We are IntechOpen, the world's leading publisher of Open Access books Built by scientists, for scientists

3,800+

Open access books available

116,000+

International authors and editors

120M+

Downloads

Our authors are among the

Top 1%

most cited scientists

12.2%

Contributors from top 500 universities

Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI)

## Interested in publishing with us? Contact book.department@intechopen.com

Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com

## **Meet the editor**

Dr. Shailendra Saxena is leading a research group on human infectious diseases at the CSIR–Centre for Cellular and Molecular Biology (CCMB) in India. The main research interests of his group are to understand the epidemiology and molecular mechanisms of host defense during human viral infections and to develop new predictive, preventive, and therapeutic strategies for them using

JEV and HIV as a model. His research work has been published in various high-impact--factor journalswith high citation. He has received many awards and honors in India and abroad, including various *Young Scientist Awards* and *BBSRC India Partnering Award* and named as *Global Leader in Science* by *The Scientist* magazine (USA) and *International Opinion Leader/Expert* involved in the vaccination for JE by IPIC (UK).

## Contents

#### **Preface XI**


## Preface

The name "retro" in retroviruses not only reversed the concept of molecular biology but also has shown its role in the evolution of modern humans from their primitive ancestor-pri‐ mates group. In the beginning years of molecular biology, we only know the unidirectional flow of genetic information from DNA to RNA to protein that is known as transcription. This irreversible flow came to be known as the "Central Dogma", which was proposed by Sir Crick. But in case of "retroviruses" this flow is completely new, where RNA genetic in‐ formation is converted into DNA first and followed by its integration into host by virus-cod‐ ed specific enzymes and finally reversing the flow of genetic information. For this reverse action, the name "retrovirus" was given to this particular group of viruses. The central dog‐ ma had to be revised when the replication of retroviruses (reverse transcription) was under‐ stood. The studies on retroviruses have opened broader areas of modern biology and medicine, which includes: the discovery of proto-oncogenes, study on cellular growth con‐ trols and carcinogenesis, and understanding of mechanisms that regulate eukaryotic gene expression and molecular genetics.

It is believed that retrovirus evolution has been nothing short of revolutionary. Considerable amount of the mammalian genome appears to be the product of reverse transcription (RT). During the course of evolution it is believed that retroviruses incorporated its genome into the human DNA, and these viral sequences have been suggested to play important roles in numerous physiological and pathological processes. The activity of RT and integrase en‐ zymes makes it feasible for genetic material from retrovirus to become permanently inte‐ grated into the DNA genome of an infected cell.

The presence of two copies of the genome that too with the most unstable form, along the two unique enzymes, makes this group of virus exclusive from the rest of RNA viruses. Sur‐ prisingly, in host, retroviruses do not appear to straightforwardly activate host innate de‐ fenses. Retroviruses such as HIV directly targets T cells of immune cells, which plays an important role in innate and adaptive responses. It is still a million dollar question that how these ultramicroscopic obligatory viruses could sense specifically only those cells from which viruses have potential harmful effects and finally targets specifically those cells and escape from host immune recognition.

On the other hand, attention to these viruses extends beyond their disease-causing capabili‐ ties because of their unique qualities. cDNA copies can be generated from RT and these cDNAs can be manipulated according to use for cloning, sequencing, etc. Retroviruses' ge‐ netic material is extensively applied in transient and stable expression of cloned genes in vertebrate cells. Ongoing investigation on application of retroviruses in gene therapy and anti-cancer agents makes these types a widely studying group. If we understand retrovirus‐ es completely, not only we can employ these viruses as model for biological research but it also gives us idea where gained knowledge could be applied in other fields such as engi‐ neering and material sciences and to develop new technologies.

This book covers a collection of articles by brilliant researchers who have devoted their time for combat against retroviruses. This book gives a comprehensive overview of recent advan‐ ces in *Retrovirology* , as well as general concepts of molecular biology of retroviral infections, immunopathology, diagnosis, treatment, epidemiology, and etiology to current clinical rec‐ ommendations in management of retroviruses, including endogenous retroviruses, high‐ lighting the ongoing issues, recent advances, with future directions in diagnostic approaches and therapeutic strategies. The book focuses on various aspects and properties of retrovirus‐ es, whose deep understanding is very important for safeguarding human race from more loss of resources and economies due to pathogens.

The authors and editors of the book hope that this work might increase the interest in this field of research and that the readers will find it useful for their investigations, management, and clinical usage. Also I would like to thank the Council of Scientific and Industrial Re‐ search (CSIR-CCMB), Director CCMB Dr. Amitabha Chattopadhyay, colleagues, family, and parents, who gave me a lot of encouragement and support during the work on this book.

> **Shailendra K. Saxena, PhD, DCAP, FAEB, FIVS, FBRS** CSIR-Centre for Cellular and Molecular Biology, Hyderabad India

**Retrovirus - Molecular Biology and Pathogenesis**

es completely, not only we can employ these viruses as model for biological research but it also gives us idea where gained knowledge could be applied in other fields such as engi‐

This book covers a collection of articles by brilliant researchers who have devoted their time for combat against retroviruses. This book gives a comprehensive overview of recent advan‐ ces in *Retrovirology* , as well as general concepts of molecular biology of retroviral infections, immunopathology, diagnosis, treatment, epidemiology, and etiology to current clinical rec‐ ommendations in management of retroviruses, including endogenous retroviruses, high‐ lighting the ongoing issues, recent advances, with future directions in diagnostic approaches and therapeutic strategies. The book focuses on various aspects and properties of retrovirus‐ es, whose deep understanding is very important for safeguarding human race from more

The authors and editors of the book hope that this work might increase the interest in this field of research and that the readers will find it useful for their investigations, management, and clinical usage. Also I would like to thank the Council of Scientific and Industrial Re‐ search (CSIR-CCMB), Director CCMB Dr. Amitabha Chattopadhyay, colleagues, family, and parents, who gave me a lot of encouragement and support during the work on this book.

**Shailendra K. Saxena, PhD, DCAP, FAEB, FIVS, FBRS**

CSIR-Centre for Cellular and Molecular Biology,

Hyderabad India

neering and material sciences and to develop new technologies.

loss of resources and economies due to pathogens.

VIII Preface

## **Molecular Biology and Pathogenesis of Retroviruses**

Shailendra K. Saxena and Sai V. Chitti

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62885

#### **Abstract**

Retroviruses consist of a varied family of enveloped RNA viruses with positive-sense RNAs that replicate in a host cell through the process of reverse transcription. Retrovirus‐ es belong to the Retroviridae family that typically carries their genetic material in the form of ribonucleic acid, while the genetic material of their hosts is in the form of deoxy‐ ribonucleic acid. Infections with a number of retroviruses can lead to serious conditions, such as AIDS, a range of malignancies, neurological diseases, and added clinical condi‐ tions. In addition, some can even become integrated as DNA in the germ line and passed as endogenous viruses from generation to generation. Surprisingly, retroviruses do not appear to straightforwardly activate host innate defenses. On the other hand, attention in these viruses extends beyond their disease causing capabilities. For example, studies on the retroviruses led to the discovery of oncogenes, understanding of mechanisms that regulate eukaryotic gene expression, and these are proving to be valuable research tools in molecular biology and have been used successfully in gene therapy. The central goals of retrovirology today are the treatment and the prevention of human and non-human diseases and to use this virus in research.

**Keywords:** retrovirus, Retroviridae, reverse transcriptase, replication, immune responses, ART

#### **1. Introduction**

During the past few decades retrovirus has done an adequate amount of harm to the human life and became a big threat globally. These are the group of viruses that belong to the family Retroviridae and that typically carry their genetic material in the form of ribonucleic acid (RNA), while the genetic material of their hosts is in the form of deoxyribonucleic acid (DNA). Retroviruses are named for an enzyme known as reverse transcriptase (RT), which was discovered independently in 1971 by American virologists Howard Temin and David Baltimore for which they have received Nobel Prize in physiology and medicine in the year

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1975. Retroviridae is a family of enveloped, obligate parasites with single-stranded positivesense RNA (ssRNA) that replicate in a host cell through the process of reverse transcription. The activity of RT makes it feasible for genetic material from a retrovirus to become perma‐ nently integrated into the DNA genome (provirus) of an infected cell.

**Figure 1.** Classification of *Retroviridae* family of viruses.

*Retroviridae* is subdivided into *Orthoretrovirinae* and *Spumaretrovirinae* (Figure 1). Under *Orthoretrovirinae* the various genus are *Alpharetrovirus* (Rous sarcoma virus, avian sarcoma leukosis virus), *Betaretrovirus* (Mouse mammary tumor virus, Jaagsiekte sheep retrovirus), *Gammaretrovirus* (murine leukemia virus, Abelson murine leukemia virus, Friend virus, koala retrovirus, xenotropic murine leukemia-related virus), *Deltaretrovirus* (Human T-lymphotrop‐ ic virus (HTLV) types 1–4, simian T-lymphotropic virus types 1–4, Bovine leukemia virus), *Epsilonretrovirus* (Walleye epidermal hyperplasia virus), and *Lentivirus* (human immunodefi‐ ciency virus (HIV), simian immunodeficiency viruses (SIV), feline immunodeficiency virus, puma lentiviruses, bovine immunodeficiency virus, caprine arthritis encephalitis virus, visna virus) are present, whereas under *Spumaretrovirinae* only one genus is present *spumavirus* (simian foamy virus, human foamy virus) [1]. The retroviruses host range include human, murine, feline (cat), avian (birds), and bovine (pig), and it is dependent upon the viral envelope, glycoproteins and structural proteins, involved in integration. Infections with a number of retroviruses can lead to serious conditions, such as AIDS, a range of malignancies, neurological diseases, and added clinical conditions [2]. In addition, some retroviruses can even become integrated as DNA in the germ line and passed as endogenous viruses from generation to generation. Using retrovirus in research has built up the need to advance the investigation in detail regarding the viral particles and genomes, their modes of replication, integration, and host immune evasion. The basic replication of retroviruses includes that (Figure 2) the ssRNA become double-stranded DNA (dsDNA) and gets into the host genetic material and employs host machinery for the synthesis of new virions.

1975. Retroviridae is a family of enveloped, obligate parasites with single-stranded positivesense RNA (ssRNA) that replicate in a host cell through the process of reverse transcription. The activity of RT makes it feasible for genetic material from a retrovirus to become perma‐

*Retroviridae* is subdivided into *Orthoretrovirinae* and *Spumaretrovirinae* (Figure 1). Under *Orthoretrovirinae* the various genus are *Alpharetrovirus* (Rous sarcoma virus, avian sarcoma leukosis virus), *Betaretrovirus* (Mouse mammary tumor virus, Jaagsiekte sheep retrovirus), *Gammaretrovirus* (murine leukemia virus, Abelson murine leukemia virus, Friend virus, koala retrovirus, xenotropic murine leukemia-related virus), *Deltaretrovirus* (Human T-lymphotrop‐ ic virus (HTLV) types 1–4, simian T-lymphotropic virus types 1–4, Bovine leukemia virus), *Epsilonretrovirus* (Walleye epidermal hyperplasia virus), and *Lentivirus* (human immunodefi‐ ciency virus (HIV), simian immunodeficiency viruses (SIV), feline immunodeficiency virus, puma lentiviruses, bovine immunodeficiency virus, caprine arthritis encephalitis virus, visna virus) are present, whereas under *Spumaretrovirinae* only one genus is present *spumavirus* (simian foamy virus, human foamy virus) [1]. The retroviruses host range include human, murine, feline (cat), avian (birds), and bovine (pig), and it is dependent upon the viral envelope, glycoproteins and structural proteins, involved in integration. Infections with a number of retroviruses can lead to serious conditions, such as AIDS, a range of malignancies, neurological diseases, and added clinical conditions [2]. In addition, some retroviruses can even become integrated as DNA in the germ line and passed as endogenous viruses from generation to generation. Using retrovirus in research has built up the need to advance the investigation in detail regarding the viral particles and genomes, their modes of replication, integration, and

nently integrated into the DNA genome (provirus) of an infected cell.

