *AIDS (London, England),* vol. 20, no. 15, pp. 1984-1986. Van Damme, N., Goff, D., Katsura, C., Jorgenson, R.L., Mitchell, R., Johnson, M.C., Stephens,

E.B. & Guatelli, J. 2008, The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated from the cell surface by the viral Vpu protein, *Cell host &* 

2008, The effect of Trim5 polymorphisms on the clinical course of HIV-1 infection,

counteracts a human host cell restriction that inhibits HIV-1 particle production, *Proceedings of the National Academy of Sciences of the United States of America,* vol. 100,

APOBEC3G mRNA expression in exposed seronegative and early stage HIV infected individuals decreases with removal of exposure and with disease

2001, Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L

Emini, E.A. 2002, Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity, *Nature,* vol. 415, no. 6869, pp. 331-335.


Simon, J.H. & Malim, M.H. 1996, The human immunodeficiency virus type 1 Vif protein

Singh, R., Gaiha, G., Werner, L., McKim, K., Mlisana, K., Luban, J., Walker, B.D., Karim, S.S.,

Skasko, M., Tokarev, A., Chen, C.C., Fischer, W.B., Pillai, S.K. & Guatelli, J. 2011, BST-2 is

for a post-ER mechanism of Vpu-action, *Virology,* vol. 411, no. 1, pp. 65-77. Sokolskaja, E. & Luban, J. 2006, Cyclophilin, TRIM5, and innate immunity to HIV-1, *Curr* 

Soumelis, V., Scott, I., Gheyas, F., Bouhour, D., Cozon, G., Cotte, L., Huang, L., Levy, J.A. &

Stenglein, M.D. & Harris, R.S. 2006, APOBEC3B and APOBEC3F inhibit L1

Strack, B., Calistri, A., Craig, S., Popova, E. & Gottlinger, H.G. 2003, AIP1/ALIX is a binding

Stremlau, M., Perron, M., Lee, M., Li, Y., Song, B., Javanbakht, H., Diaz-Griffero, F.,

Stremlau, M., Owens, C.M., Perron, M.J., Kiessling, M., Autissier, P. & Sodroski, J. 2004, The

Su, Y., Popik, W. & Pitha, P.M. 1995, Inhibition of human immunodeficiency virus type 1

Tavel, J.A., Huang, C.Y., Shen, J., Metcalf, J.A., Dewar, R., Shah, A., Vasudevachari, M.B.,

Terwilliger, E.F., Cohen, E.A., Lu, Y.C., Sodroski, J.G. & Haseltine, W.A. 1989, Functional

HIV-1 infection, *Journal of virology,* vol. 85, no. 1, pp. 208-216.

HIV-infected AIDS patients, *Blood,* vol. 98, no. 4, pp. 906-912.

*biological chemistry,* vol. 281, no. 25, pp. 16837-16841.

*Proc Natl Acad Sci U S A,* vol. 103, no. 14, pp. 5514-5519.

*Interferon and Cytokine Research,* vol. 30, no. 7, pp. 461-464.

1400.

6, pp. 689-699.

1, pp. 110-121.

*virology,* vol. 70, no. 8, pp. 5297-5305.

*Opin Microbiol,* vol. 9, no. 4, pp. 404-408.

monkeys, *Nature,* vol. 427, pp. 848-853.

Emini, E.A. 2002, Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity, *Nature,* vol. 415, no. 6869, pp. 331-335. Simon, J.H., Gaddis, N.C., Fouchier, R.A. & Malim, M.H. 1998, Evidence for a newly

discovered cellular anti-HIV-1 phenotype, *Nature medicine,* vol. 4, no. 12, pp. 1397-

modulates the postpenetration stability of viral nucleoprotein complexes, *Journal of* 

Brass, A.L., Ndung'u, T. & CAPRISA Acute Infection Study Team 2011, Association of TRIM22 with the type 1 interferon response and viral control during primary

rapidly down-regulated from the cell surface by the HIV-1 protein Vpu: evidence

Liu, Y.J. 2001, Depletion of circulating natural type 1 interferon-producing cells in

retrotransposition by a DNA deamination-independent mechanism, *The Journal of* 

partner for HIV-1 p6 and EIAV p9 functioning in virus budding, *Cell,* vol. 114, no.

Anderson, D.J., Sundquist, W.I. & Sodroski, J. 2006, Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor,

cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World

replication by a Tat-activated, transduced interferon gene: targeted expression to human immunodeficiency virus type 1-infected cells, *Journal of virology,* vol. 69, no.

Follmann, D.A., Herpin, B., Davey, R.T., Polis, M.A., Kovacs, J., Masur, H. & Lane, H.C. 2010, Interferon-alpha produces significant decreases in HIV load, *Journal of interferon & cytokine research : the official journal of the International Society for* 

role of human immunodeficiency virus type 1 vpu, *Proceedings of the National Academy of Sciences of the United States of America,* vol. 86, no. 13, pp. 5163-5167.


**7** 

*Japan* 

**Retroviral Host Cell Factors: TRIM5,** 

*Department of Molecular Virology, Tokyo Medical and Dental University,* 

The conventional innate and adaptive immune systems are very effective at viral infections. However, for retroviral infections, there is another immune system that can recognize at multiple levels e.g. expression of internal host factors with antiviral activity. This is a component of viral recognition and subsequent restriction that has been called "intrinsic immunity"(Bieniasz, 2004). Intrinsic immunity can distinguish from innate and adaptive immunity, and it does not need to be induced by viral infections. Retrovirus replication has many steps in common with other retroviruses. Upon entry into the cytoplasm of target cells, some host factors are required for efficient retroviral replication cycle, and others act as restriction factors that block reverse transcription and ligation of viral cDNA to chromosomal DNA. Recently, several host factors have been identified such as the proline isomerase cyclophilin A (CypA), ApoB mRNA editing catalytic subunit (APOBEC) and tripartite motif protein 5 alpha (TRIM5) against retrovirus infection. This review will focus on how these host factors modulate retroviral activity. It will then present our current understanding of the mechanism that may explain zoonotic

**1.1 Fv1 and Fv4: Restriction factors that block infection by Friend-MLV in murine cells**  The most intensively studied anti-cellular gene is Friend virus susceptibility (Fv) gene in laboratory mice. Fv1 and Fv4 were of special interest in Fv alleles because cultured murine cells containing them were resistant to infection by Friend murine leukemia virus (MLV)(Gardner et al., 1980; Hartley et al., 1970; Pincus et al., 1971; Rasheed and Gardner, 1983; Suzuki, 1975). Fv1-mediated restriction of MLV, for instance, is a well-studied representative of a class of restriction factors that act after membrane fusion, are highly virus-specific (Goff, 2004). Fv1 has two alleles, Fv1n and Fv1b, targeting B- and N-tropic MLV, respectively (Rein et al., 1976). Fv4 was shown to encode an ecotropic MLV-like *env* gene and recent report showed that Fv4 inhibits infection by exerting dominant negative effect on MLV Env (Takeda and Matano, 2007). Although the precise mechanism of Fv1 restriction remains unclear, the important point is that the viral determinants for this type of restriction have been mapped to the capsid protein (MLV amino acid 110) and as a target of host factors that can modulate retroviral life cycle (Gautsch et al., 1978; Kozak and

**1. Introduction** 

transmission of retroviruses.

Chakraborti, 1996).

**APOBEC3G and Cyclophilins** 

Ryuta Sakuma and Hiroaki Takeuchi

domain in HIV type 1 Pr55(Gag), *Proc Natl Acad Sci U S A,* vol. 98, no. 1421332273, pp. 7724-779.


## **Retroviral Host Cell Factors: TRIM5, APOBEC3G and Cyclophilins**

Ryuta Sakuma and Hiroaki Takeuchi *Department of Molecular Virology, Tokyo Medical and Dental University, Japan* 

## **1. Introduction**

182 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Vigan, R. & Neil, S.J. 2010, Determinants of tetherin antagonism in the transmembrane

Vyas, T.K., Shah, L. & Amiji, M.M. 2006, Nanoparticulate drug carriers for delivery of

Wang, B., Mikhail, M., Dyer, W.B., Zaunders, J.J., Kelleher, A.D. & Saksena, N.K. 2003, First

Woo, J.S., Imm, J.H., Min, C.K., Kim, K.J., Cha, S.S. & Oh, B.H. 2006, Structural and

Yamashita, T., Kamada, K., Hatcho, K., Adachi, A. & Nomaguchi, M. 2008, Identification of

Yang, B., Chen, K., Zhang, C., Huang, S. & Zhang, H. 2007, Virion-associated uracil DNA

Yao, S., Liu, M.S., Masters, S.L., Zhang, J.G., Babon, J.J., Nicola, N.A., Nicholson, S.E. &

Yap, M.W., Nisole, S. & Stoye, J.P. 2005, A single amino acid change in the SPRY domain of human Trim5alpha leads to HIV-1 restriction, *Curr Biol,* vol. 15, no. 1, pp. 73-78. Yu, X., Yu, Y., Liu, B., Luo, K., Kong, W., Mao, P. & Yu, X.F. 2003, Induction of APOBEC3G

pp. 7724-779.

5, pp. 613-628.

pp. 1353-1363.

1149.

*virology,* vol. 84, no. 24, pp. 12958-12970.

*reviews.Molecular cell biology,* vol. 8, no. 5, pp. 355-368.

*chemistry,* vol. 282, no. 16, pp. 11667-11675.