**Figure 1.** Classification of *Retroviridae* family of viruses.

4 Advances in Molecular Retrovirology

**Figure 2.** The reverse of Cricks' central dogma that occurs in retroviruses. RNA genome is converted by reverse tran‐ scriptase into double-stranded DNA, followed by integration into the host genome, transcription and translation of vi‐ ral proteins occurs along with the host.


The typical retrovirus structure is enveloped, spherical to pleomorphic in shape, and they have diameter of 80–100 nm. The different genuses of retrovirus virions (Figure 3) have diverse morphology, but they have their same virion component, which includes the outer envelope coat, two copies of the genetic material, and the viral proteins. Envelope consists of lipids that are obtained from the host plasma membrane during budding process and the glycoprotein such as gp120 and gp41 in case of HIV [3]. The retroviral envelope serves three separate functions that includes the outer lipid bilayer protects from the extracellular environment, it also aids in the entry and way out of host cells through endosomal membrane trafficking, and the facility to straightforwardly enter cells by fusing with their

**2. Retrovirus structure, genome, and proteins** 

membranes.

#### **2. Retrovirus structure, genome, and proteins**

The typical retrovirus structure is enveloped, spherical to pleomorphic in shape, and they have diameter of 80–100 nm. The different genuses of retrovirus virions (Figure 3) have diverse morphology, but they have their same virion component, which includes the outer envelope coat, two copies of the genetic material, and the viral proteins. Envelope consists of lipids that are obtained from the host plasma membrane during budding process and the glycoprotein such as gp120 and gp41 in case of HIV [3]. The retroviral envelope serves three separate functions that includes the outer lipid bilayer protects from the extracellular environment, it also aids in the entry and way out of host cells through endosomal membrane trafficking, and the facility to straightforwardly enter cells by fusing with their membranes.

**Figure 3.** Schematic cross section through a retroviral particle: Showing retrovirus components.

The genome of retrovirus is monopartite, linear, dimeric, ssRNA (+) of about 8–10 kb, with a 5'-cap and a 3'poly-A tail (Figure 4). The group-specific gene (*gag*), *pol*, *pro*, envelope (*env*) genes are flanked between the R regions. The 5'-long terminal repeats (LTRs) consist of U3 (unique sequence), R primer binding site (PBS), and U5 regions. The 3' end consists of a polypurine tract (PPT), U3, and R regions. The R region is a short repeated sequence at each end of the genome used during the reverse transcription to ensure correct end-to-end transfer in the growing chain. U5, on the other hand, is a short exceptional arrangement in the middle of R and PBS [4]. PBS consists of 18 bases corresponding to 3' end of tRNA primer. L region is an untranslated leader region that gives the sign for packaging of the genome RNA. The retroviral protein includes gag, protease, pol, and env proteins. Gag is the primary retroviral structural protein responsible for orchestrating the majority of steps in viral assembly. Most of these assembly steps occur through interactions with three gag subdomains—matrix (MA), capsid (CA), and nucleocapsid (NC). The gag subdomains are structurally discrete but have functionally overlapping roles in the viral assembly process [5, 6].

**Figure 4.** Retrovirus structure and genome: The three significant protein coding genes include group-specific gene (gag), pol, envelope (env) genes, which are flanked between the R regions and codes for capsid, reverse transcriptase, integrase, protease, and envelope proteins, respectively. Retroviruses are characterized by the 5′ and 3′ long terminal repeat (LTR) sequences, which are thought to control and gene expression. The LTRs each contain two unique nonprotein-coding sequences, called U5 at the 5′ end and U3 at the 3′ end, which encodes controlling elements. PBS con‐ sists of 18 bases corresponding to 3' end of tRNA primer. The R region is a short repeated sequence at each end of the genome used during the reverse transcription to ensure correct end-to-end transfer in the growing chain.

#### **3. Genetic variations and retroviruses**

**2. Retrovirus structure, genome, and proteins**

6 Advances in Molecular Retrovirology

The typical retrovirus structure is enveloped, spherical to pleomorphic in shape, and they have diameter of 80–100 nm. The different genuses of retrovirus virions (Figure 3) have diverse morphology, but they have their same virion component, which includes the outer envelope coat, two copies of the genetic material, and the viral proteins. Envelope consists of lipids that are obtained from the host plasma membrane during budding process and the glycoprotein such as gp120 and gp41 in case of HIV [3]. The retroviral envelope serves three separate functions that includes the outer lipid bilayer protects from the extracellular environment, it also aids in the entry and way out of host cells through endosomal membrane trafficking, and

the facility to straightforwardly enter cells by fusing with their membranes.

**Figure 3.** Schematic cross section through a retroviral particle: Showing retrovirus components.

functionally overlapping roles in the viral assembly process [5, 6].

The genome of retrovirus is monopartite, linear, dimeric, ssRNA (+) of about 8–10 kb, with a 5'-cap and a 3'poly-A tail (Figure 4). The group-specific gene (*gag*), *pol*, *pro*, envelope (*env*) genes are flanked between the R regions. The 5'-long terminal repeats (LTRs) consist of U3 (unique sequence), R primer binding site (PBS), and U5 regions. The 3' end consists of a polypurine tract (PPT), U3, and R regions. The R region is a short repeated sequence at each end of the genome used during the reverse transcription to ensure correct end-to-end transfer in the growing chain. U5, on the other hand, is a short exceptional arrangement in the middle of R and PBS [4]. PBS consists of 18 bases corresponding to 3' end of tRNA primer. L region is an untranslated leader region that gives the sign for packaging of the genome RNA. The retroviral protein includes gag, protease, pol, and env proteins. Gag is the primary retroviral structural protein responsible for orchestrating the majority of steps in viral assembly. Most of these assembly steps occur through interactions with three gag subdomains—matrix (MA), capsid (CA), and nucleocapsid (NC). The gag subdomains are structurally discrete but have

Retroviruses, similar to all RNA viruses, show a high mutation rate. The real component for creating genetic variation within retroviral populations is because of the polymerization error during DNA synthesis by RT, which does not have a proofreading activity [7]. The reason for this high genetic variation is because of viral mutation rate, recombination rate, rate of replication, size of the viral population, and selective forces [8, 9]. Genetic variation has been documented extensively in populations of HIV type 1. This genetic adaptability has significant consequences for the evolution of HIV-1 and other retroviruses and their impact on human health [8]. Because of the genetic variations, retroviruses are expanding its host range; for example, HIV-1 can switch from using the CCR5 co-receptor to using the CXCR4 co-receptor [10]. This difference in retroviral populations provides a mechanism for retroviruses to escape host immune responses and expand resistance to all known antiretroviral drugs [7, 11].

#### **4. Replication of retroviruses**

Replication is a multistep process; each step is crucial for the virus entry and multiplies itself in the host cell. The study of retroviruses particle assembly, budding, and release has been especially rich in terms of the exchange of concepts and techniques with related areas of cell biology [12]. There are seven steps in the replication cycle of the retrovirus (Figure 5). The initial step is attachment, in which the retrovirus utilizes one of its glycoproteins to attach to one or more particular cell-surface receptors on the host cell. Some retroviruses likewise utilize an optional receptor, referred to as the co-receptor. The second and third steps are penetration and uncoating, individually. Retroviruses infiltrate the host cell by direct fusion of the virion envelope with the plasma membrane of the host. The fourth step is replication, which happens after the retrovirus undergoes partial uncoating thereby releasing its genome and three essential enzymes (RT, integrase, and pol gene coding enzymes). At this stage, the RNA genome is converted by RT into double-stranded DNA, followed by integration into the host genome, transcription and translation of viral proteins along with the host. The fifth step is assembly, in which retrovirus capsids are assembled in an immature form. The sixth step is budding, in which the immature viral particle acquires the host plasma membrane, and the final step is maturation and release, in which the gag and pol proteins of the retrovirus are cleaved by the retroviral protease, thus forming the mature and infectious form of the virus [13]. The retrovirus replication is well studied in case of HIV virus. HIV replicates million of time per day, destroying the host immune cells and eventually causing disease progression. During HIV replication the virus recognizes host cell such as CD4+ T- lymphocyte. Entry of HIV into the cells requires certain substances on the cell surface such as CD4 receptor and coreceptors such as CCR5 and CXCR4 [14]. These receptors interact with protein complexes that are embedded in the viral envelope. The viral proteins consist of extracellular gp120 and transmembrane gp41 proteins. When HIV approaches the target cell, the gp120 binds with the cell surface receptor, this process is termed as attachment. Following co-receptor binding results in a conformational change in gp120, this allows gp41 to unfold and extend its hydrophobic terminal into the cell membrane. gp41 then folds back on itself, this causes the virus to move close toward the cell and facilitates the fusion of their membranes. The viral nucleocapsid then enters the cell and releasing two viral RNA strands and three essential replication enzymes; integrase, protease, and RT. HIV RT is a heterodimer composed of two subunits (p66 and p51). At first, RT begins the reverse transcription of viral RNA; it consists of two catalytic domains —ribonuclease H active site and polymerase active site. In the polymerase active site, singlestranded viral RNA is transcribed into an RNA-DNA double helix. These RNA-DNA hybrids are cleaved into individual stands by ribonuclease H. The polymerase then completes the remaining strand into DNA double helix (dsDNA). After the formation of dsDNA, integrase moves into action, it cleaves each dinucleotide from 3' end of the DNA creating two sticky ends. Integrase then transfers the viral DNA into the cell nucleus and viral dsDNA is covalently and randomly integrated into the cell's genome [15]. The host cell genome now contains the genetic information of HIV virus. Activation of the host cell induces the transcription of proviral DNA by Pol II produces viral spliced and unspliced messenger RNAs. This messenger RNA now migrates into the cytoplasm, where building blocks for a new virus were synthe‐ sized. Some of the building blocks have to be processed by the viral protease where longer proteins are cleaved into small core proteins. The processing of viral proteins is crucial to create an infectious virus. Translation of unspliced viral RNAs produces env, gag, and gag-pol polyproteins. The two viral RNA strands with three enzymes come together and core proteins assemble around them forming a capsid, which is an immature virus particle. Capsid leaves the host cell by acquiring new envelope of host and viral proteins (mature virus) such as gp120 and gp41; this process is known as budding. Recent reports suggest that during this process of budding, clathrin is recruited into the HIV particle with high specificity [16]. These matured virus become ready to infect the other cells [15]. A critical aspect of viral replication is the assembly of virus particles, which are subsequently released as progeny virus. While a great deal of attention has been focused on better understanding this phase of the viral lifecycle, many aspects of the molecular details remain poorly understood.

especially rich in terms of the exchange of concepts and techniques with related areas of cell biology [12]. There are seven steps in the replication cycle of the retrovirus (Figure 5). The initial step is attachment, in which the retrovirus utilizes one of its glycoproteins to attach to one or more particular cell-surface receptors on the host cell. Some retroviruses likewise utilize an optional receptor, referred to as the co-receptor. The second and third steps are penetration and uncoating, individually. Retroviruses infiltrate the host cell by direct fusion of the virion envelope with the plasma membrane of the host. The fourth step is replication, which happens after the retrovirus undergoes partial uncoating thereby releasing its genome and three essential enzymes (RT, integrase, and pol gene coding enzymes). At this stage, the RNA genome is converted by RT into double-stranded DNA, followed by integration into the host genome, transcription and translation of viral proteins along with the host. The fifth step is assembly, in which retrovirus capsids are assembled in an immature form. The sixth step is budding, in which the immature viral particle acquires the host plasma membrane, and the final step is maturation and release, in which the gag and pol proteins of the retrovirus are cleaved by the retroviral protease, thus forming the mature and infectious form of the virus [13]. The retrovirus replication is well studied in case of HIV virus. HIV replicates million of time per day, destroying the host immune cells and eventually causing disease progression. During HIV replication the virus recognizes host cell such as CD4+ T- lymphocyte. Entry of HIV into the cells requires certain substances on the cell surface such as CD4 receptor and coreceptors such as CCR5 and CXCR4 [14]. These receptors interact with protein complexes that are embedded in the viral envelope. The viral proteins consist of extracellular gp120 and transmembrane gp41 proteins. When HIV approaches the target cell, the gp120 binds with the cell surface receptor, this process is termed as attachment. Following co-receptor binding results in a conformational change in gp120, this allows gp41 to unfold and extend its hydrophobic terminal into the cell membrane. gp41 then folds back on itself, this causes the virus to move close toward the cell and facilitates the fusion of their membranes. The viral nucleocapsid then enters the cell and releasing two viral RNA strands and three essential replication enzymes; integrase, protease, and RT. HIV RT is a heterodimer composed of two subunits (p66 and p51). At first, RT begins the reverse transcription of viral RNA; it consists of two catalytic domains —ribonuclease H active site and polymerase active site. In the polymerase active site, singlestranded viral RNA is transcribed into an RNA-DNA double helix. These RNA-DNA hybrids are cleaved into individual stands by ribonuclease H. The polymerase then completes the remaining strand into DNA double helix (dsDNA). After the formation of dsDNA, integrase moves into action, it cleaves each dinucleotide from 3' end of the DNA creating two sticky ends. Integrase then transfers the viral DNA into the cell nucleus and viral dsDNA is covalently and randomly integrated into the cell's genome [15]. The host cell genome now contains the genetic information of HIV virus. Activation of the host cell induces the transcription of proviral DNA by Pol II produces viral spliced and unspliced messenger RNAs. This messenger RNA now migrates into the cytoplasm, where building blocks for a new virus were synthe‐ sized. Some of the building blocks have to be processed by the viral protease where longer proteins are cleaved into small core proteins. The processing of viral proteins is crucial to create an infectious virus. Translation of unspliced viral RNAs produces env, gag, and gag-pol polyproteins. The two viral RNA strands with three enzymes come together and core proteins

8 Advances in Molecular Retrovirology

**Figure 5.** Replication of retroviruses: There are seven steps in the replication cycle of the retrovirus. The initial step is attachment, in which the retrovirus utilizes one of its glycoproteins to attach to one or more particular cell-surface re‐ ceptors on the host cell. Some retroviruses likewise utilize an optional receptor, referred to as the co-receptor. The sec‐ ond step is penetration and uncoating, individually. Retroviruses infiltrate the host cell by direct fusion of the virion envelope with the plasma membrane of the host. The third step is replication, which happens after the retrovirus un‐ dergoes partial uncoating thereby releasing its genome and three essential enzymes. At this stage, the RNA genome is converted by reverse transcriptase into double-stranded DNA (dsDNA). The fourth step is integration, in which retro‐ virus dsDNA integrates into the host genome followed by transcription and translation of the viral proteins occurs. The fifth step includes proteolytic processing of viral proteins. The sixth step includes assembly of viral proteins and RNA. The seventh step is budding, in which the immature viral particle acquires the host plasma membrane and final step is maturation and release, in which the gag and pol proteins of the retrovirus are cleaved by the retroviral pro‐ tease, thus forming the mature and infectious form of the virus.

A provirus can be transmitted through the germ line from parents to offspring as an endoge‐ nous retrovirus [17, 18]. Human endogenous retroviruses (HERVs) account for about 8% of the human genome [19]. Exogenous retroviruses seem to have arisen from endogenous retrotransposons by acquisition of a cellular envelope gene [20]. The existence of HERVs has been identified for many years, but their abundance in the genome was not predicted by earlier studies [21]. Retroviral genome gets into human genome and by de novo insertion followed by activation of downstream proto-oncogenes, or by gene disruption [22]. Retrovirus integra‐ tion does not occur in resting (Go phase) cells yet rather requires that cells be in the S phase (DNA synthesis) of their mitotic cycle. Since the mitotic phase is induced by a binary recog‐ nition event, integration and virus reproduction perhaps require that the invaded target T-cell interact with the appropriate B-cell-processed antigen complex. Once the viral cDNA integra‐ tes, transcription to mRNA proceeds at some rate that depends on the details of the infecting virus and the invaded cell [23]. It is also reported that enhancer and promoter elements in retroviral LTRs can influence the transcription of next genes that can result in transcriptional activation or gene silencing and which may result in abnormal expression of tissue-specific proteins [24]. The human genome contains many endogenous retroviral sequences, and these have been suggested to play important roles in a number of physiological and pathological processes. Researchers also found that ERVs also take part in the body's immune defense against regular bacterial and viral pathogens. HERVs are classified into three broad classes (I, II, III). Analysis of the draft human genome has so far found only three HERV proviruses with complete open reading frames for gag, pol, and env, which are considered as essential viral genes, and at least one of these HERVs is mutated at a critical residue in the reverse transcrip‐ tase domain of pol [25]. HERVs have frequently been reported as etiological cofactors in chronic diseases such as cancer, autoimmunity, and neurological disease.

#### **5. Mode of transmission**

Most of the retroviruses transmission occurs through cell to cell, mother to fetus transmission, and through biological fluids. Cell-to-cell transmission of retroviruses is much more efficient as compared with cell-free conditions, and as retroviruses reach through the tight cell-cell interface, they are out of reach of the immune system [26]. Retrovirus employs various mechanisms of immune evasion, however, and can destroy the immune system or subvert it to enable successful transmission [27].

#### **6. Immune system and retroviruses**

The human immune system needs to manage with various pathogens, ranging from RNA viruses to 30-foot-long tapeworms [28]. Although we have gained much understanding of innate immune recognition of many microbial pathogens, currently we have very little knowledge about innate immune responses against retroviral infections [29]. The immune system retroviruses (ISRV) are defined as a retrovirus (HIV) whose target is T4-positive T- helper cells of the immune system that requires stimulation by antigens to reproduce. T-cells are part of the response mechanism that defends the body against attacking agents and are stimulated to reproduce by such agents [23]. Antiviral responses are characteristically marked by stimulation of type I interferon through various mechanisms that recognize viral nucleic acids. These responses restrain the viral replication by various mechanisms and activate the adaptive immune responses with the help of antigen presenting cells and also aids in devel‐ oping memory and viral clearance. But, quite number of reports suggests that viruses also have developed a variety of means for circumventing innate immune responses, ensuring their survival and transmission. Surprisingly retroviruses do not appear to straightforwardly activate host innate defenses. It was observed that generalized immune activation and increased amount of cytokines and immunoglobulins along with the progressive loss of CD4+ T-cells was reported during HIV-1 infection [30].

Generally, viral infection triggers innate immune sensors to produce type I interferon. However, this is not the case with retroviruses; the reason is still not known. The recent reports suggest various molecules it includes: TREX1, which is a cytosolic exonuclease that degrades DNA [31] derived from HIV or endogenous retroelements, thereby preventing the accumula‐ tion of cytosolic DNA, which would otherwise trigger innate immunity. In a study on Trex1(-/-) mouse cells and human CD4(+) T-cells and macrophages in which TREX1 was inhibited by RNA-mediated interference, cytosolic HIV-DNA accumulated and HIV infection induced type I interferon that inhibited HIV replication and spreading [32]. The recent study on innate immune sensors during retroviral infection has identified the enzyme cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) synthase (cGAS) which triggers the cytosolic DNA and activates the production of type I interferons and other cytokines. The mechanism in which these sensors act by includes firstly viral DNA binds and activates cGAS, which catalyzes the synthesis of a cGAMP isomer from adenosine triphosphate (ATP) and guanosine triphosphate (GTP). This cGAMP isomer that is termed has 2′3′-cGAMP contains both 2′-5′ and 3′-5′ phosphodiester linkages. cGAMP then binds and activates the endoplasmic reticulum protein stimulator of IFN genes (STING) and functions as a second messenger. STING activates the NF- κB, interferon regulatory factor 3 (IRF3) to induce interferons and other cytokines through the activation of protein kinases IκB kinase (IKK) and TANK-binding kinase 1 (TBK1) [32–35]. In our view, there is currently insufficient understanding about how the retroviral infection, in general, is sensed by the innate immune system. If the innate immune part is traced, that will aid in understanding the activation of adaptive immunity and devel‐ opment of antiviral against retroviruses.

#### **7. Antiretroviral therapy**

A provirus can be transmitted through the germ line from parents to offspring as an endoge‐ nous retrovirus [17, 18]. Human endogenous retroviruses (HERVs) account for about 8% of the human genome [19]. Exogenous retroviruses seem to have arisen from endogenous retrotransposons by acquisition of a cellular envelope gene [20]. The existence of HERVs has been identified for many years, but their abundance in the genome was not predicted by earlier studies [21]. Retroviral genome gets into human genome and by de novo insertion followed by activation of downstream proto-oncogenes, or by gene disruption [22]. Retrovirus integra‐ tion does not occur in resting (Go phase) cells yet rather requires that cells be in the S phase (DNA synthesis) of their mitotic cycle. Since the mitotic phase is induced by a binary recog‐ nition event, integration and virus reproduction perhaps require that the invaded target T-cell interact with the appropriate B-cell-processed antigen complex. Once the viral cDNA integra‐ tes, transcription to mRNA proceeds at some rate that depends on the details of the infecting virus and the invaded cell [23]. It is also reported that enhancer and promoter elements in retroviral LTRs can influence the transcription of next genes that can result in transcriptional activation or gene silencing and which may result in abnormal expression of tissue-specific proteins [24]. The human genome contains many endogenous retroviral sequences, and these have been suggested to play important roles in a number of physiological and pathological processes. Researchers also found that ERVs also take part in the body's immune defense against regular bacterial and viral pathogens. HERVs are classified into three broad classes (I, II, III). Analysis of the draft human genome has so far found only three HERV proviruses with complete open reading frames for gag, pol, and env, which are considered as essential viral genes, and at least one of these HERVs is mutated at a critical residue in the reverse transcrip‐ tase domain of pol [25]. HERVs have frequently been reported as etiological cofactors in

chronic diseases such as cancer, autoimmunity, and neurological disease.

Most of the retroviruses transmission occurs through cell to cell, mother to fetus transmission, and through biological fluids. Cell-to-cell transmission of retroviruses is much more efficient as compared with cell-free conditions, and as retroviruses reach through the tight cell-cell interface, they are out of reach of the immune system [26]. Retrovirus employs various mechanisms of immune evasion, however, and can destroy the immune system or subvert it

The human immune system needs to manage with various pathogens, ranging from RNA viruses to 30-foot-long tapeworms [28]. Although we have gained much understanding of innate immune recognition of many microbial pathogens, currently we have very little knowledge about innate immune responses against retroviral infections [29]. The immune system retroviruses (ISRV) are defined as a retrovirus (HIV) whose target is T4-positive T-

**5. Mode of transmission**

10 Advances in Molecular Retrovirology

to enable successful transmission [27].

**6. Immune system and retroviruses**

There are about 34 million HIV-1–infected people in the world [36], and this number plainly says that there is an urgent unmet need for investigation of antiretroviral therapy (ART), and management of this worldwide risk is highly desired [37]. ART is treatment of people infected with the retroviruses using anti-retroviral drugs. The goal of antiretroviral therapy is to reduce the amount of virus in infected individual body (viral load) to a level that can no longer be detected with ongoing treatment blood test. Considerable advances in ART have been made since the introduction of zidovudine (3'-azido-3'-deoxythymidine—AZT) in 1987 [38]. The regular treatment consists of a grouping of at least three drugs called as highly active antire‐ troviral therapy (HAART) that hold back the viral replication within the host cell and thereby reduces the viral load.