*York, N.Y.),* vol. 302, no. 5647, pp. 1056-1060.

vol. 15, no. 12, pp. 2761-2772.

domain in HIV type 1 Pr55(Gag), *Proc Natl Acad Sci U S A,* vol. 98, no. 1421332273,

domain of the human immunodeficiency virus type 1 Vpu protein, *Journal of* 

HIV/AIDS therapy to viral reservoir sites, *Expert opinion on drug delivery,* vol. 3, no.

demonstration of a lack of viral sequence evolution in a nonprogressor, defining replication-incompetent HIV-1 infection, *Virology,* vol. 312, no. 1, pp. 135-150. Whitehead, K.A., Langer, R. & Anderson, D.G. 2009, Knocking down barriers: advances in siRNA delivery, *Nature reviews.Drug discovery,* vol. 8, no. 2, pp. 129-138. Williams, R.L. & Urbe, S. 2007, The emerging shape of the ESCRT machinery, *Nature* 

functional insights into the B30.2/SPRY domain, *The EMBO journal,* vol. 25, no. 6,

amino acid residues in HIV-1 Vif critical for binding and exclusion of APOBEC3G/F, *Microbes and infection / Institut Pasteur,* vol. 10, no. 10-11, pp. 1142-

glycosylase-2 and apurinic/apyrimidinic endonuclease are involved in the degradation of APOBEC3G-edited nascent HIV-1 DNA, *The Journal of biological* 

Norton, R.S. 2006, Dynamics of the SPRY domain-containing SOCS box protein 2: flexibility of key functional loops, *Protein science : a publication of the Protein Society,* 

ubiquitination and degradation by an HIV-1 Vif-Cul5-SCF complex, *Science (New* 

The conventional innate and adaptive immune systems are very effective at viral infections. However, for retroviral infections, there is another immune system that can recognize at multiple levels e.g. expression of internal host factors with antiviral activity. This is a component of viral recognition and subsequent restriction that has been called "intrinsic immunity"(Bieniasz, 2004). Intrinsic immunity can distinguish from innate and adaptive immunity, and it does not need to be induced by viral infections. Retrovirus replication has many steps in common with other retroviruses. Upon entry into the cytoplasm of target cells, some host factors are required for efficient retroviral replication cycle, and others act as restriction factors that block reverse transcription and ligation of viral cDNA to chromosomal DNA. Recently, several host factors have been identified such as the proline isomerase cyclophilin A (CypA), ApoB mRNA editing catalytic subunit (APOBEC) and tripartite motif protein 5 alpha (TRIM5) against retrovirus infection. This review will focus on how these host factors modulate retroviral activity. It will then present our current understanding of the mechanism that may explain zoonotic transmission of retroviruses.

**1.1 Fv1 and Fv4: Restriction factors that block infection by Friend-MLV in murine cells**  The most intensively studied anti-cellular gene is Friend virus susceptibility (Fv) gene in laboratory mice. Fv1 and Fv4 were of special interest in Fv alleles because cultured murine cells containing them were resistant to infection by Friend murine leukemia virus (MLV)(Gardner et al., 1980; Hartley et al., 1970; Pincus et al., 1971; Rasheed and Gardner, 1983; Suzuki, 1975). Fv1-mediated restriction of MLV, for instance, is a well-studied representative of a class of restriction factors that act after membrane fusion, are highly virus-specific (Goff, 2004). Fv1 has two alleles, Fv1n and Fv1b, targeting B- and N-tropic MLV, respectively (Rein et al., 1976). Fv4 was shown to encode an ecotropic MLV-like *env* gene and recent report showed that Fv4 inhibits infection by exerting dominant negative effect on MLV Env (Takeda and Matano, 2007). Although the precise mechanism of Fv1 restriction remains unclear, the important point is that the viral determinants for this type of restriction have been mapped to the capsid protein (MLV amino acid 110) and as a target of host factors that can modulate retroviral life cycle (Gautsch et al., 1978; Kozak and Chakraborti, 1996).

Retroviral Host Cell Factors: TRIM5, APOBEC3G and Cyclophilins 185

infection (Pacheco et al., 2010). Another consideration is the clinical significance of TRIM5 against acquired immunodeficiency syndrome (AIDS) in human. Moreover several reports showed that the efficacy of TRIM5-mediated suppression of HIV-1 replication might interfere with disease progression of AIDS in humans (Cagliani et al., 2010; van Manen et al., 2008). Thus, TRIM5-mediated restriction may occur multi step in retrovirus replication

Recently, the lab of Dr. Yasuhiro Ikeda reported that rhesus macaque TRIM5 also inhibits HIV-1 production by inducing the degradation of a viral precursor Gag protein (Sakuma et al., 2007). To restrict HIV-1 production, amino acid residues in B-box 2 and coiled-coil domains dictated the specificity of the restriction. In the late restriction, the accumulation of HIV-1 RNA was not affected but the accumulation of precursor Gag was inhibited in an ubiqutine-proteasome independent manner. This TRIM5-mediated late-restriction is still controversial (Zhang et al., 2008), yet it is presumable that TRIM5 restricts HIV-1 infection and production in two distinct mechanisms. Although TRIM5 restricts HIV-1 infection in

Here is another notable class of the TRIM family called TRIM-Cyp isolated from new wold monkeys (NWM). A report from the laboratory of Dr. Jeremy Luban demonstrated that owl monkey has TRIM-Cyp that restricts HIV-1 infection (Sayah et al., 2004). Although TRIM-Cyp has a cyclophilin A sequence in its C-terminal region instead of B30.2(PRYSPRY) domain that dictates the specificity and the magnitude of post entry restriction in OWM-TRIM5-mediated post-entry restriction, it recognizes incoming core structure and restricts HIV-1 infection (Stremlau et al., 2006). Recently, TRIM-Cyp mRNA was also detected in a rhesus macaque cell, and over-expressed rhesus TRIM-Cyp restricts HIV-1 infection and production (Brennan et al., 2008; Dietrich et al., 2010; Sakuma et al., 2010; Wilson et al.,

Not like other restriction factors, the counter part of TRIM5-mediated restrictions is not accessory gene product of HIV-1, and human TRIM5 has just a modest restriction activity. NWM cell doesn't have TRIM5, yet even without B30.2(PRYSPRY), TRIM5-Cyp can be a defense against viral infection. These evidences suggest that TRIM5 could be a key molecule to explain the species-species barrier. And if so, TRIM5's dual antiviral activities can block the viral transmission even from closer species like to human from monkeys.

Replication of HIV-1 in primary CD4+ T cells, monocyte and some immortalized T cell lines depends on the presence of HIV-1 accessory gene product, Vif (stands for virus infectivity factor)(Fisher et al., 1987; Strebel et al., 1987), and it works in a host cell-specific manner. Vif is required for enhanced HIV-1 replication in some cell types called non-permissive cells, in contrast HIV-1 replication is Vif-independent in permissive cells (Akari et al., 1992; Blanc et al., 1993; Borman et al., 1995; Fan and Peden, 1992; Gabuzda et al., 1992; Sakai et al., 1993; von Schwedler et al., 1993). Recently, some cytidine deaminases were identified as a new class of host restriction factors that target retroviruses such as HIV-1 or SIV (Cullen, 2006; Harris and Liddament, 2004). APOBEC3G (Apo3G), a member of the APOBEC family of cytidine deaminases, is the first identified enzymatic restriction factor and the determinant that makes cells permissive or non-permissive. Unlike TRIM5 nor Fv1, Apo3G does not exert its antiviral activity by targeting the viral capsid protein, but it has to be incorporated into a newly synthesized virion during a production step, and then inhibits virus replication

**1.4 APOBEC: Enzymatic restriction factor that target retroviruses** 

broad range of cells, its late restriction depends on a cell line (Sakuma et al., 2007).

with the relationship between other host factor(s).

2008).

#### **1.2 Ref1 and Lv1: Fv1-type restriction factors in human or primate cells**

A host factor that belongs to the same category of Fv1-type restriction factors is Ref1 (restriction factor 1). Ref1 is expressed in human and other non-murine cells and imposes a similar restriction of Fv1 that is controlled by relationship between the same capsid residue (MLV CA 110) and Fv1 (Towers et al., 2000). The difference between Ref1 and Fv1 function is that Ref1 restricts retroviral replication at a step prior to reverse transcription while Fv1 seems to impose a post-reverse transcription block (Goff, 2004). Another restriction factor, lentivirus susceptibility factor 1 (Lv1), was found to be responsible for restricting HIV-1 and N-tropic MLV but not rhesus macaque simian immunodeficiency virus (SIVmac) replication in Old World monkey cells (Besnier et al., 2002; Cowan et al., 2002; Munk et al., 2002).

#### **1.3 TRIM5: Fv1-type host factor restricting HIV-1 in primate cells**

Recently, the host protein which dictates Ref1 activity was identified as an -isoform of rhesus macaque TRIM5 protein by the laboratory of Dr. Joseph Sodroski (Stremlau et al., 2004). TRIM5 is a member of the tripartite motif (TRIM) family of proteins, and has RING, B-box 2 and coiled-coil as common and conserved domains among the family and B30.2(PRYSPRY) domain on its c-terminal region (Nisole et al., 2005). Subsequently, the human and non-human primates homologues of TRIM5 were shown to explain restriction activity against retroviruses, N-MLV, and equine anemia virus (Hatziioannou et al., 2004b; Keckesova et al., 2004; Perron et al., 2004; Si et al., 2006; Song et al., 2005; Yap et al., 2004; Ylinen et al., 2005). Rhesus monkey TRIM5 has strong anti-HIV-1 activity, only modest restriction against SIVmac, and does not block MLV infection, whereas its human homologue does not active against HIV-1 infection.