Six classes of antiretroviral agents (Table 1) currently exist (specifically towards HIV); they include the following:


**6.** Chemokine receptor antagonist. This small molecule such as maraviroc [49] selectively and reversibly binds the CCR5 coreceptor, blocking the V3 loop interaction and inhibiting fusion of the cellular membranes. Using these inhibitors individually or in combination the virus replication process is slowed and the retroviruses find it more difficult to overcome this combined attack. ART has the potential both to reduce mortality and morbidity rates among infected people, and to improve their quality of life [50].


**Table 1.** Classes of antiretroviral agents and their mode of action

#### **8. Conclusions**

detected with ongoing treatment blood test. Considerable advances in ART have been made since the introduction of zidovudine (3'-azido-3'-deoxythymidine—AZT) in 1987 [38]. The regular treatment consists of a grouping of at least three drugs called as highly active antire‐ troviral therapy (HAART) that hold back the viral replication within the host cell and thereby

Six classes of antiretroviral agents (Table 1) currently exist (specifically towards HIV); they

**1.** Nucleoside reverse transcriptase inhibitors (NRTIs) such as abacavir, emtricitabine, and tenofovir [39] takes action by interfering with the HIV replication cycle through compet‐ itive inhibition of reverse transcriptase enzyme a key enzyme in replication and thereby terminates the DNA formation. These can also terminate the DNA formation by incorpo‐ rating into the proviral DNA; the reason is that NRTIs are structurally similar to the DNA

**2.** Non-nucleoside reverse transcriptase inhibitors (NNRTIs) includes niverapine, efavirenz, and etravirine, which acts by non-competitive binding of NNRTIs at the hydrophobic pocket of p66 subunit of the enzyme results in a conformational change and alters the active site and limits RT activity. The limitation of these drugs is that it has a low genetic barrier, i.e., a single mutation in RT genome induces a high-level of phenotypic resistance

**3.** Protease inhibitors (PIs) include indinavir, atazanavir, darunavir, and tipranavir. This retrovirus protease is a 99-amino-acid, aspartic acid protein, which plays an important role in the maturation of virus particles late in the viral life cycle. During or immediately after viral budding from an infected cell, proteases systematically cleaves individual proteins from the gag and gag-pol polypeptide precursors into functional subunits for viral capsid formation. Protease inhibitors act as competitive inhibitors that directly bind to protease and put off the subsequent cleavage of polypeptides. It has recently been suggested that PIs can directly inhibit lymphocyte apoptosis and this effect may contribute

**4.** Integrase inhibitors (INSTIs) such as dolutegravir and raltegravir are used in combination with a protease inhibitor and target the strand transfer step of retroviral DNA integration. These are approved by FDA in 2007. Integration is essential for viral replication and is thus an attractive target for novel chemotherapy [46]. The integrase enzyme is responsible for transfer of virus-encoded DNA to the host cell chromosome, a necessary event in retrovirus replication [15] INSTIs active against a wide range, including both CCR5 co-

**5.** Fusion inhibitors (FIs) include enfuvirtide—act extracellularly to prevent the fusion. It is a peptide based on the gp41 sequence that specifically prevents membrane fusion by competitively binds to gp41 and preventing the conformational change of gp41 required

to an immunologic benefit independently of an antiviral effect [43–45].

receptor and CXCR4 coreceptor–using strains [47].

to complete the final step in the fusion process [48].

reduces the viral load.

12 Advances in Molecular Retrovirology

include the following:

nucleoside bases.

and prevents its use [40–42].

The central goals of retrovirology nowadays are the treatment and the prevention of human and non-human diseases and to use this virus in research. Recent studies have shown that retroviruses can be used in a number of ways such as model for biological research, for understanding of genes, molecular and cell biology studies. On the other hand, attention in these viruses extends beyond their disease causing capabilities, discovery of oncogenes, understanding of mechanisms that regulate eukaryotic gene expression was possible because of the study on retroviruses. The complete understanding of retrovirus could help the researchers and clinicians to use them in various fields of biology and medicine for the development of new methodologies and techniques. Ongoing investigation on application of retroviruses in gene therapy and anti-cancer agents makes these type a widely studying group. The way retroviruses enter and target the specific cells and integrate itself into the host genome was very fascinating to the scientists globally, and these can be used as models to develop new vectors that could be employed in research. Collaborative international project needs to be taken up to understand the complete life cycle of retroviruses. The reason is that this not only aids in developing antiviral, but also gives us idea where gained knowledge could be applied in other fields such as engineering and material sciences and to develop new technologies.

#### **Acknowledgements**

Authors are grateful to the Director, CCMB and Council of Scientific and Industrial Research (CSIR-CCMB), India, for the encouragement and support for this work. SK Saxena is also supported by US National Institute of Health Grants: R37DA025576 and R01MH085259.

#### **Author details**

Shailendra K. Saxena\* and Sai V. Chitti

\*Address all correspondence to: shailen@ccmb.res.in; shailen1@gmail.com

CSIR–Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India

#### **References**


[5] Rabson AB, Graves BJ. Synthesis and processing of viral RNA. In: Coffin JM, Hughes SH, Varmus HE, editors. Retroviruses. Cold Spring Harbor (NY): Cold Spring Har‐ bor Laboratory Press; 1997.

retroviruses can be used in a number of ways such as model for biological research, for understanding of genes, molecular and cell biology studies. On the other hand, attention in these viruses extends beyond their disease causing capabilities, discovery of oncogenes, understanding of mechanisms that regulate eukaryotic gene expression was possible because of the study on retroviruses. The complete understanding of retrovirus could help the researchers and clinicians to use them in various fields of biology and medicine for the development of new methodologies and techniques. Ongoing investigation on application of retroviruses in gene therapy and anti-cancer agents makes these type a widely studying group. The way retroviruses enter and target the specific cells and integrate itself into the host genome was very fascinating to the scientists globally, and these can be used as models to develop new vectors that could be employed in research. Collaborative international project needs to be taken up to understand the complete life cycle of retroviruses. The reason is that this not only aids in developing antiviral, but also gives us idea where gained knowledge could be applied in other fields such as engineering and material sciences and to develop new technologies.

Authors are grateful to the Director, CCMB and Council of Scientific and Industrial Research (CSIR-CCMB), India, for the encouragement and support for this work. SK Saxena is also supported by US National Institute of Health Grants: R37DA025576 and R01MH085259.

[1] Zhang W, Cao S, Martin JL, Mueller JD, Mansky LM. Morphology and ultrastructure

[3] Wyatt R, Sodroski J. The HIV-1 envelope glycoproteins: Fusogens, antigens, and im‐

[4] Coffin JM. Structure and classification of retroviruses. In: Levy, JA. The *Retroviridae* 1

**Acknowledgements**

14 Advances in Molecular Retrovirology

**Author details**

**References**

Shailendra K. Saxena\*

and Sai V. Chitti

\*Address all correspondence to: shailen@ccmb.res.in; shailen1@gmail.com

of retrovirus particles. AIMS Biophys. 2015; 2(3): 343-369.

[2] Varmus H. Retroviruses. Science. 1988; 240(4858): 1427-1435.

(1st ed.). New York: Plenum; 1992. p. 20. ISBN 0-306-44074-1.

munogens. Science. 1998; 280(5371): 1884-1888.

CSIR–Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India


[33] Yan N, Regalado-Magdos AD, Stiggelbout B, Lee-Kirsch MA, Lieberman J. The cyto‐ solic exonuclease TREX1 inhibits the innate immune response to human immunode‐ ficiency virus type 1. Nat Immunol. 2010; 11(11): 1005-1013.

[19] Black SG, Arnaud F, Palmarini M, Spencer TE. Endogenous retroviruses in tropho‐ blast differentiation and placental development. Am J Reprod Immunol. 2010; 64(4):

[20] Gim JA, Han K, Kim HS. Identification and expression analysis of human endoge‐ nous retrovirus Y (HERV-Y) in various human tissues. Arch Virol. 2015; 160(9):

[21] Malik HS, Henikoff S, Eickbush TH. Poised for contagion: evolutionary origins of the infectious abilities of invertebrate retroviruses. Genome Res. 2000; 10(9): 1307-1318.

[22] Boeke JD, Stoye JP. Retrotransposons, endogenous retroviruses, and the evolution of retroelements. In: Coffin JM, Hughes SH, Varmus HE, editors. Retroviruses. Cold

[23] Löwer R. The pathogenic potential of endogenous retroviruses: Facts and fantasies.

[24] Cooper LN. Theory of an immune system retrovirus. Proc Natl Acad Sci U S A. 1986;

[25] Griffiths DJ. Endogenous retroviruses in the human genome sequence. Genome Biol.

[26] Mayer J, Sauter M, Rácz A, Scherer D, Mueller-Lantzsch N, Meese E. An almost-in‐ tact human endogenous retrovirus K on human chromosome 7. Nat Genet. 1999;

[27] Jin J, Sherer NM, Heidecker G, Derse D, Mothes W. Assembly of the murine leuke‐ mia virus is directed towards sites of cell-cell contact. PLoS Biol. 2009; 7(7).

[28] Kane M, Case LK, Kopaskie K, Kozlova A, MacDearmid C, Chervonsky AV, Golov‐ kina T. Successful transmission of a retrovirus depends on the commensal microbio‐

[29] Medzhitov R, Littman D. HIV immunology needs a new direction. Nature. 2008;

[30] Gao D, Wu J, Wu YT, Du F, Aroh C, Yan N, Sun L, Chen ZJ. Cyclic GMP-AMP syn‐ thase is an innate immune sensor of HIV and other retroviruses. Science. 2013;

[31] Manel N, Littman DR. Hiding in plain sight: How HIV evades innate immune re‐

[32] Stetson DB, Ko JS, Heidmann T, Medzhitov R. Trex1 prevents cell-intrinsic initiation

Spring Harbor (NY): Cold Spring Harbor Laboratory Press: 1997, pp. 343-435.

255-264.

16 Advances in Molecular Retrovirology

2161-2168.

83(23): 9159-9163.

2001; 2(6).

21(3): 257-258.

455(7213): 591.

341(6148): 903-906.

Trends Microbiol. 1999; 7(9): 350-356.

ta. Science. 2011; 334(6053): 245-249.

sponses. Cell. 2011; 147(2): 271-274.

of autoimmunity. Cell. 2008; 134(4): 587-598.


**Retrovirus Replication, Gene Expression, Latency and Reactivation**

[46] Chavan S, Kodoth S, Pahwa R, Pahwa S. The HIV protease inhibitor Indinavir inhib‐ its cell-cycle progression in vitro in lymphocytes of HIV-infected and uninfected in‐

[47] Hazuda DJ, Felock P, Witmer M, Wolfe A, Stillmock K, Grobler JA, Espeseth A, Gab‐ ryelski L, Schleif W, Blau C, Miller MD. Inhibitors of strand transfer that prevent in‐ tegration and inhibit HIV-1 replication in cells. Science. 2000; 287(5453): 646-650. [48] Hicks C, Gulick RM. Raltegravir: The first HIV type 1 integrase inhibitor. Clin Infect

[49] Eggink D, Berkhout B, Sanders RW. Inhibition of HIV-1 by fusion inhibitors. Curr

[50] Tilton JC, Doms RW. Entry inhibitors in the treatment of HIV-1 infection. Antiviral

[51] Sagar V, Pilakka-Kanthikeel S, Pottathil R, Saxena SK, Nair M. Towards nanomedi‐

cines for neuro AIDS. Rev Med Virol. 2014; 24(2): 103-124.

dividuals. Blood. 2001; 98(2): 383-389.

Pharm Des. 2010; 16(33): 3716-3728.

Dis. 2009; 48(7): 931-939.

18 Advances in Molecular Retrovirology

Res. 2010; 85(1): 91-100.