TRIM5 recognizes incoming viral core, but not a monomeric capsid protein, thorough its B30.2(PRYSPRY) domain. B-box2 and coiled-coil domains are required for TRIM5 multimerization, and both coiled-coil and B30.2(PRYSPRY) domains are essential for viral core binding (Reymond et al., 2001; Stremlau et al., 2006). TRIM5 captures HIV-1 core at a very early step(s) after infection, immediately after the release of core into cytoplasm. To restrict HIV-1 infection and to recognize viral core, TRIM5 must be oligomerized through its B-box 2 and coiled-coil domains. Its RING domain has E3 ubiqutin ligase activity, and self-ubiqutination is occurred, then TRIM5 is quickly degraded. This quick degradation of TRIM5 is not necessary for post-entry restriction, since replacement of TRIM5 RING domain with the corresponding domain of TRIM21 which has lower self-ubiqutination activity and longer half life than TRIM5 didn't alter the antiviral activity. When TRIM5 was over expressed, cytoplasmic body is formed, and the cytoplasmic body is supposed to be required for its antiviral activity. During TRIM5-mediated post-entry restriction, disassembly of viral core is induced too quickly and the accumulation of viral RT-products is reduced. MG132 treatment inhibits to induce quick-disassembly, but still HIV-1 infectivity was restricted. Two reports showed that TRIM5 could block not only viral cDNA accumulation but also the nuclear import of viral cDNA (Berthoux et al., 2004; Wu et al., 2006). Thus TRIM5-mediated post-entry restriction is thought to have at least two phases: (i) TRIM5 induces quick-disassembly of viral core in a proteasome dependent manner and (ii) TRIM5 degrades HIV-1 cDNAs in a proteasome independent manner. The determinant of specificity and magnitude of the post-entry restriction lies on B30.2(PRYSPRY) domain. Recently, Pacheco *et al.* reported that new world monkey TRIM5 restricts foamy virus

A host factor that belongs to the same category of Fv1-type restriction factors is Ref1 (restriction factor 1). Ref1 is expressed in human and other non-murine cells and imposes a similar restriction of Fv1 that is controlled by relationship between the same capsid residue (MLV CA 110) and Fv1 (Towers et al., 2000). The difference between Ref1 and Fv1 function is that Ref1 restricts retroviral replication at a step prior to reverse transcription while Fv1 seems to impose a post-reverse transcription block (Goff, 2004). Another restriction factor, lentivirus susceptibility factor 1 (Lv1), was found to be responsible for restricting HIV-1 and N-tropic MLV but not rhesus macaque simian immunodeficiency virus (SIVmac) replication in Old World monkey cells (Besnier et al., 2002; Cowan et al.,

Recently, the host protein which dictates Ref1 activity was identified as an -isoform of rhesus macaque TRIM5 protein by the laboratory of Dr. Joseph Sodroski (Stremlau et al., 2004). TRIM5 is a member of the tripartite motif (TRIM) family of proteins, and has RING, B-box 2 and coiled-coil as common and conserved domains among the family and B30.2(PRYSPRY) domain on its c-terminal region (Nisole et al., 2005). Subsequently, the human and non-human primates homologues of TRIM5 were shown to explain restriction activity against retroviruses, N-MLV, and equine anemia virus (Hatziioannou et al., 2004b; Keckesova et al., 2004; Perron et al., 2004; Si et al., 2006; Song et al., 2005; Yap et al., 2004; Ylinen et al., 2005). Rhesus monkey TRIM5 has strong anti-HIV-1 activity, only modest restriction against SIVmac, and does not block MLV infection, whereas its human

TRIM5 recognizes incoming viral core, but not a monomeric capsid protein, thorough its B30.2(PRYSPRY) domain. B-box2 and coiled-coil domains are required for TRIM5 multimerization, and both coiled-coil and B30.2(PRYSPRY) domains are essential for viral core binding (Reymond et al., 2001; Stremlau et al., 2006). TRIM5 captures HIV-1 core at a very early step(s) after infection, immediately after the release of core into cytoplasm. To restrict HIV-1 infection and to recognize viral core, TRIM5 must be oligomerized through its B-box 2 and coiled-coil domains. Its RING domain has E3 ubiqutin ligase activity, and self-ubiqutination is occurred, then TRIM5 is quickly degraded. This quick degradation of TRIM5 is not necessary for post-entry restriction, since replacement of TRIM5 RING domain with the corresponding domain of TRIM21 which has lower self-ubiqutination activity and longer half life than TRIM5 didn't alter the antiviral activity. When TRIM5 was over expressed, cytoplasmic body is formed, and the cytoplasmic body is supposed to be required for its antiviral activity. During TRIM5-mediated post-entry restriction, disassembly of viral core is induced too quickly and the accumulation of viral RT-products is reduced. MG132 treatment inhibits to induce quick-disassembly, but still HIV-1 infectivity was restricted. Two reports showed that TRIM5 could block not only viral cDNA accumulation but also the nuclear import of viral cDNA (Berthoux et al., 2004; Wu et al., 2006). Thus TRIM5-mediated post-entry restriction is thought to have at least two phases: (i) TRIM5 induces quick-disassembly of viral core in a proteasome dependent manner and (ii) TRIM5 degrades HIV-1 cDNAs in a proteasome independent manner. The determinant of specificity and magnitude of the post-entry restriction lies on B30.2(PRYSPRY) domain. Recently, Pacheco *et al.* reported that new world monkey TRIM5 restricts foamy virus

**1.2 Ref1 and Lv1: Fv1-type restriction factors in human or primate cells** 

**1.3 TRIM5: Fv1-type host factor restricting HIV-1 in primate cells** 

homologue does not active against HIV-1 infection.

2002; Munk et al., 2002).

infection (Pacheco et al., 2010). Another consideration is the clinical significance of TRIM5 against acquired immunodeficiency syndrome (AIDS) in human. Moreover several reports showed that the efficacy of TRIM5-mediated suppression of HIV-1 replication might interfere with disease progression of AIDS in humans (Cagliani et al., 2010; van Manen et al., 2008). Thus, TRIM5-mediated restriction may occur multi step in retrovirus replication with the relationship between other host factor(s).

Recently, the lab of Dr. Yasuhiro Ikeda reported that rhesus macaque TRIM5 also inhibits HIV-1 production by inducing the degradation of a viral precursor Gag protein (Sakuma et al., 2007). To restrict HIV-1 production, amino acid residues in B-box 2 and coiled-coil domains dictated the specificity of the restriction. In the late restriction, the accumulation of HIV-1 RNA was not affected but the accumulation of precursor Gag was inhibited in an ubiqutine-proteasome independent manner. This TRIM5-mediated late-restriction is still controversial (Zhang et al., 2008), yet it is presumable that TRIM5 restricts HIV-1 infection and production in two distinct mechanisms. Although TRIM5 restricts HIV-1 infection in broad range of cells, its late restriction depends on a cell line (Sakuma et al., 2007).

Here is another notable class of the TRIM family called TRIM-Cyp isolated from new wold monkeys (NWM). A report from the laboratory of Dr. Jeremy Luban demonstrated that owl monkey has TRIM-Cyp that restricts HIV-1 infection (Sayah et al., 2004). Although TRIM-Cyp has a cyclophilin A sequence in its C-terminal region instead of B30.2(PRYSPRY) domain that dictates the specificity and the magnitude of post entry restriction in OWM-TRIM5-mediated post-entry restriction, it recognizes incoming core structure and restricts HIV-1 infection (Stremlau et al., 2006). Recently, TRIM-Cyp mRNA was also detected in a rhesus macaque cell, and over-expressed rhesus TRIM-Cyp restricts HIV-1 infection and production (Brennan et al., 2008; Dietrich et al., 2010; Sakuma et al., 2010; Wilson et al., 2008).

Not like other restriction factors, the counter part of TRIM5-mediated restrictions is not accessory gene product of HIV-1, and human TRIM5 has just a modest restriction activity. NWM cell doesn't have TRIM5, yet even without B30.2(PRYSPRY), TRIM5-Cyp can be a defense against viral infection. These evidences suggest that TRIM5 could be a key molecule to explain the species-species barrier. And if so, TRIM5's dual antiviral activities can block the viral transmission even from closer species like to human from monkeys.

## **1.4 APOBEC: Enzymatic restriction factor that target retroviruses**

Replication of HIV-1 in primary CD4+ T cells, monocyte and some immortalized T cell lines depends on the presence of HIV-1 accessory gene product, Vif (stands for virus infectivity factor)(Fisher et al., 1987; Strebel et al., 1987), and it works in a host cell-specific manner. Vif is required for enhanced HIV-1 replication in some cell types called non-permissive cells, in contrast HIV-1 replication is Vif-independent in permissive cells (Akari et al., 1992; Blanc et al., 1993; Borman et al., 1995; Fan and Peden, 1992; Gabuzda et al., 1992; Sakai et al., 1993; von Schwedler et al., 1993). Recently, some cytidine deaminases were identified as a new class of host restriction factors that target retroviruses such as HIV-1 or SIV (Cullen, 2006; Harris and Liddament, 2004). APOBEC3G (Apo3G), a member of the APOBEC family of cytidine deaminases, is the first identified enzymatic restriction factor and the determinant that makes cells permissive or non-permissive. Unlike TRIM5 nor Fv1, Apo3G does not exert its antiviral activity by targeting the viral capsid protein, but it has to be incorporated into a newly synthesized virion during a production step, and then inhibits virus replication

Retroviral Host Cell Factors: TRIM5, APOBEC3G and Cyclophilins 187

steps following reverse transcription (Iwatani et al., 2007). Therefore, precise mechanism of

**1.5 Cyclophilin A: positive factor against retrovirus replication (or restriction factor?)**  Cyclophilins are ubiquitous proteins and first identified as the target of cyclosporine A (CsA), an immunosuppressive reagent (Takahashi et al., 1989). CypA has proline-isomerase activity that catalyzes the cis-trans isomerization of proline residue (Fischer et al., 1989). The binding of cyclosporine A to cyclophilin A inhibits this isomerase activity (Takahashi et al., 1989). In retrovirus replication, CypA was found to bind HIV-1 capsid (CA) in the yeast two-hybrid system (Luban et al., 1993). The sequence Ala88-Gly89-Pro90-Ile91 of CA protein is the major fragment bound to the active site of CypA (Franke et al., 1994; Gamble et al., 1996; Zhao et al., 1997). Interestingly, The peptidyl-prolyl bond between Gly89 and Pro90 of the CA fragment has a trans conformation, in contrast to the cis conformation observed in other known CypA-peptide complexes (Bosco et al., 2002; Zhao et al., 1997), and Gly89 preceding Pro90 has an unfavorable backbone formation usually only adopted by glycine, suggesting that special Gly89-Pro90 sequence but not other Gly-Pro motif is required for the binding of CA protein to CypA. Therefore, CypA might be likely to act as a molecular chaperone but not a cis-trans isomerase (Zhao et al., 1997). However, one report showed that CypA does not only bind CA protein but also catalyzes efficiently cic-trans isomerization of Gly89-Pro90 peptidyl-prolyl bond (Bosco et al., 2002). The relationship between the Gly89-

Pro90 bond and catalysis of cis-trans isomerization by CypA still remain unclear.