## **Role of Host Proteins in HIV-1 Early Replication**

Lokeswara S. Balakrishna and Anand K. Kondapi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62108

#### **Abstract**

After 33 years of the identification of HIV-1 infection, very little is known about the role of host cellular proteins. Till now considerable work has been done in the area of hostpathogen interactions facilitated by the viral proteins and host receptors. The role of the main receptor CD4 and co-receptors like CCR5, CXCR4 and their alternative receptors were well studied in disease progression. But the intracellular events during the hostpathogen interactions were poorly understood. Much data is available based on the glob‐ al analysis of genome-wide RNA interference screens, yeast two-hybrid system and coimmunoprecipitation studies but their exact roles are not yet characterized. There are very few host proteins like APOBEC3G, LEDGF/p75, INI1, HMG I(Y), BAF which are well studied and characterized. Majority of the reported proteins are attributed to multi‐ ple functions. It will be useful to study such proteins to develop as future candidates in HIV-1 therapeutics.

**Keywords:** HIV, Reverse transcription, Host proteins, CD4, CCR5, CXCR4, Topoisomer‐ ase, PICs

#### **1. Introduction**

Host cell responses are key determinants of infection pertaining to infectious diseases. Different kind of host cell responses are exerted during the course of the infection, either a host defense response to restrict the invasion of pathogen or may promote the invasion. During the course of evolution, pathogens have acquired capability to protect themselves from host defense. This protection is mainly by modulating the key regulators of the host signal trans‐ duction mechanism. Unlike other pathogens, viruses are very small with a small genome which codes for the essential structural proteins and enzymes, a reason to consider them as primitive. These proteins are enough to takeover host cell and to control majority of the cellular processes. This takeover property may be due to its dependence on the host mechanisms to fulfill its

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

needs to establish itself and for replication in the host. For this they have to suppress certain mechanisms and promote others. It is very important to know the host factors involved the host-pathogen interactions, establishment of the infection and pathogenicity. In retrovirus especially in HIV infection, host proteins requirement starts from the beginning of attachment of the virus to its target host cell, reverse transcription, integration, transcription and transla‐ tion of viral proteins, carrying of viral proteins to plasma membrane and release of viral particles. Overall knowledge on host proteins involvement in any infection would increase the possibilities of invention of the inhibitors for the host proteins which may be candidates for the future drug inventions.

### **2. Binding of virus to cell surface**

Viruses have remarkable specificity for the host species and the cell types that they can infect. Similar to other viruses, human immunodeficiency virus (HIV) also has host and exhibits target cell specificity. This feature is based on the properties of a cell which can fulfill the virus catch and grab mechanism to complete its infection cycle (from attachment to the production of progeny viruses is considered as an infection cycle).

HIV as a single particle is called as virion. This virion consists of an outer envelope and inner capsid. Outer envelope is host derived plasma membrane with host cell surface molecules and viral transmembrane glycoprotein called gp41, which connects outer surface gp120 glycopro‐ tein expressed from *env* gene of virus. Capsid is a cone shaped structure made up of a viral protein p24 (named based on its molecular weight 24 kDa) which is a processed poly-protein product of virus gene called *gag*. Envelope glycoprotein is translated as a 160 kDa polyprotein, later processed into two subunits of 120 kDa and another is 41 kDa by a cellular endo-protease [1]. One gp120 and one gp41 collectively form a unit and a trimer of this unit enmeshed in the viral envelope [2, 3]. The gp41 is the transmembrane portion and gp120 is the extracellular region which works as an anchor to grab the specific host cells. The primary/preliminary target for this gp120 is CD4 (receptor) and a secondary target is a chemokine receptor (co-receptor) of host cells.

#### **2.1. Role of CD4**

Leucocyte differentiation antigen, CD4 is a cellular receptor for HIV-1, HIV-2 and Simian immune deficiency virus (SIV). These viruses share CD4 as the common primary receptor and the binding sites on these viruses are highly conserved [4]. The CD4 receptor is found on CD4 T-cells (high expression) macrophages and dendritic cells (DCs; low expression). A CD4 binding site present on each monomer of gp120. Recruitment of one CD4 molecule on a single gp120 in the trimeric anchor can induce conformational changes in all three glycoprotein monomers of the trimer [5]. This binding of gp120 to CD4 can be blocked by the host antibodies produced against the gp120 of the virus which are called neutralizing antibodies. But due to variation in gp120 among virus population, there is a lag time in producing enough antibodies to block virus attachment. Furthermore, the conformational change occurs in the gp120, which avoid recognition by the neutralizing antibodies, a process known as conformational masking. The conformational changes in gp120 allow it to bind to a second receptor on the CD4 cell surface [6].

needs to establish itself and for replication in the host. For this they have to suppress certain mechanisms and promote others. It is very important to know the host factors involved the host-pathogen interactions, establishment of the infection and pathogenicity. In retrovirus especially in HIV infection, host proteins requirement starts from the beginning of attachment of the virus to its target host cell, reverse transcription, integration, transcription and transla‐ tion of viral proteins, carrying of viral proteins to plasma membrane and release of viral particles. Overall knowledge on host proteins involvement in any infection would increase the possibilities of invention of the inhibitors for the host proteins which may be candidates for

Viruses have remarkable specificity for the host species and the cell types that they can infect. Similar to other viruses, human immunodeficiency virus (HIV) also has host and exhibits target cell specificity. This feature is based on the properties of a cell which can fulfill the virus catch and grab mechanism to complete its infection cycle (from attachment to the production of

HIV as a single particle is called as virion. This virion consists of an outer envelope and inner capsid. Outer envelope is host derived plasma membrane with host cell surface molecules and viral transmembrane glycoprotein called gp41, which connects outer surface gp120 glycopro‐ tein expressed from *env* gene of virus. Capsid is a cone shaped structure made up of a viral protein p24 (named based on its molecular weight 24 kDa) which is a processed poly-protein product of virus gene called *gag*. Envelope glycoprotein is translated as a 160 kDa polyprotein, later processed into two subunits of 120 kDa and another is 41 kDa by a cellular endo-protease [1]. One gp120 and one gp41 collectively form a unit and a trimer of this unit enmeshed in the viral envelope [2, 3]. The gp41 is the transmembrane portion and gp120 is the extracellular region which works as an anchor to grab the specific host cells. The primary/preliminary target for this gp120 is CD4 (receptor) and a secondary target is a chemokine receptor (co-receptor)

Leucocyte differentiation antigen, CD4 is a cellular receptor for HIV-1, HIV-2 and Simian immune deficiency virus (SIV). These viruses share CD4 as the common primary receptor and the binding sites on these viruses are highly conserved [4]. The CD4 receptor is found on CD4 T-cells (high expression) macrophages and dendritic cells (DCs; low expression). A CD4 binding site present on each monomer of gp120. Recruitment of one CD4 molecule on a single gp120 in the trimeric anchor can induce conformational changes in all three glycoprotein monomers of the trimer [5]. This binding of gp120 to CD4 can be blocked by the host antibodies produced against the gp120 of the virus which are called neutralizing antibodies. But due to variation in gp120 among virus population, there is a lag time in producing enough antibodies to block virus attachment. Furthermore, the conformational change occurs in the gp120, which

the future drug inventions.

22 Advances in Molecular Retrovirology

of host cells.

**2.1. Role of CD4**

**2. Binding of virus to cell surface**

progeny viruses is considered as an infection cycle).

The second docking area on the CD4+ cell surface is a chemokine receptor, a seven transmem‐ brane (7TM) co-receptor namely C-C chemokine receptor type 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR4). The viral preference of using one co-receptor among others is called 'viral tropism'. The virus which can infect the macrophages predominantly uses CCR5 as their co-receptor. About 90% of all HIV infections involve the M-tropic HIV strain. CXCR4, also called fusin, which is a glycoprotein-linked chemokine receptor used by T-cell infecting (Ttropic) HIV to attach to the T cell. Indeed some HIV are reported to use co-receptors other than these two co-receptors like CCR1, CCR2b, CCR3, CCR8, CCR9, CXCR4, CX3CR1/V28, STRL-33/BONZO/CXCR6, GPR1, GPR15/BOB, APJ, ChemR23, RDC1, and Leukotriene B4 receptor though the mechanisms are unknown.

Once the HIV gp120 has attached to the CD4 molecule, it undergoes conformational changes which enables the binding of the gp120 to a co-receptor leads to the further structural rear‐ rangements in the gp41 to fuse with the cell membrane and entry of the virions core into the cell's cytoplasm (Fig. 1). Once within a cell, virus is safe from neutralizing antibodies, but vulnerable to attack by CD8 cells (cytotoxic T-lymphocytes or CTLs).

**Figure 1. Virus and cell surface receptors interactions**. In panel (A), schematic diagram represents virion particle and host cell surface receptors before attachment of the virion to the host. gp120, gp41 and CD4 binding region on gp120 of virus and CD4 and co-receptors (CCR5/CXCR4) were labeled. In panel (B), the attachment of CD4 in the CD4 binding site of gp120 and also binding of the co-receptor were shown.

In the absence of CD4, infection is inefficient and its significance *in vivo* is controversial. More than this binding and internalization, CD4 is involved in the signal transduction. Binding of gp120 to the CD4 induces rapid activation of the ERK/mitogen-activated protein (MAP) kinase pathway and stimulates expression of cytokine and chemokine genes by the bind‐ ing of nuclear transcription factors (AP-1, NF-kB, and C/EBP) in both T-cell tropic and macrophage tropic strain. The activation of this signaling pathway requires functional CD4 receptors which is independent of binding to CXCR4 [7, 8]. Signal transduction by CD4 depends on its association with Lck, a src-family tyrosine kinase. Lck, interacts with CD4 with its unique NH2-terminal domain, and interacts with other intracellular signaling proteins with its SH2 and SH3 domains. Given the necessity of Lck kinase activity for T lymphocyte development and for mature T cell functions, perhaps Lck may function at different stages during T cell activation [9].

#### **2.2. Role of chemokine receptors**

Chemokine receptors are seven transmembrane (7TM) domain, G protein–coupled molecules that mediate the chemotaxis of T cells and phagocytic cells to the areas of inflammation [10]. Chemokine receptors have four domains exposed on the cell surface: the N terminus and three extracellular loops (E1, E2 and E3). Co-receptors take up different conformations on cell surface and on different cell types [11, 12], influencing their ability to support HIV infection. For X4 strains, E2 is critical. Deletion of the N terminus of CXCR4 affects some of the strains but not all [13], although, when present, participates in binding gp120 [14]. The gp120 of HIV-1 is structurally divided into five regions called Variable regions and represented with V1,V2 etc. The highly conserved region present between the variable region of gp120 functions as a coreceptor binding site [15]. *In vitro*, envelope glycoproteins in soluble form are even capable of co-receptor mediated signal transduction [16-18] which involves rapid phosphorylation of GPCRs at the carboxyl-terminal tail [19]. There are 21 potential phosphorylation sites in CXCR4 and only seven in CCR5. Chemokines, small low-molecular weight proteins, are the ligands that activate and signal through CCR5 and CXCR4 to mediate several cellular functions including development, leukocyte trafficking, angiogenesis, and immune response [20].

The extracellular loop2 (E2) which is present on the N terminus of the co-receptor, is respon‐ sible for the gp120 binding and HIV entry. After binding of the viral extracellular gp120 to the host cell surface, a cellular kinase called Focal adhesion kinase interacts with CCR5 [21]. The affinity of the gp120 and the co-receptors influences the strength of the signal transduction which can be correlated with the successes of post-fusion events of the virus [22]. This signal transduction from the co-receptor converts the host non permissive environment to the permissive environment for the virus establishment in the host. The binding capacity of the viral gp120 to the host co-receptor has been considered as the viral infectivity. Even though, the early and late events after co-receptor-gp120 interaction are not completely elucidated, yet these interactions have an important role in very early events of HIV-1 lifecycle.