Towers et al., 2003).

It has been well established that CypA promotes an early step of HIV-1 infection in human cells (Braaten et al., 1996a; Braaten et al., 1996c; Braaten and Luban, 2001; Franke and Luban, 1996; Franke et al., 1994; Hatziioannou et al., 2005; Sokolskaja et al., 2004; Thali et al., 1994). CypA is efficiently encapsidated into HIV-1 produced from infected cells through interaction with the CA domains of the Gag polyprotein and disruption of CypA incorporation into virions by CsA or HIV-1 Gag mutants caused a decrease in replication efficiency (Ackerson et al., 1998; Braaten et al., 1996a; Braaten and Luban, 2001; Bukovsky et al., 1997; Franke et al., 1994; Ott et al., 1995; Thali et al., 1994). It is still unclear how CypA is efficiently packaged into HIV-1 virion, but several report showed that both dimerization of CA and multimerization of CypA is required for efficient binding each other (Colgan et al., 1996; Javanbakht et al., 2007). Although CA-CypA interaction is required for infectivity, the important point is that CypA interacts with incoming HIV-1 cores in newly target cells than occurring as core assemble during HIV-1 budding from the virion producer cells, indicated that target cell CypA promotes HIV-1 infectivity (Kootstra et al., 2003; Sokolskaja et al., 2004;

CypA-dependent virus replication is only limited the retroviruses which encode CA that binds CypA. In fact, only those retroviruses are dependent upon CypA for replication (Braaten et al., 1996c; Franke and Luban, 1996; Franke et al., 1994; Luban et al., 1993; Thali et al., 1994). These observations suggested that CA-CypA interaction might contribute tropism determinants for retroviruses. HIV-1 infection in non-human primate cells inhibits prior to reverse transcription after virus entry (Besnier et al., 2002; Cowan et al., 2002; Hatziioannou et al., 2003; Himathongkham and Luciw, 1996; Hofmann et al., 1999; Munk et al., 2002; Shibata et al., 1995; Towers et al., 2003). This restriction is thought to be the same step in the retrovirus life cycle where CypA works (Braaten et al., 1996b). Indeed, Analysis of CypAbinding region of CA with chimeric viruses of HIV-1 and SIV showed the viral determinant for species-specificity (Berthoux et al., 2004; Bukovsky et al., 1997; Cowan et al., 2002;

Apo3G-dependent restriction of retroviral infection still remains unclear.

by targeting single-stranded viral cDNA during an infection step. HIV-1 counteracts Apo3G with Vif expression. During the production of progeny virions, Vif binds to Apo3G and induces Apo3G's proteosomal degradation, resulting in the decreased steady-state levels of human Apo3G (hApo3G) (Yu et al., 2003).

There are several antiretroviral mechanisms of Apo3G against HIV-1 infection. First, Apo3G-containing virus can be resulted in a large number substitution that register as cytidine (C) to thymine (T) in a virus minus-strand during reverse transcription, resulting guanine (G) to adenine (A) mutations in a viral plus strand, known as 'G to A hypermutaion'(Harris et al., 2003; Lecossier et al., 2003; Mangeat et al., 2003; Mariani et al., 2003; Yu et al., 2004; Zhang et al., 2003). Second, Apo3G can inhibit tRNA annealing or tRNA processing during reverse transcription (Guo et al., 2006; Guo et al., 2007; Mbisa et al., 2007). Third, Apo3G inhibits DNA strand transfer or integration (Li et al., 2007; Luo et al., 2007; Mbisa et al., 2007). Although Apo3G has the most potent anti-HIV-1 activity among the APOBEC family of proteins, another member of the family, APOBEC3F (Apo3F) was shown to inhibit HIV-1 infection in the absence of Vif (Bishop et al., 2004a; Liddament et al., 2004; Wiegand et al., 2004; Zheng et al., 2004), whereas APOBEC3B (Apo3B) can inhibit HIV-1 infection in both the presence and absence of Vif (Bishop et al., 2004a; Doehle et al., 2005; Rose et al., 2005).

Although we can imagine the broad range of antiretroviral activity of APOBEC family because APOBEC proteins from non-human species can also inhibit HIV-1 infection (Bishop et al., 2004a; Bishop et al., 2004b; Cullen, 2006; Mariani et al., 2003; Wiegand et al., 2004), the Vif-Apo3G interaction is thought to be species specific (Mariani et al., 2003; Simon et al., 1998). Accordingly, hApo3G is insensitive to SIVagm Vif while african green monkey Apo3G (agmApo3G) is insensitive to HIV-1 Vif and the determinant of this species specificity depends on amino acid 128 of hApo3G and agmApo3G (Bogerd et al., 2004; Mangeat et al., 2004; Mariani et al., 2003; Schrofelbauer et al., 2004; Xu et al., 2004). However, such species specificity is not strictly controlled, for example a report from the laboratory of Klaus Strebel demonstrated that SIVagm Vif supported replication of SIVagm virus in the hApo3G-positive human A3.01 T cell line. Replication of *vif*-defective SIVagm in A3.01 cells was severely restricted, resulted in an accumulation of cytidine deaminase-induced G-to-A mutations in SIVagm genome (Takeuchi et al., 2005). Therefore, it is probable that SIV Vif has evolved to counteract hApo3G restriction and this might contribute zoonotic transmission of SIV.

Although the antiviral activity of Apo3G is clearly correlated with its deaminase activity (Iwatani et al., 2006; Mangeat et al., 2003; Navarro et al., 2005; Opi et al., 2006; Shindo et al., 2003; Zhang et al., 2003), some members of APOBEC family have additional anti-retrovirus activities that do not require catalytically activity of itself (Li et al., 2007; Luo et al., 2007). In fact, several reports showed that deaminase-defective Apo3G and Apo3F have antiviral activity, and some antiviral-inactive mutants of both Apo3G and Apo3F have cytidine deaminase activity (Bishop et al., 2006; Holmes et al., 2007; Newman et al., 2005; Shindo et al., 2003).

However, deaminase-defective Apo3G mutant with C288S/C291A substitutions did not show any anti-viral actibity and over-expression of the mutant could work as a dominant negative agent of wild-type Apo3G, suggesting a tightly-relationship between antiviral and deaminase activities (Miyagi et al., 2007; Opi et al., 2006). Recently, it was demonstrated that hApo3G has an intrinsic immune effect on viral DNA synthesis, which may account for cytidine deaminase-independent antiviral activity of Apo3G, and did not abort replication

by targeting single-stranded viral cDNA during an infection step. HIV-1 counteracts Apo3G with Vif expression. During the production of progeny virions, Vif binds to Apo3G and induces Apo3G's proteosomal degradation, resulting in the decreased steady-state levels of

There are several antiretroviral mechanisms of Apo3G against HIV-1 infection. First, Apo3G-containing virus can be resulted in a large number substitution that register as cytidine (C) to thymine (T) in a virus minus-strand during reverse transcription, resulting guanine (G) to adenine (A) mutations in a viral plus strand, known as 'G to A hypermutaion'(Harris et al., 2003; Lecossier et al., 2003; Mangeat et al., 2003; Mariani et al., 2003; Yu et al., 2004; Zhang et al., 2003). Second, Apo3G can inhibit tRNA annealing or tRNA processing during reverse transcription (Guo et al., 2006; Guo et al., 2007; Mbisa et al., 2007). Third, Apo3G inhibits DNA strand transfer or integration (Li et al., 2007; Luo et al., 2007; Mbisa et al., 2007). Although Apo3G has the most potent anti-HIV-1 activity among the APOBEC family of proteins, another member of the family, APOBEC3F (Apo3F) was shown to inhibit HIV-1 infection in the absence of Vif (Bishop et al., 2004a; Liddament et al., 2004; Wiegand et al., 2004; Zheng et al., 2004), whereas APOBEC3B (Apo3B) can inhibit HIV-1 infection in both the presence and absence of Vif (Bishop et al., 2004a; Doehle et al., 2005;

Although we can imagine the broad range of antiretroviral activity of APOBEC family because APOBEC proteins from non-human species can also inhibit HIV-1 infection (Bishop et al., 2004a; Bishop et al., 2004b; Cullen, 2006; Mariani et al., 2003; Wiegand et al., 2004), the Vif-Apo3G interaction is thought to be species specific (Mariani et al., 2003; Simon et al., 1998). Accordingly, hApo3G is insensitive to SIVagm Vif while african green monkey Apo3G (agmApo3G) is insensitive to HIV-1 Vif and the determinant of this species specificity depends on amino acid 128 of hApo3G and agmApo3G (Bogerd et al., 2004; Mangeat et al., 2004; Mariani et al., 2003; Schrofelbauer et al., 2004; Xu et al., 2004). However, such species specificity is not strictly controlled, for example a report from the laboratory of Klaus Strebel demonstrated that SIVagm Vif supported replication of SIVagm virus in the hApo3G-positive human A3.01 T cell line. Replication of *vif*-defective SIVagm in A3.01 cells was severely restricted, resulted in an accumulation of cytidine deaminase-induced G-to-A mutations in SIVagm genome (Takeuchi et al., 2005). Therefore, it is probable that SIV Vif has evolved to counteract hApo3G restriction and this might contribute zoonotic

Although the antiviral activity of Apo3G is clearly correlated with its deaminase activity (Iwatani et al., 2006; Mangeat et al., 2003; Navarro et al., 2005; Opi et al., 2006; Shindo et al., 2003; Zhang et al., 2003), some members of APOBEC family have additional anti-retrovirus activities that do not require catalytically activity of itself (Li et al., 2007; Luo et al., 2007). In fact, several reports showed that deaminase-defective Apo3G and Apo3F have antiviral activity, and some antiviral-inactive mutants of both Apo3G and Apo3F have cytidine deaminase activity (Bishop et al., 2006; Holmes et al., 2007; Newman et al., 2005; Shindo et

However, deaminase-defective Apo3G mutant with C288S/C291A substitutions did not show any anti-viral actibity and over-expression of the mutant could work as a dominant negative agent of wild-type Apo3G, suggesting a tightly-relationship between antiviral and deaminase activities (Miyagi et al., 2007; Opi et al., 2006). Recently, it was demonstrated that hApo3G has an intrinsic immune effect on viral DNA synthesis, which may account for cytidine deaminase-independent antiviral activity of Apo3G, and did not abort replication

human Apo3G (hApo3G) (Yu et al., 2003).

Rose et al., 2005).

transmission of SIV.

al., 2003).

steps following reverse transcription (Iwatani et al., 2007). Therefore, precise mechanism of Apo3G-dependent restriction of retroviral infection still remains unclear.

#### **1.5 Cyclophilin A: positive factor against retrovirus replication (or restriction factor?)**

Cyclophilins are ubiquitous proteins and first identified as the target of cyclosporine A (CsA), an immunosuppressive reagent (Takahashi et al., 1989). CypA has proline-isomerase activity that catalyzes the cis-trans isomerization of proline residue (Fischer et al., 1989). The binding of cyclosporine A to cyclophilin A inhibits this isomerase activity (Takahashi et al., 1989). In retrovirus replication, CypA was found to bind HIV-1 capsid (CA) in the yeast two-hybrid system (Luban et al., 1993). The sequence Ala88-Gly89-Pro90-Ile91 of CA protein is the major fragment bound to the active site of CypA (Franke et al., 1994; Gamble et al., 1996; Zhao et al., 1997). Interestingly, The peptidyl-prolyl bond between Gly89 and Pro90 of the CA fragment has a trans conformation, in contrast to the cis conformation observed in other known CypA-peptide complexes (Bosco et al., 2002; Zhao et al., 1997), and Gly89 preceding Pro90 has an unfavorable backbone formation usually only adopted by glycine, suggesting that special Gly89-Pro90 sequence but not other Gly-Pro motif is required for the binding of CA protein to CypA. Therefore, CypA might be likely to act as a molecular chaperone but not a cis-trans isomerase (Zhao et al., 1997). However, one report showed that CypA does not only bind CA protein but also catalyzes efficiently cic-trans isomerization of Gly89-Pro90 peptidyl-prolyl bond (Bosco et al., 2002). The relationship between the Gly89- Pro90 bond and catalysis of cis-trans isomerization by CypA still remain unclear.

It has been well established that CypA promotes an early step of HIV-1 infection in human cells (Braaten et al., 1996a; Braaten et al., 1996c; Braaten and Luban, 2001; Franke and Luban, 1996; Franke et al., 1994; Hatziioannou et al., 2005; Sokolskaja et al., 2004; Thali et al., 1994). CypA is efficiently encapsidated into HIV-1 produced from infected cells through interaction with the CA domains of the Gag polyprotein and disruption of CypA incorporation into virions by CsA or HIV-1 Gag mutants caused a decrease in replication efficiency (Ackerson et al., 1998; Braaten et al., 1996a; Braaten and Luban, 2001; Bukovsky et al., 1997; Franke et al., 1994; Ott et al., 1995; Thali et al., 1994). It is still unclear how CypA is efficiently packaged into HIV-1 virion, but several report showed that both dimerization of CA and multimerization of CypA is required for efficient binding each other (Colgan et al., 1996; Javanbakht et al., 2007). Although CA-CypA interaction is required for infectivity, the important point is that CypA interacts with incoming HIV-1 cores in newly target cells than occurring as core assemble during HIV-1 budding from the virion producer cells, indicated that target cell CypA promotes HIV-1 infectivity (Kootstra et al., 2003; Sokolskaja et al., 2004; Towers et al., 2003).

CypA-dependent virus replication is only limited the retroviruses which encode CA that binds CypA. In fact, only those retroviruses are dependent upon CypA for replication (Braaten et al., 1996c; Franke and Luban, 1996; Franke et al., 1994; Luban et al., 1993; Thali et al., 1994). These observations suggested that CA-CypA interaction might contribute tropism determinants for retroviruses. HIV-1 infection in non-human primate cells inhibits prior to reverse transcription after virus entry (Besnier et al., 2002; Cowan et al., 2002; Hatziioannou et al., 2003; Himathongkham and Luciw, 1996; Hofmann et al., 1999; Munk et al., 2002; Shibata et al., 1995; Towers et al., 2003). This restriction is thought to be the same step in the retrovirus life cycle where CypA works (Braaten et al., 1996b). Indeed, Analysis of CypAbinding region of CA with chimeric viruses of HIV-1 and SIV showed the viral determinant for species-specificity (Berthoux et al., 2004; Bukovsky et al., 1997; Cowan et al., 2002;

Retroviral Host Cell Factors: TRIM5, APOBEC3G and Cyclophilins 189

Bishop, K.N., Holmes, R.K., and Malim, M.H. (2006). Antiviral potency of APOBEC proteins does not correlate with cytidine deamination. *Journal of virology 80*, 8450-8458. Bishop, K.N., Holmes, R.K., Sheehy, A.M., Davidson, N.O., Cho, S.J., and Malim, M.H.

Bishop, K.N., Holmes, R.K., Sheehy, A.M., and Malim, M.H. (2004b). APOBEC-mediated

Blanc, D., Patience, C., Schulz, T.F., Weiss, R., and Spire, B. (1993). Transcomplementation of

Bogerd, H.P., Doehle, B.P., Wiegand, H.L., and Cullen, B.R. (2004). A single amino acid

Borman, A.M., Quillent, C., Charneau, P., Dauguet, C., and Clavel, F. (1995). Human

Braaten, D., Aberham, C., Franke, E.K., Yin, L., Phares, W., and Luban, J. (1996a).

Braaten, D., Franke, E.K., and Luban, J. (1996b). Cyclophilin A is required for an early step in

Braaten, D., Franke, E.K., and Luban, J. (1996c). Cyclophilin A is required for the replication

Braaten, D., and Luban, J. (2001). Cyclophilin A regulates HIV-1 infectivity, as demonstrated by gene targeting in human T cells. *The EMBO journal 20*, 1300-1309. Brennan, G., Kozyrev, Y., and Hu, S.L. (2008). TRIMCyp expression in Old World primates

Bukovsky, A.A., Weimann, A., Accola, M.A., and Gottlinger, H.G. (1997). Transfer of the

Cagliani, R., Fumagalli, M., Biasin, M., Piacentini, L., Riva, S., Pozzoli, U., Bonaglia, M.C.,

reverse transcription. *Journal of virology 70*, 3551-3560.

*Sciences of the United States of America 105*, 3569-3574.

immunodeficiency viruses. *Journal of virology 70*, 4220-4227.

editing of viral RNA. *Science* (New York, NY *305*, 645.

*Curr Biol 14*, 1392-1396.

cycle. *Virology 193*, 186-192.

5252.

*virology 70*, 5170-5176.

10943-10948.

577-588.

*the United States of America 101*, 3770-3774.

(2004a). Cytidine deamination of retroviral DNA by diverse APOBEC proteins.

VIF- HIV-1 mutants in CEM cells suggests that VIF affects late steps of the viral life

difference in the host APOBEC3G protein controls the primate species specificity of HIV type 1 virion infectivity factor. *Proceedings of the National Academy of Sciences of* 

immunodeficiency virus type 1 Vif- mutant particles from restrictive cells: role of Vif in correct particle assembly and infectivity. *Journal of virology 69*, 2058-2067. Bosco, D.A., Eisenmesser, E.Z., Pochapsky, S., Sundquist, W.I., and Kern, D. (2002). Catalysis

of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A. *Proceedings of the National Academy of Sciences of the United States of America 99*, 5247-

Cyclosporine A-resistant human immunodeficiency virus type 1 mutants demonstrate that Gag encodes the functional target of cyclophilin A. *Journal of* 

the life cycle of human immunodeficiency virus type 1 before the initiation of

of group M human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus SIV(CPZ)GAB but not group O HIV-1 or other primate

Macaca nemestrina and Macaca fascicularis. *Proceedings of the National Academy of* 

HIV-1 cyclophilin-binding site to simian immunodeficiency virus from Macaca mulatta can confer both cyclosporin sensitivity and cyclosporin dependence. *Proceedings of the National Academy of Sciences of the United States of America 94*,

Bresolin, N., Clerici, M., and Sironi, M. (2010). Long-term balancing selection maintains trans-specific polymorphisms in the human TRIM5 gene. *Hum Genet 128*,

Dorfman and Gottlinger, 1996; Hatziioannou et al., 2004a; Hatziioannou et al., 2006; Ikeda et al., 2004; Kamada et al., 2006; Kootstra et al., 2003; Owens et al., 2004; Owens et al., 2003; Sayah et al., 2004; Shibata et al., 1991; Shibata et al., 1995; Stremlau et al., 2004; Towers et al., 2003).