#### **2.3. CCR5**

Virus using the CCR5 as co-receptor, infects the macrophages called M-tropic virus. This viral strain has ability to infect other cell types like dendritic cells and CD4 T-cells. Majority of the viral isolates utilize CCR5 co-receptors for their transmission. M-tropic HIV replicates in peripheral blood lymphocytes are less virulent and does not form syncytia. Syncytia are multinuclear cells which are result of cellular fusion.

The expression of CCR5 was observed on widely diverged cell types [23] and modulated by pro-inflammatory cytokines. A number of inflammatory CC-chemokines, like MIP-1, RANTES etc. [24] act as CCR5 agonists while, MCP-3 functions as antagonist. The cytosolic domain of the CCR5 bound with GPCRs (G-protein coupled receptors) transduces signals upon binding of ligands to CCR5. The signal transduction include different secondary messengers like cAMP, Ca2+, PI3-kinase, MAP kinases, as well as other tyrosine kinase cascades [7, 25-31]. The importance of the CCR5 mediated signaling [32] in HIV-1 infection was observed in CCR5 Δ 32 [33] mutant populations. Homozygous CCR5 Δ 32 hampers HIV's ability to infiltrate immune cells. Not only this, many other genetic mutations in CCR5 have effect on the progression and transmission of the HIV-1 infection. This mutation exerts resistance to many modes of HIV-1 infection [34].

Variation in the levels of chemokines was observed from person to person. In long-term nonprogressors and seronegative individuals (people with repeated exposure to the virus but who do not become infected), unusually high levels of the CCR5 ligands were observed. These chemokines could function as natural competitive inhibitors to HIV-1 infection.[35].

#### **2.4. CXCR4**

receptors which is independent of binding to CXCR4 [7, 8]. Signal transduction by CD4 depends on its association with Lck, a src-family tyrosine kinase. Lck, interacts with CD4 with its unique NH2-terminal domain, and interacts with other intracellular signaling proteins with its SH2 and SH3 domains. Given the necessity of Lck kinase activity for T lymphocyte development and for mature T cell functions, perhaps Lck may function at

Chemokine receptors are seven transmembrane (7TM) domain, G protein–coupled molecules that mediate the chemotaxis of T cells and phagocytic cells to the areas of inflammation [10]. Chemokine receptors have four domains exposed on the cell surface: the N terminus and three extracellular loops (E1, E2 and E3). Co-receptors take up different conformations on cell surface and on different cell types [11, 12], influencing their ability to support HIV infection. For X4 strains, E2 is critical. Deletion of the N terminus of CXCR4 affects some of the strains but not all [13], although, when present, participates in binding gp120 [14]. The gp120 of HIV-1 is structurally divided into five regions called Variable regions and represented with V1,V2 etc. The highly conserved region present between the variable region of gp120 functions as a coreceptor binding site [15]. *In vitro*, envelope glycoproteins in soluble form are even capable of co-receptor mediated signal transduction [16-18] which involves rapid phosphorylation of GPCRs at the carboxyl-terminal tail [19]. There are 21 potential phosphorylation sites in CXCR4 and only seven in CCR5. Chemokines, small low-molecular weight proteins, are the ligands that activate and signal through CCR5 and CXCR4 to mediate several cellular functions including development, leukocyte trafficking, angiogenesis, and immune response [20].

The extracellular loop2 (E2) which is present on the N terminus of the co-receptor, is respon‐ sible for the gp120 binding and HIV entry. After binding of the viral extracellular gp120 to the host cell surface, a cellular kinase called Focal adhesion kinase interacts with CCR5 [21]. The affinity of the gp120 and the co-receptors influences the strength of the signal transduction which can be correlated with the successes of post-fusion events of the virus [22]. This signal transduction from the co-receptor converts the host non permissive environment to the permissive environment for the virus establishment in the host. The binding capacity of the viral gp120 to the host co-receptor has been considered as the viral infectivity. Even though, the early and late events after co-receptor-gp120 interaction are not completely elucidated, yet

Virus using the CCR5 as co-receptor, infects the macrophages called M-tropic virus. This viral strain has ability to infect other cell types like dendritic cells and CD4 T-cells. Majority of the viral isolates utilize CCR5 co-receptors for their transmission. M-tropic HIV replicates in peripheral blood lymphocytes are less virulent and does not form syncytia. Syncytia are

these interactions have an important role in very early events of HIV-1 lifecycle.

multinuclear cells which are result of cellular fusion.

different stages during T cell activation [9].

**2.2. Role of chemokine receptors**

24 Advances in Molecular Retrovirology

**2.3. CCR5**

T-tropic HIV uses CXCR4 as a co-receptor which belongs to the family of α-chemokine receptor. CXCR4 (known as fusin or X4) is also a GPCR with natural ligand CXCL12, known as Stromal Cell-Derived Factor 1 (SDF-1) [36, 37]. T-tropic virus can induce syncytium (SI) and are responsible for the rapid disease progression in HIV-positive individuals. During HIV-1 infections X4-tropic virus has the tendency of emergence and maintains higher viral loads and much lower CD4 cell counts in infected persons. Even though highly virulent, X4 infections are susceptible to antiretroviral therapy [7]. Mutational effect of CXCR4 in HIV-1 infection is not known due to its significant role in development and knockout mutant in mice for this gene is lethal at the embryonic stage [38].

CXCR4 also involved in cell death of CD4+ T-cells which was induced by gp120 indicate an important *in vivo* role for CXCR4 mediated signaling. The interaction of gp120 with CXCR4 triggers a cell death pathway of Fas independent, mitochondrial dependent, cytochrome c mediated activation of caspase-9 and -3 [39]. Membrane fusion dependent CD4+ T-cell death was observed in the virus strains of X4 and dual tropic (R5X4) [40].

However, majority of *in vivo* HIV-1 infection is mediated by M-tropic (R5) viruses could able to lyse their target cells and X4 viruses can kill CXCR4+ . It shows that, CD4 has no role in gp120 induced cell death. Moreover, cell lysis and syncytia formation were inhibited in the cells with high levels of CD4 expression. Interestingly, Glycol protein from non-infectious strains of X4 or R5X4 could not induce cell death [41].

Based on the capability to support infection of CD4+ cell lines, other than CCR5 and CXCR4 more than 14 potential co-receptors were identified *in vitro* [6] (Table 1). These receptors are members of (or closely related to) the chemokine receptor family. The significance of other coreceptors for HIV-1 replication *in vivo* and pathogenesis remains unclear.

Recently STRL-33 (CXCR6) functions as co-receptor for HIV-1 infection in primary T-cells [42], and in thymocytes, CCR8 were identified *in vitro* [43].


**Table 1.** HIV-1 receptors and cell tropism

### **3. CD4-independent infection**

CD4 expression is not uniform in all hematopoietic cells. While, some cell types (T-cells) express high levels of CD4, others, including macrophages and dendritic cells (DCs), express barely detectable amounts. But T-cells as well as macrophages are susceptible to the HIV-1 infection *in vivo.* This susceptibility reveals the existence of alternative host cell surface receptors to which, HIV-1 may attach to cells by CD4-independent manner. Sugar groups present on the both virus and host cell surface (like mannose-specific macrophage endocytosis receptor) mediate the host pathogen interactions and helps in the HIV-1 infection [44]. Apart from the sugars, a cell surface protein (DC-SIGN) of dendritic cells [45, 46]. A closely related receptor to this (DC-SIGNR) on endothelial cells [47], Glycolipids namely galactocerebroside (GalC), galactosulfatide (sGalC) which express on neurons and glia in the brain, colon epithelial cell lines and, importantly, on macrophages [48-50] helps in HIV-1 infection. GalC supports suboptimal entry of particular HIV-1 strains without CD4, although infection requires a co-receptor [51]. Glycosaminoglycans like heparansulphate involved in the infection of HeLa cells [52]. Besides cell surface receptors, cell derived molecules incorporated onto virions such as integrin ICAM-1 (intercellular adhesion molecule-1) and LFA-1 (lymphocyte function-associated antigen-1) [53, 54] enhance the overall efficiency of virus entry.

Primary HIV-2 isolates generally infect CD4<sup>−</sup> co-receptor+ cells more efficiently than HIV-1 [55, 56]. In these infections, CXCR4 plays a crucial role [56].

In glioma cell line (D-54 cells) binding of recombinant gp120 to the GalC or sGalC and a 180 kDa receptor activates signal transduction by a tyrosine-kinase which phosphorylates 130- and 115-kDa proteins [57]. It shows that not only main receptors but also the alternative receptors involved in signal transduction and host protein modifications in HIV-1 infections but the differences were observed in efficiency of infection and disease progression. It also conveys that HIV envelope glycoprotein can specifically bind to the different host cell surface receptors which can function as receptor or co-receptor and this feature provides flexibility to virus to infect wide variety of host cell types [56].

#### **3.1. Fusion and internalization of viral particles**

Recently STRL-33 (CXCR6) functions as co-receptor for HIV-1 infection in primary T-cells [42],

CCR1 MIP-1α, RANTES, MPIF-1, MCP-3 + CCR2b MCP-1, MCP-2, MCP-3 + CCR3 Eotaxin, Eotaxin-2, MCP-3, MCP-4, RANTES ++

CCR8 I-309 + CCR9 TECK +

CX3CR1/V28 Fractalkine + STRL-33/BONZO/CXCR6 CXCL16 +

GPR1 ? + GPR15/BOB ? + APJ Apelin + ChemR23 ? + RDC1 ? +

Leukotriene B4 receptor Leukotriene B<sup>4</sup> +

CD4 expression is not uniform in all hematopoietic cells. While, some cell types (T-cells) express high levels of CD4, others, including macrophages and dendritic cells (DCs), express barely detectable amounts. But T-cells as well as macrophages are susceptible to the HIV-1 infection *in vivo.* This susceptibility reveals the existence of alternative host cell surface receptors to which, HIV-1 may attach to cells by CD4-independent manner. Sugar groups present on the both virus and host cell surface (like mannose-specific macrophage endocytosis receptor) mediate the host pathogen interactions and helps in the HIV-1 infection [44]. Apart from the sugars, a cell surface protein (DC-SIGN) of dendritic cells [45, 46]. A closely related receptor to this (DC-SIGNR) on endothelial cells [47], Glycolipids namely galactocerebroside (GalC), galactosulfatide (sGalC) which express on neurons and glia in the brain, colon epithelial cell lines and, importantly, on macrophages [48-50] helps in HIV-1 infection. GalC supports suboptimal entry of particular HIV-1 strains without CD4, although infection requires a co-receptor [51]. Glycosaminoglycans like heparansulphate involved in the infection

**Table 1.** HIV-1 receptors and cell tropism

**3. CD4-independent infection**

**Co-receptors Ligands Role in viral replication**

CCR5 MIP-1α, MIP-1β, RANTES, MCP-2 ++++ ++++

CXCR4 SDF-1 +++ ++

*In vitro In vivo*

and in thymocytes, CCR8 were identified *in vitro* [43].

26 Advances in Molecular Retrovirology

The HIV envelope glycoprotein is responsible not only for the virus attachment to the cell surface but also mediates viral entry. The two parts of the envelope; gp41 and gp120 trimer forms a functional unit, which under goes a series of structural changes (Fig. 2) upon binding to the CD4 and an appropriate chemokine receptors. These interactions promote conforma‐ tional changes in gp120 and gp41, respectively. These changes exposes the fusion domain of the gp41 and allows to undergo fusion [58-60]. In the viral infection co-receptor has a crucial role in fusion. Fusion of viral particles in the absence of CD4 was observed but in the absence of co-receptor was not yet identified. Following attachment to the receptors, some virus particles enters into endosomes (Fig. 3) later the low pH of the endosomes promotes fusion [61]. However, HIV uses a co-receptor dependent and independent of pH [62].

**Figure 2. Schematic representation of structural changes in gp120 and gp41.** Different conformational changes in gp120 and gp41 up on binding to the host cell surface receptors were represented schematically. From left, structures of the virion envelope gp120 before binding. Once gp120 bound to CD4 of host cell, structural change in the gp120 region in the pink dotted circle. The extreme right represents the changes in the gp120 and especially gp41 once recep‐ tor and co-receptor of the host cell interacts with gp120 of virus labeled in pink dotted circle.

**Figure 3. HIV-1 entry strategies.** Two well reported strategies of HIV-1 entry into host cell. The direct membrane fu‐ sion upon binding to the CD4 and co-receptors like CCR5/CXCR4 and endocytosis mediated entry upon receptor and co-receptors binding were represented schematically.

CD4 is the primary receptor for HIVs, but virus penetration requires further interactions with chemokine receptor CCR5 or CXCR4. Earlier studies proposed that binding to co-receptor initiate fusion directly at the plasma membrane. The ability of formation of syncytia, and evidences of putative fusion events at the cell surface have supported the direct fusion at the plasma membrane [63]. However, recent evidence with endocytosis inhibitors and singleparticle tracking revealed fusion and infection occur after endocytic uptake [64, 65]. Moreover, in macrophages HIV infection occurs through macropinocytosis [66, 67]. Based on the receptor density and mobility, the mode of viral entry to the host cell whether fusion on cell surface or after endocytosis will be determined [68].

#### **3.2. Receptor-mediated endocytosis**

Adhesion to the receptor initiates later events which enable viruses to enter the cytosol. The cortex is the potential barrier to prevent entry of large molecules [69]. Receptor-mediated signaling induced by envelope of the viruses allow viruses that undergo penetration at the cell surface and transit the cortex [70-72]. In HIV, Gαi (a heterotrimeric G protein subunit that inhibits the production of cAMP from ATP [73]) and CXCR4 on resting CD4+ve T cells activate cofilin which induces reorganization of the cortex that facilitates infection [71]. This evolve‐ ment of use of different mechanisms of viral invasion into host cell may have distinct advan‐ tages for virus, which provides broader range of cell types to infect or different ways to infect a same cell type [68] to bypass the host restriction mechanisms.

Immediately after its release into the cytoplasm, the viral core undergoes a partial and progressive disassembly, known as uncoating, that leads to the generation of sub viral particles called reverse-transcription complexes (RTCs) and pre-integration complexes (PICs). In HIV-1 the uncoating of capsid is coupled to the initiation of reverse transcription [74].

### **4. Reverse transcription of viral RNA**

#### **4.1. Uncoating of capsid**

**Figure 3. HIV-1 entry strategies.** Two well reported strategies of HIV-1 entry into host cell. The direct membrane fu‐ sion upon binding to the CD4 and co-receptors like CCR5/CXCR4 and endocytosis mediated entry upon receptor and

CD4 is the primary receptor for HIVs, but virus penetration requires further interactions with chemokine receptor CCR5 or CXCR4. Earlier studies proposed that binding to co-receptor initiate fusion directly at the plasma membrane. The ability of formation of syncytia, and evidences of putative fusion events at the cell surface have supported the direct fusion at the plasma membrane [63]. However, recent evidence with endocytosis inhibitors and singleparticle tracking revealed fusion and infection occur after endocytic uptake [64, 65]. Moreover, in macrophages HIV infection occurs through macropinocytosis [66, 67]. Based on the receptor density and mobility, the mode of viral entry to the host cell whether fusion on cell surface or

Adhesion to the receptor initiates later events which enable viruses to enter the cytosol. The cortex is the potential barrier to prevent entry of large molecules [69]. Receptor-mediated signaling induced by envelope of the viruses allow viruses that undergo penetration at the cell surface and transit the cortex [70-72]. In HIV, Gαi (a heterotrimeric G protein subunit that inhibits the production of cAMP from ATP [73]) and CXCR4 on resting CD4+ve T cells activate cofilin which induces reorganization of the cortex that facilitates infection [71]. This evolve‐ ment of use of different mechanisms of viral invasion into host cell may have distinct advan‐ tages for virus, which provides broader range of cell types to infect or different ways to infect

Immediately after its release into the cytoplasm, the viral core undergoes a partial and progressive disassembly, known as uncoating, that leads to the generation of sub viral particles called reverse-transcription complexes (RTCs) and pre-integration complexes (PICs). In HIV-1

the uncoating of capsid is coupled to the initiation of reverse transcription [74].

co-receptors binding were represented schematically.

28 Advances in Molecular Retrovirology

after endocytosis will be determined [68].

a same cell type [68] to bypass the host restriction mechanisms.

**3.2. Receptor-mediated endocytosis**

The events in retrovirus infection that occur between entry into the host cell and reverse transcription are less understood. Uncoating is the process of disintegration of the capsid and release of its components into the cytoplasm of the host cell. The uncoating of the HIV-1 capsid is thought to precede reverse transcription, whereas MLV capsid proteins remain associated with the reverse transcription and pre-integration complexes [75-77]. The uncoating of capsid is a temporarily regulated [78] and the host cell factors involved are poorly understood. There are few known host proteins function as restriction factors and block the HIV-1 infection. One of them is TRIM5α, which prevents retrovirus infection by disrupting an early, post entry events by associating with the retroviral capsid [79, 80]. In owl monkeys, RBCC domains of TRIM5 fused with cyclophilin A (CypA), a known capsid ligand, was identified. [81-87]. Mechanism of Cyp A in capsid uncoating or some other step in the post-entry phases of the HIV-1 life cycle not clearly understood [88]. TRIM5 has multiple roles in early infection. It can interfere with the uncoating process, intercept reverse transcription and viral genome trans‐ port to the nucleus by binding to the capsid proteins [89].

*In vitro,* uncoating of HIV-1 capsid required activated CD4+ lymphocytes. Two distinct cellular factors with molecular mass of approximately 60 and 160 kDa were found to be involved in capsid uncoating [90]. In contrary, cyclophilins mediated activation of T-cells by regulating the activity of calcineurin, a phosphatase necessary for T-cell activation was reported. [91]

#### **4.2. Reverse transcription (Reverse transcriptase and cellular factors: regulators of HIV-1 reverse transcription)**

Reverse transcription takes place in a complex organization called reverse transcription complex (RTC), is a nucleoprotein complex comprising viral RNA, a tRNA primer and newly synthesized DNA, along with these nucleic acids, viral factors and host factors present. Reverse transcription complex (RTC) is a composite organization of nucleoprotein complex comprising of viral RNA, tRNA primer and synthesized DNA where the reverse transcription takes place. RTC comprises of several other nucleic acids, host and viral factors.

The first host factors in association with reverse transcription which are packaged in virus particles is cellular tRNALys3, which is bound to the aminoacyl-tRNALys3, synthetase (LysRS) [92]. tRNALys3 works as a primer by binding to the primer binding site (PBS) [93] of the HIV-1 genomic mRNA and is the first step in reverse transcription. Initiation of reverse transcription refers to the addition of the first five deoxynucleotides to the tRNALys3 primer [94]. Several cellular factors which bound to Integrase (IN) and effects reverse transcription were identified [95] but their direct role in reverse transcription not yet illustrated. Some of the known cellular proteins are integrase interactor 1 (INI1, hSNF5) [96, 97], sin3A-associated protein (SAP18), histone deactylase 1 (HDAC1) [98] and survival motor neuron (SMN)-interacting protein 2 (Gemin2) [99].

INI1 is a component of the SWI/SNF chromatin remodeling complex of host cell [100] and is associated with virus. INI 1 Packaging into virus is specific for HIV-1 and is regulated by a direct interaction with the IN domain of the HIV-1 Gag-Pol protein [101]. INI1 associates with viral RTC/PIC [102] and stimulates IN [96] activity moreover involved in the regulation of reverse transcription.

Like INI1 of SWI/SNF complex, Sin3a-HDAC1 complex members Sin3a, Sap18, Sap30, and HDAC1 were found in virion [98]. HDAC1 is required for the initiation of reverse transcription and involved in a step between uncoating and reverse transcription which results in defective viral cDNA synthesis [98]. Another IN binding protein is Gemin2 is shown to be required for an early reverse transcription product (negative strand strong stop) or integration of viral DNA, suggesting that Gemin2 association with either the reverse transcription or preintegration complexes [99]. It can recruit other cellular factors like DHX9 (RNA helicase A) [103], which intern associated with the SMN complex [104]. But the precise mechanism of how Gemin2 affects early replication remains to be determined.

*Human antigen R* (HuR) is a nuclear protein with nucleocytoplasmic shuttling capabilities [105]. It is a RNA binding protein with 3 RNA binding domains and exhibits high specificity and affinity for AU-rich elements (AREs) [106, 107]. HuR is required for optimal reverse transcrip‐ tion. While the mechanism behind this activity remains unclear, it appears to be due to an interaction with the RNase H domain of RT [108]. APOBEC3G (hA3G), is a host protein with negative effects on reverse transcription [109-112]. Furthermore, hA3G was a member of several ribonucleoproteins including DHX9 [103], hnRNP U [113], PABPC1 [114], YB-1 and SNRPA [115] which can affect HIV-1 replication including reverse transcription. A direct interaction between hA3G and HuR was reported [116]. By formation of protein complex, hA3G and HuR could regulate the functions of RTC in the cytoplasm.

*A kinase anchor protein 1 (AKAP1)* which bind the regulatory subunits of cAMP-dependent protein kinase A (PKA) and anchors them to various membranes throughout the cell [117]. Interaction between AKAP149 and HIV-1 RT was also reported [108]. Like HuR, it interacts with the RNase H region of RT but the mechanism of RT regulation was not yet clearly understood. DNA topoisomerases 1 (TOP1) is another host protein which interact with HIV-1 NC and participate in the initiation of the cDNA synthesis by enhancing the activity of HIV-1 RT [118, 119].

Several studies were conducted on the role of Topoisomerase II in HIV-1 infection. In eukaryotes two isoforms of topoisomerase II (Topo II) was identified. The smaller one is 170kDa topoisomerase II alpha (Topo IIα) and the bigger one is 180 kDa topoisomerase II beta (Topo IIβ) [120]. Recent evidence has proven their key role in viral infections [121, 122]. In response to HIV-1 infection, increased protein levels of Topo IIα and β were observed [123, 124]. Both of these isoforms were reported to undergo phosphorylation in HIV-1 infection [125-127]. These isoforms are phosphorylated by serine kinase present in the purified HIV-1 virion and are associated with pre-integration complexes (PICs) [126]. Topo II inhibitors abrogate HIV-1 replication cycle by interfering with the PICs formation [128]. Down regulation if these isoforms using siRNA resulting in impaired HIV-1 replication [124,129, 130] due to the incomplete reverse transcription. Co-localization studies reveled the associa‐

tion of these isoforms with HIV-1 reverse transcriptase [124]. Similar to this, another host protein HMGI(Y)reported to be with involvement in covalent strand transferin HIV-1 reverse transcription. [131].

APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G, (A3G)) belong to a group of interferon-stimulated gene [132] and is an editing enzyme for nucleic acids. It blocks virus replication by deamination of viral minus-strand DNA, resulting in Gto-A hyper mutation. In addition to the deaminase activity, A3G has also been shown to directly inhibit HIV-1 reverse transcription by a non-editing mechanism [133, 134]. A3G may also reduce viral DNA synthesis and can inducing viral DNA degradation by interacting physically with HIV-1 reverse transcriptase [135]. Its action was blocked by the viral protein Vif and another host protein apoptosis signal-regulating kinase 1 (ASK1) by binding to Vif restores A3G function [136]. Cyclin-dependent kinase (CDK) 2 is a host protein which regulates the reverse transcriptase by phosphorylating on threonine which improves the increased efficiency and stability of reverse transcriptase and enhanced viral fitness. p21, a cell-intrinsic CDK inhibitor, counteracts the CDK2-dependent phosphorylation and significantly reduced the efficacy of viral reverse transcription [137].