Human CypA is required for efficient HIV-1 infection but not SIV. There is no known role for CypA in SIV infection in human cells. Recently, the first report from the laboratory of Klaus Strebel showed that human CypA acts as restriction factor against SIV infection in human cells, and SIV Vif counteracts a CypA-imposed inhibition against SIV infection with exclusion of CypA from SIV vision (Takeuchi et al., 2007). This phenomenon could distinguish from the function of SIV Vif against hApo3G previously reported from same laboratory (Takeuchi et al., 2005) because they used human cells lacking detectable deaminase activity. This observation raised the possibility that SIV Vif is crucial for zoonotic transmission of SIV from monkey to human.

## **2. Conclusion**

Viral replication requires a lot of host cell factors, whose species specificity may affect viral tropism. On the other hand, there exist host factors that restrict viral replication. The antiviral system mediated by some of these restriction factors, termed intrinsic immunity, which is distinguished from the conventional innate and adaptive immunity has been indicated to play an important role in making species-specific barriers against viral infection. As discussed in this chapter, we describe the current progress in understanding of such restriction factors against retroviral replication, especially focusing on TRIM5 and APOBEC whose anti-retroviral effects have recently been recognized. Additionally, we mentioned CypA that is essential for HIV-1 replication in human cells and may affect viral tropism. Understanding of these host factors would contribute to identification of the determinants for viral tropism. Finally, understanding of the factors mediating intrinsic immunity may lead to the development of antiviral agents that can boost their potency and thereby lead to treatments for viral disease.

## **3. References**


Dorfman and Gottlinger, 1996; Hatziioannou et al., 2004a; Hatziioannou et al., 2006; Ikeda et al., 2004; Kamada et al., 2006; Kootstra et al., 2003; Owens et al., 2004; Owens et al., 2003; Sayah et al., 2004; Shibata et al., 1991; Shibata et al., 1995; Stremlau et al., 2004; Towers et al.,

Human CypA is required for efficient HIV-1 infection but not SIV. There is no known role for CypA in SIV infection in human cells. Recently, the first report from the laboratory of Klaus Strebel showed that human CypA acts as restriction factor against SIV infection in human cells, and SIV Vif counteracts a CypA-imposed inhibition against SIV infection with exclusion of CypA from SIV vision (Takeuchi et al., 2007). This phenomenon could distinguish from the function of SIV Vif against hApo3G previously reported from same laboratory (Takeuchi et al., 2005) because they used human cells lacking detectable deaminase activity. This observation raised the possibility that SIV Vif is crucial for zoonotic

Viral replication requires a lot of host cell factors, whose species specificity may affect viral tropism. On the other hand, there exist host factors that restrict viral replication. The antiviral system mediated by some of these restriction factors, termed intrinsic immunity, which is distinguished from the conventional innate and adaptive immunity has been indicated to play an important role in making species-specific barriers against viral infection. As discussed in this chapter, we describe the current progress in understanding of such restriction factors against retroviral replication, especially focusing on TRIM5 and APOBEC whose anti-retroviral effects have recently been recognized. Additionally, we mentioned CypA that is essential for HIV-1 replication in human cells and may affect viral tropism. Understanding of these host factors would contribute to identification of the determinants for viral tropism. Finally, understanding of the factors mediating intrinsic immunity may lead to the development of antiviral agents that can boost their potency and

Ackerson, B., Rey, O., Canon, J., and Krogstad, P. (1998). Cells with high cyclophilin A

Berthoux, L., Sebastian, S., Sokolskaja, E., and Luban, J. (2004). Lv1 inhibition of human

or nuclear translocation of viral cDNA. *Journal of virology 78*, 11739-11750. Besnier, C., Takeuchi, Y., and Towers, G. (2002). Restriction of lentivirus in monkeys.

Bieniasz, P.D. (2004). Intrinsic immunity: a front-line defense against viral attack. *Nature* 

mononuclear cells. *Archives of virology 123*, 157-167.

content support replication of human immunodeficiency virus type 1 Gag mutants with decreased ability to incorporate cyclophilin A. *Journal of virology 72*, 303-308. Akari, H., Sakuragi, J., Takebe, Y., Tomonaga, K., Kawamura, M., Fukasawa, M., Miura, T.,

Shinjo, T., and Hayami, M. (1992). Biological characterization of human immunodeficiency virus type 1 and type 2 mutants in human peripheral blood

immunodeficiency virus type 1 is counteracted by factors that stimulate synthesis

*Proceedings of the National Academy of Sciences of the United States of America 99*,

2003).

**2. Conclusion** 

**3. References** 

11920-11925.

*immunology 5*, 1109-1115.

transmission of SIV from monkey to human.

thereby lead to treatments for viral disease.


Retroviral Host Cell Factors: TRIM5, APOBEC3G and Cyclophilins 191

Guo, F., Cen, S., Niu, M., Saadatmand, J., and Kleiman, L. (2006). Inhibition of formula-

tRNA3Lys annealing to viral RNA. *Journal of virology 81*, 11322-11331. Harris, R.S., Bishop, K.N., Sheehy, A.M., Craig, H.M., Petersen-Mahrt, S.K., Watt, I.N.,

Harris, R.S., and Liddament, M.T. (2004). Retroviral restriction by APOBEC proteins. *Nat* 

Hartley, J.W., Rowe, W.P., and Huebner, R.J. (1970). Host-range restrictions of murine leukemia viruses in mouse embryo cell cultures. *Journal of virology 5*, 221-225. Hatziioannou, T., Cowan, S., Goff, S.P., Bieniasz, P.D., and Towers, G.J. (2003). Restriction of multiple divergent retroviruses by Lv1 and Ref1. *The EMBO journal 22*, 385-394. Hatziioannou, T., Cowan, S., Von Schwedler, U.K., Sundquist, W.I., and Bieniasz, P.D.

Hatziioannou, T., Perez-Caballero, D., Cowan, S., and Bieniasz, P.D. (2005). Cyclophilin

opposing effects on infectivity in human cells. *Journal of virology 79*, 176-183. Hatziioannou, T., Perez-Caballero, D., Yang, A., Cowan, S., and Bieniasz, P.D. (2004b).

Hatziioannou, T., Princiotta, M., Piatak, M., Jr., Yuan, F., Zhang, F., Lifson, J.D., and

Himathongkham, S., and Luciw, P.A. (1996). Restriction of HIV-1 (subtype B) replication at

Hofmann, W., Schubert, D., LaBonte, J., Munson, L., Gibson, S., Scammell, J., Ferrigno, P.,

Ikeda, Y., Ylinen, L.M., Kahar-Bador, M., and Towers, G.J. (2004). Influence of gag on human

Iwatani, Y., Chan, D.S., Wang, F., Maynard, K.S., Sugiura, W., Gronenborn, A.M., Rouzina,

immunodeficiency virus infection. *Journal of virology 73*, 10020-10028. Holmes, R.K., Koning, F.A., Bishop, K.N., and Malim, M.H. (2007). APOBEC3F can inhibit

the entry step in rhesus macaque cells. *Virology 219*, 485-488.

immunity to retroviral infection. *Cell 113*, 803-809.

virus type 1 capsid. *Journal of virology 78*, 6005-6012.

*Rev Immunol 4*, 868-877.

*America 101*, 10774-10779.

*282*, 2587-2595.

11816-11822.

*Res*.

evasion. *Science* (New York, NY *314*, 95.

primed reverse transcription by human APOBEC3G during human immunodeficiency virus type 1 replication. *Journal of virology 80*, 11710-11722. Guo, F., Cen, S., Niu, M., Yang, Y., Gorelick, R.J., and Kleiman, L. (2007). The interaction of

APOBEC3G with human immunodeficiency virus type 1 nucleocapsid inhibits

Neuberger, M.S., and Malim, M.H. (2003). DNA deamination mediates innate

(2004a). Species-specific tropism determinants in the human immunodeficiency

interactions with incoming human immunodeficiency virus type 1 capsids with

Retrovirus resistance factors Ref1 and Lv1 are species-specific variants of TRIM5alpha. *Proceedings of the National Academy of Sciences of the United States of* 

Bieniasz, P.D. (2006). Generation of simian-tropic HIV-1 by restriction factor

and Sodroski, J. (1999). Species-specific, postentry barriers to primate

the accumulation of HIV-1 reverse transcription products in the absence of hypermutation. Comparisons with APOBEC3G. *The Journal of biological chemistry* 

immunodeficiency virus type 1 species-specific tropism. *Journal of virology 78*,

I., Williams, M.C., Musier-Forsyth, K., and Levin, J.G. (2007). Deaminaseindependent inhibition of HIV-1 reverse transcription by APOBEC3G. *Nucleic Acids* 


Colgan, J., Yuan, H.E., Franke, E.K., and Luban, J. (1996). Binding of the human

Cowan, S., Hatziioannou, T., Cunningham, T., Muesing, M.A., Gottlinger, H.G., and

Cullen, B.R. (2006). Role and mechanism of action of the APOBEC3 family of antiretroviral

Dietrich, E.A., Jones-Engel, L., and Hu, S.L. (2010). Evolution of the antiretroviral restriction

Doehle, B.P., Schafer, A., and Cullen, B.R. (2005). Human APOBEC3B is a potent inhibitor of HIV-1 infectivity and is resistant to HIV-1 Vif. *Virology 339*, 281-288. Dorfman, T., and Gottlinger, H.G. (1996). The human immunodeficiency virus type 1 capsid

Fan, L., and Peden, K. (1992). Cell-free transmission of Vif mutants of HIV-1. Virology *190*,

Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T., and Schmid, F.X. (1989).

Fisher, A.G., Ensoli, B., Ivanoff, L., Chamberlain, M., Petteway, S., Ratner, L., Gallo, R.C.,

Franke, E.K., and Luban, J. (1996). Inhibition of HIV-1 replication by cyclosporine A or

Franke, E.K., Yuan, H.E., and Luban, J. (1994). Specific incorporation of cyclophilin A into

Gabuzda, D.H., Lawrence, K., Langhoff, E., Terwilliger, E., Dorfman, T., Haseltine, W.A.,

Gamble, T.R., Vajdos, F.F., Yoo, S., Worthylake, D.K., Houseweart, M., Sundquist, W.I., and

Gardner, M.B., Rasheed, S., Pal, B.K., Estes, J.D., and O'Brien, S.J. (1980). Akvr-1, a dominant

Gautsch, J.W., Elder, J.H., Schindler, J., Jensen, F.C., and Lerner, R.A. (1978). Structural

Goff, S.P. (2004). Genetic control of retrovirus susceptibility in mammalian cells. *Annual* 

type 1 in CD4+ T lymphocytes. *Journal of virology 66*, 6489-6495.

terminal domain of HIV-1 capsid. *Cell 87*, 1285-1294.

*Sciences of the United States of America 99*, 11914-11919.

factor TRIMCyp in Old World primates. *PLoS One 5*, e14019.

transmission in vitro. *Science* (New York, NY *237*, 888-893.

resistance factors. *Journal of virology 80*, 1067-1076.

*of virology 70*, 5751-5757.

*Nature 337*, 476-478.

interaction. *Virology 222*, 279-282.

HIV-1 virions. *Nature 372*, 359-362.

4310.

19-29.

531-535.

*America 75*, 4170-4174.

*review of genetics 38*, 61-85.

immunodeficiency virus type 1 Gag polyprotein to cyclophilin A is mediated by the central region of capsid and requires Gag dimerization. *Journal of virology 70*, 4299-

Bieniasz, P.D. (2002). Cellular inhibitors with Fv1-like activity restrict human and simian immunodeficiency virus tropism. *Proceedings of the National Academy of* 

p2 domain confers sensitivity to the cyclophilin-binding drug SDZ NIM 811. *Journal* 

Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins.

and Wong-Staal, F. (1987). The sor gene of HIV-1 is required for efficient virus

related compounds correlates with the ability to disrupt the Gag-cyclophilin A

and Sodroski, J. (1992). Role of vif in replication of human immunodeficiency virus

Hill, C.P. (1996). Crystal structure of human cyclophilin A bound to the amino-

murine leukemia virus restriction gene, is polymorphic in leukemia-prone wild mice. *Proceedings of the National Academy of Sciences of the United States of America 77*,

markers on core protein p30 of murine leukemia virus: functional correlation with Fv-1 tropism. *Proceedings of the National Academy of Sciences of the United States of* 


Retroviral Host Cell Factors: TRIM5, APOBEC3G and Cyclophilins 193

Miyagi, E., Opi, S., Takeuchi, H., Khan, M., Goila-Gaur, R., Kao, S., and Strebel, K. (2007).

Munk, C., Brandt, S.M., Lucero, G., and Landau, N.R. (2002). A dominant block to HIV-1

Navarro, F., Bollman, B., Chen, H., Konig, R., Yu, Q., Chiles, K., and Landau, N.R. (2005).

Newman, E.N., Holmes, R.K., Craig, H.M., Klein, K.C., Lingappa, J.R., Malim, M.H., and

Nisole, S., Stoye, J.P., and Saib, A. (2005). TRIM family proteins: retroviral restriction and

Opi, S., Takeuchi, H., Kao, S., Khan, M.A., Miyagi, E., Goila-Gaur, R., Iwatani, Y., Levin, J.G.,

Ott, D.E., Coren, L.V., Johnson, D.G., Sowder, R.C., 2nd, Arthur, L.O., and Henderson, L.E.

Owens, C.M., Song, B., Perron, M.J., Yang, P.C., Stremlau, M., and Sodroski, J. (2004).

Owens, C.M., Yang, P.C., Gottlinger, H., and Sodroski, J. (2003). Human and simian

postentry replication blocks in simian cells. *Journal of virology 77*, 726-731. Pacheco, B., Finzi, A., McGee-Estrada, K., and Sodroski, J. (2010). Species-specific inhibition

Perron, M.J., Stremlau, M., Song, B., Ulm, W., Mulligan, R.C., and Sodroski, J. (2004).

Pincus, T., Hartley, J.W., and Rowe, W.P. (1971). A major genetic locus affecting resistance to

occurring viruses. *The Journal of experimental medicine 133*, 1219-1233. Rasheed, S., and Gardner, M.B. (1983). Resistance to fibroblasts and hematopoietic cells to

TRIM5{alpha} proteins. *Journal of virology 84*, 4095-4099.

immunodeficiency virus type 1. *Journal of virology 81*, 13346-13353.

*Academy of Sciences of the United States of America 99*, 13843-13848.

cytidine deaminase activity. *Curr Biol 15*, 166-170.

antiviral activity. *Journal of virology 80*, 4673-4682.

antiviral defence. *Nature reviews 3*, 799-808.

7099-7110.

374-386.

1003-1006.

*Journal of virology 78*, 5423-5437.

*America 101*, 11827-11832.

*journal of cancer 31*, 491-496.

immunodeficiency virus type 1 cDNAs produced in the presence of APOBEC3G exhibit defects in plus-strand DNA transfer and integration. *Journal of virology 81*,

Enzymatically active APOBEC3G is required for efficient inhibition of human

replication at reverse transcription in simian cells. *Proceedings of the National* 

Complementary function of the two catalytic domains of APOBEC3G. *Virology 333*,

Sheehy, A.M. (2005). Antiviral function of APOBEC3G can be dissociated from

and Strebel, K. (2006). Monomeric APOBEC3G is catalytically active and has

(1995). Analysis and localization of cyclophilin A found in the virions of human immunodeficiency virus type 1 MN strain. *AIDS research and human retroviruses 11*,

Binding and susceptibility to postentry restriction factors in monkey cells are specified by distinct regions of the human immunodeficiency virus type 1 capsid.

immunodeficiency virus capsid proteins are major viral determinants of early,

of foamy viruses from South American monkeys by New World Monkey

TRIM5alpha mediates the postentry block to N-tropic murine leukemia viruses in human cells. *Proceedings of the National Academy of Sciences of the United States of* 

infection with murine leukemia viruses. I. Tissue culture studies of naturally

ecotropic murine leukemia virus infection; an Akvr-1R gene effect. *International* 


Iwatani, Y., Takeuchi, H., Strebel, K., and Levin, J.G. (2006). Biochemical activities of highly

Javanbakht, H., Diaz-Griffero, F., Yuan, W., Yeung, D.F., Li, X., Song, B., and Sodroski, J.

Kamada, K., Igarashi, T., Martin, M.A., Khamsri, B., Hatcho, K., Yamashita, T., Fujita, M.,

Keckesova, Z., Ylinen, L.M., and Towers, G.J. (2004). The human and African green monkey

Kootstra, N.A., Munk, C., Tonnu, N., Landau, N.R., and Verma, I.M. (2003). Abrogation of

Kozak, C.A., and Chakraborti, A. (1996). Single amino acid changes in the murine leukemia virus capsid protein gene define the target of Fv1 resistance. *Virology 225*, 300-305. Lecossier, D., Bouchonnet, F., Clavel, F., and Hance, A.J. (2003). Hypermutation of HIV-1 DNA in the absence of the Vif protein. *Science* (New York, NY *300*, 1112. Li, X.Y., Guo, F., Zhang, L., Kleiman, L., and Cen, S. (2007). APOBEC3G inhibits DNA strand

Liddament, M.T., Brown, W.L., Schumacher, A.J., and Harris, R.S. (2004). APOBEC3F

Luban, J., Bossolt, K.L., Franke, E.K., Kalpana, G.V., and Goff, S.P. (1993). Human

Luo, K., Wang, T., Liu, B., Tian, C., Xiao, Z., Kappes, J., and Yu, X.F. (2007). Cytidine

Mangeat, B., Turelli, P., Caron, G., Friedli, M., Perrin, L., and Trono, D. (2003). Broad

Mangeat, B., Turelli, P., Liao, S., and Trono, D. (2004). A single amino acid determinant

Mariani, R., Chen, D., Schrofelbauer, B., Navarro, F., Konig, R., Bollman, B., Munk, C.,

Mbisa, J.L., Barr, R., Thomas, J.A., Vandegraaff, N., Dorweiler, I.J., Svarovskaia, E.S., Brown,

*Academy of Sciences of the United States of America 103*, 16959-16964.

*Journal of virology 80*, 5992-6002.

*Virology 367*, 19-29.

10780-10785.

1298-1303.

32065-32074.

1067-1078.

7238-7248.

*Curr Biol 14*, 1385-1391.

reverse transcripts. *Nature 424*, 99-103.

*biological chemistry 279*, 14481-14483.

APOBEC3G from HIV-1 virions by Vif. *Cell 114*, 21-31.

purified, catalytically active human APOBEC3G: correlation with antiviral effect.

(2007). The ability of multimerized cyclophilin A to restrict retrovirus infection.

Uchiyama, T., and Adachi, A. (2006). Generation of HIV-1 derivatives that productively infect macaque monkey lymphoid cells. *Proceedings of the National* 

TRIM5alpha genes encode Ref1 and Lv1 retroviral restriction factor activities. *Proceedings of the National Academy of Sciences of the United States of America 101*,

postentry restriction of HIV-1-based lentiviral vector transduction in simian cells. *Proceedings of the National Academy of Sciences of the United States of America 100*,

transfer during HIV-1 reverse transcription. *The Journal of biological chemistry 282*,

properties and hypermutation preferences indicate activity against HIV-1 in vivo.

immunodeficiency virus type 1 Gag protein binds to cyclophilins A and B. *Cell 73*,

deaminases APOBEC3G and APOBEC3F interact with human immunodeficiency virus type 1 integrase and inhibit proviral DNA formation. *Journal of virology 81*,

antiretroviral defence by human APOBEC3G through lethal editing of nascent

governs the species-specificsensitivity of APOBEC3G to Vif action. *The Journal of* 

Nymark-McMahon, H., and Landau, N.R. (2003). Species-specific exclusion of

W.L., Mansky, L.M., Gorelick, R.J., Harris, R.S.*, et al.* (2007). Human

immunodeficiency virus type 1 cDNAs produced in the presence of APOBEC3G exhibit defects in plus-strand DNA transfer and integration. *Journal of virology 81*, 7099-7110.


Retroviral Host Cell Factors: TRIM5, APOBEC3G and Cyclophilins 195

Song, B., Javanbakht, H., Perron, M., Park, D.H., Stremlau, M., and Sodroski, J. (2005).

Strebel, K., Daugherty, D., Clouse, K., Cohen, D., Folks, T., and Martin, M.A. (1987). The HIV 'A' (sor) gene product is essential for virus infectivity. *Nature 328*, 728-730. Stremlau, M., Owens, C.M., Perron, M.J., Kiessling, M., Autissier, P., and Sodroski, J. (2004).

Stremlau, M., Perron, M., Lee, M., Li, Y., Song, B., Javanbakht, H., Diaz-Griffero, F.,

Suzuki, S. (1975). FV-4: a new gene affecting the splenomegaly induction by Friend leukemia

Takahashi, N., Hayano, T., and Suzuki, M. (1989). Peptidyl-prolyl cis-trans isomerase is the

Takeda, A., and Matano, T. (2007). Inhibition of infectious murine leukemia virus

Takeuchi, H., Buckler-White, A., Goila-Gaur, R., Miyagi, E., Khan, M.A., Opi, S., Kao, S.,

Takeuchi, H., Kao, S., Miyagi, E., Khan, M.A., Buckler-White, A., Plishka, R., and Strebel, K.

Thali, M., Bukovsky, A., Kondo, E., Rosenwirth, B., Walsh, C.T., Sodroski, J., and Gottlinger,

Towers, G., Bock, M., Martin, S., Takeuchi, Y., Stoye, J.P., and Danos, O. (2000). A conserved

Towers, G.J., Hatziioannou, T., Cowan, S., Goff, S.P., Luban, J., and Bieniasz, P.D. (2003).

van Manen, D., Rits, M.A., Beugeling, C., van Dort, K., Schuitemaker, H., and Kootstra, N.A.

von Schwedler, U., Song, J., Aiken, C., and Trono, D. (1993). Vif is crucial for human

Wiegand, H.L., Doehle, B.P., Bogerd, H.P., and Cullen, B.R. (2004). A second human

*of Sciences of the United States of America 97*, 12295-12299.

production by Fv-4 env gene products exerting dominant negative effect on viral

Sokolskaja, E., Pertel, T., Luban, J.*, et al.* (2007). Vif counteracts a cyclophilin Aimposed inhibition of simian immunodeficiency viruses in human cells. *Journal of* 

(2005). Production of infectious SIVagm from human cells requires functional inactivation but not viral exclusion of human APOBEC3G. *The Journal of biological* 

H.G. (1994). Functional association of cyclophilin A with HIV-1 virions. *Nature* 

mechanism of retrovirus restriction in mammals. *Proceedings of the National Academy* 

Cyclophilin A modulates the sensitivity of HIV-1 to host restriction factors. *Nature* 

(2008). The effect of Trim5 polymorphisms on the clinical course of HIV-1 infection.

immunodeficiency virus type 1 proviral DNA synthesis in infected cells. *Journal* 

antiretroviral factor, APOBEC3F, is suppressed by the HIV-1 and HIV-2 Vif

virus. *The Japanese journal of experimental medicine 45*, 473-478.

cyclosporin A-binding protein cyclophilin. *Nature 337*, 473-475.

primates. *Journal of virology 79*, 3930-3937.

World monkeys. *Nature 427*, 848-853.

envelope glycoprotein. *Microbes Infect*.

*virology 81*, 8080-8090.

*chemistry 280*, 375-382.

*medicine 9*, 1138-1143.

*PLoS Pathog 4*, e18.

*of virology 67*, 4945-4955.

proteins. *The EMBO journal 23*, 2451-2458.

*372*, 363-365.

5514-5519.

Retrovirus restriction by TRIM5alpha variants from Old World and New World

The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old

Anderson, D.J., Sundquist, W.I., and Sodroski, J. (2006). Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor. *Proceedings of the National Academy of Sciences of the United States of America 103*,


Rein, A., Kashmiri, S.V., Bassin, R.H., Gerwin, B.L., and Duran-Troise, G. (1976). Phenotypic

Reymond, A., Meroni, G., Fantozzi, A., Merla, G., Cairo, S., Luzi, L., Riganelli, D., Zanaria,

Rose, K.M., Marin, M., Kozak, S.L., and Kabat, D. (2005). Regulated production and anti-

Sakai, H., Shibata, R., Sakuragi, J., Sakuragi, S., Kawamura, M., and Adachi, A. (1993). Cell-

Sakuma, R., Noser, J.A., Ohmine, S., and Ikeda, Y. (2007). Rhesus monkey TRIM5alpha

Sakuma, R., Ohmine, S., and Ikeda, Y. (2010). Determinants for the rhesus monkey

Sayah, D.M., Sokolskaja, E., Berthoux, L., and Luban, J. (2004). Cyclophilin A

Schrofelbauer, B., Chen, D., and Landau, N.R. (2004). A single amino acid of APOBEC3G

Shibata, R., Kawamura, M., Sakai, H., Hayami, M., Ishimoto, A., and Adachi, A. (1991).

Shindo, K., Takaori-Kondo, A., Kobayashi, M., Abudu, A., Fukunaga, K., and Uchiyama, T.

Si, Z., Vandegraaff, N., O'Huigin, C., Song, B., Yuan, W., Xu, C., Perron, M., Li, X., Marasco,

Simon, J.H., Miller, D.L., Fouchier, R.A., Soares, M.A., Peden, K.W., and Malim, M.H. (1998).

Sokolskaja, E., Sayah, D.M., and Luban, J. (2004). Target cell cyclophilin A modulates human immunodeficiency virus type 1 infectivity. *Journal of virology 78*, 12800-12808.

maturation of virus particles. *Journal of virology 67*, 1663-1666.

with dual sensitivity to Fv-1 restriction. *Cell 7*, 373-379.

compartments. *The EMBO journal 20*, 2140-2151.

*and human retroviruses 21*, 611-619.

*Nature medicine 13*, 631-635.

*430*, 569-573.

3927-3932.

*biological chemistry 285*, 3784-3793.

*general virology 76 ( Pt 11)*, 2723-2730.

*The Journal of biological chemistry 278*, 44412-44416.

transmission. *The EMBO journal 17*, 1259-1267.

*of Sciences of the United States of America 103*, 7454-7459.

mixing between N- and B-tropic murine leukemia viruses: infectious particles

E., Messali, S., Cainarca, S.*, et al.* (2001). The tripartite motif family identifies cell

HIV type 1 activities of cytidine deaminases APOBEC3B, 3F, and 3G. *AIDS research* 

dependent requirement of human immunodeficiency virus type 1 Vif protein for

restricts HIV-1 production through rapid degradation of viral Gag polyproteins.

TRIM5alpha-mediated block of the late phase of HIV-1 replication. *The Journal of* 

retrotransposition into TRIM5 explains owl monkey resistance to HIV-1. *Nature* 

controls its species-specific interaction with virion infectivity factor (Vif). *Proceedings of the National Academy of Sciences of the United States of America 101*,

Generation of a chimeric human and simian immunodeficiency virus infectious to monkey peripheral blood mononuclear cells. *Journal of virology 65*, 3514-3520. Shibata, R., Sakai, H., Kawamura, M., Tokunaga, K., and Adachi, A. (1995). Early replication

block of human immunodeficiency virus type 1 in monkey cells. *The Journal of* 

(2003). The enzymatic activity of CEM15/Apobec-3G is essential for the regulation of the infectivity of HIV-1 virion but not a sole determinant of its antiviral activity.

W.A., Engelman, A.*, et al.* (2006). Evolution of a cytoplasmic tripartite motif (TRIM) protein in cows that restricts retroviral infection. *Proceedings of the National Academy* 

The regulation of primate immunodeficiency virus infectivity by Vif is cell species restricted: a role for Vif in determining virus host range and cross-species


**Part 2** 

**From the Laboratory to the Clinic:** 

**HIV and the Immune System** 