#### **4.3. Pre-integration complex formation**

INI1 is a component of the SWI/SNF chromatin remodeling complex of host cell [100] and is associated with virus. INI 1 Packaging into virus is specific for HIV-1 and is regulated by a direct interaction with the IN domain of the HIV-1 Gag-Pol protein [101]. INI1 associates with viral RTC/PIC [102] and stimulates IN [96] activity moreover involved in the regulation of

Like INI1 of SWI/SNF complex, Sin3a-HDAC1 complex members Sin3a, Sap18, Sap30, and HDAC1 were found in virion [98]. HDAC1 is required for the initiation of reverse transcription and involved in a step between uncoating and reverse transcription which results in defective viral cDNA synthesis [98]. Another IN binding protein is Gemin2 is shown to be required for an early reverse transcription product (negative strand strong stop) or integration of viral DNA, suggesting that Gemin2 association with either the reverse transcription or preintegration complexes [99]. It can recruit other cellular factors like DHX9 (RNA helicase A) [103], which intern associated with the SMN complex [104]. But the precise mechanism of how

*Human antigen R* (HuR) is a nuclear protein with nucleocytoplasmic shuttling capabilities [105]. It is a RNA binding protein with 3 RNA binding domains and exhibits high specificity and affinity for AU-rich elements (AREs) [106, 107]. HuR is required for optimal reverse transcrip‐ tion. While the mechanism behind this activity remains unclear, it appears to be due to an interaction with the RNase H domain of RT [108]. APOBEC3G (hA3G), is a host protein with negative effects on reverse transcription [109-112]. Furthermore, hA3G was a member of several ribonucleoproteins including DHX9 [103], hnRNP U [113], PABPC1 [114], YB-1 and SNRPA [115] which can affect HIV-1 replication including reverse transcription. A direct interaction between hA3G and HuR was reported [116]. By formation of protein complex,

*A kinase anchor protein 1 (AKAP1)* which bind the regulatory subunits of cAMP-dependent protein kinase A (PKA) and anchors them to various membranes throughout the cell [117]. Interaction between AKAP149 and HIV-1 RT was also reported [108]. Like HuR, it interacts with the RNase H region of RT but the mechanism of RT regulation was not yet clearly understood. DNA topoisomerases 1 (TOP1) is another host protein which interact with HIV-1 NC and participate in the initiation of the cDNA synthesis by enhancing the activity of HIV-1

Several studies were conducted on the role of Topoisomerase II in HIV-1 infection. In eukaryotes two isoforms of topoisomerase II (Topo II) was identified. The smaller one is 170kDa topoisomerase II alpha (Topo IIα) and the bigger one is 180 kDa topoisomerase II beta (Topo IIβ) [120]. Recent evidence has proven their key role in viral infections [121, 122]. In response to HIV-1 infection, increased protein levels of Topo IIα and β were observed [123, 124]. Both of these isoforms were reported to undergo phosphorylation in HIV-1 infection [125-127]. These isoforms are phosphorylated by serine kinase present in the purified HIV-1 virion and are associated with pre-integration complexes (PICs) [126]. Topo II inhibitors abrogate HIV-1 replication cycle by interfering with the PICs formation [128]. Down regulation if these isoforms using siRNA resulting in impaired HIV-1 replication [124,129, 130] due to the incomplete reverse transcription. Co-localization studies reveled the associa‐

Gemin2 affects early replication remains to be determined.

hA3G and HuR could regulate the functions of RTC in the cytoplasm.

reverse transcription.

30 Advances in Molecular Retrovirology

RT [118, 119].

Immediately after disintegration of the capsid into the host cytosol, the viral +stand genomic RNA converted into double stranded DNA. This newly synthesized viral genomic DNA wrapped around the host and viral proteins in a protective manner and protected from nuclease degradation [138]. The viral single-stranded RNA genome is converted into a linear double-stranded DNA. The viral DNA intermediate then migrates to the cell nucleus and is covalently integrated into a host chromosome. The integration of reverse transcribed HIV-1 cDNA into a host cell chromosome is an essential step in the viral replication cycle [108, 109]. Retroviral integration *in vivo* is mediated by pre-integration complexes (PICs). PICs are be formed with viral and host cell proteins like high-mobility group protein A1 (HMGA1) and the barrier-to-auto-integration factor (BAF) which were well studied and identified their functions as cofactors for integration [139]. HMG I(Y) is another host protein required for the proper function of the PICs *in vitro* [131]. Other proteins like XRCC6, TFRC and HSP70 were identified as in association with viral DNA [140]. Topoisomerase IIα and β isoforms were also identified as nucleoprotein components of PICs, which suggesting their significant role in HIV-1 replication [128].

#### **5. Integration of viral DNA into host genome**

Integration of the viral DNA is mediated by PICs formation. These PICs are capable of performing integration *in vitro*. Even though specific (HMG I(Y)) and non-specific (bovine RNase A) could able to restore the activity, but only BAF can restore the native structure of the HIV-1 protein–DNA intasome from salt stripped PICs [141]. HIV-1 integration in host genome is not a random event. In majority of cases, it takes place in AT rich, euchromatin region. Even though PIC formation can protect the DNA and helps in the nuclear transport, it cannot guide the integration in a proper location in the host genome. A host protein called cellular lens epithelium-derived growth factor (LEDGF/p75) which binds both chromosomal DNA and HIV integrase [142], directs the integration to a location where active transcription takes place under its control [143]. The interaction between the integrase and INI1 stimulates the DNA-joining activity of the integrase and helps to target the viral DNA towards active genes [96, 102, 144]. Presence of a Topo II cleavage site in the HIV-1 promoter and also at 180 bp upstream of the HIV-1 integration site [145, 146] and association of these Topo II isoforms with HIV-1 PICs [128]. Based on the available information, it can be derived that, IN alone can carry out the integration reaction but for the selection of the proper location in the host genome for the integration and success full HIV-1 gene transcription, Integrase required the support of the host factors.

#### **5.1. Transport of PICs into nucleus**

Transportation of the PICs to the nucleus can be divided in two parts. One is transportation to the nuclear periphery and second is from periphery to inside the nucleus. After completion of the successful cell surface attachment, capsid internalization and degradation in cytoplasm which leads to the reverse transcription of the viral genome RNA to DNA and the formation of the pre-integration complex to protect the DNA from the host nucleases takes place. This nucleoprotein PICs should travel from the cytosol to the nucleus to form provirus which is an integrated viral DNA in host chromatin and can produce progeny virus [147, 148].

The PICs in the cytoplasm translocate to the perinuclear compartment by the cytoplasmic movements by the cytoskeleton. Actin and microtubule [149] selectively plays a role in this transport with the help of Myosin VI and Dynein [148]. The dynein complex proteins such as dynein light chain1 (DYNLL1), Tctex1 and Dynactin have been shown to be involved in this process. But very little is known about how HIV-1 targets cytoskeleton. Many of the viral elements found in association with the PIC have been proposed to be important for HIV-1 nuclear import.

Studies on the HIV-1 infection in dividing and non-dividing cells provided enough evidences to believe that PICs enter the nucleoplasm by crossing the nuclear envelope through nuclear pore complexes (NPCs), which form stable channels through the nuclear envelope and gatekeep the trafficking of molecules between the nucleus and cytoplasm [150]. The RTC/PIC with the size of~100-250nm [149, 151, 152] cannot cross the nuclear pore. So, only few important components of the PIC may enter into the nucleus [149, 152].

For nuclear transport, KPNs and NUPs functions as carrier proteins by binding to the integrase. KPN α adaptor proteins importin α1 (Rch1) [153] and importin α3 (KPNA4) [154], to which KPN β1 proteins bound additionally. Importin 7 [155, 156] and transportin 3 [157, 158] are recruited by the nuclear localization signal (NLS) present on IN. In both dividing and nondividing cells Impα3 which interacts with IN is found to be essential nuclear import and replication [154]. In addition to these, IN can directly interact with the KPN β1, NUP153 [159], Pom121 [160] or hCG1 [161] who has possible interactions with IN and Vpr and can facilitate nuclear import [162].

#### **6. Summary**

region. Even though PIC formation can protect the DNA and helps in the nuclear transport, it cannot guide the integration in a proper location in the host genome. A host protein called cellular lens epithelium-derived growth factor (LEDGF/p75) which binds both chromosomal DNA and HIV integrase [142], directs the integration to a location where active transcription takes place under its control [143]. The interaction between the integrase and INI1 stimulates the DNA-joining activity of the integrase and helps to target the viral DNA towards active genes [96, 102, 144]. Presence of a Topo II cleavage site in the HIV-1 promoter and also at 180 bp upstream of the HIV-1 integration site [145, 146] and association of these Topo II isoforms with HIV-1 PICs [128]. Based on the available information, it can be derived that, IN alone can carry out the integration reaction but for the selection of the proper location in the host genome for the integration and success full HIV-1 gene transcription, Integrase required the support

Transportation of the PICs to the nucleus can be divided in two parts. One is transportation to the nuclear periphery and second is from periphery to inside the nucleus. After completion of the successful cell surface attachment, capsid internalization and degradation in cytoplasm which leads to the reverse transcription of the viral genome RNA to DNA and the formation of the pre-integration complex to protect the DNA from the host nucleases takes place. This nucleoprotein PICs should travel from the cytosol to the nucleus to form provirus which is an

The PICs in the cytoplasm translocate to the perinuclear compartment by the cytoplasmic movements by the cytoskeleton. Actin and microtubule [149] selectively plays a role in this transport with the help of Myosin VI and Dynein [148]. The dynein complex proteins such as dynein light chain1 (DYNLL1), Tctex1 and Dynactin have been shown to be involved in this process. But very little is known about how HIV-1 targets cytoskeleton. Many of the viral elements found in association with the PIC have been proposed to be important for HIV-1

Studies on the HIV-1 infection in dividing and non-dividing cells provided enough evidences to believe that PICs enter the nucleoplasm by crossing the nuclear envelope through nuclear pore complexes (NPCs), which form stable channels through the nuclear envelope and gatekeep the trafficking of molecules between the nucleus and cytoplasm [150]. The RTC/PIC with the size of~100-250nm [149, 151, 152] cannot cross the nuclear pore. So, only few important

For nuclear transport, KPNs and NUPs functions as carrier proteins by binding to the integrase. KPN α adaptor proteins importin α1 (Rch1) [153] and importin α3 (KPNA4) [154], to which KPN β1 proteins bound additionally. Importin 7 [155, 156] and transportin 3 [157, 158] are recruited by the nuclear localization signal (NLS) present on IN. In both dividing and nondividing cells Impα3 which interacts with IN is found to be essential nuclear import and replication [154]. In addition to these, IN can directly interact with the KPN β1, NUP153 [159], Pom121 [160] or hCG1 [161] who has possible interactions with IN and Vpr and can facilitate

components of the PIC may enter into the nucleus [149, 152].

integrated viral DNA in host chromatin and can produce progeny virus [147, 148].

of the host factors.

32 Advances in Molecular Retrovirology

nuclear import.

nuclear import [162].

**5.1. Transport of PICs into nucleus**

After 33 years of the identification of HIV-1 infection, very little is known about the role of host cellular proteins. Till now considerable work has been done in the area of host–pathogen interactions facilitated by the viral proteins and host receptors. The role of the main receptor like CD4 and co-receptors like CCR5, CXCR4 and their alternative receptors were well studied with the role of their signaling in disease progression. But the intracellular events of the host– pathogen interactions were poorly understood. Much data is available based on the global wide analysis of genome-wide RNA interference screens, yeast two-hybrid system and coimmunoprecipitation studies but their exact roles were not yet characterized. There are very few host proteins like APOBEC3G, LEDGF/p75, INI1, HMG I(Y) and BAF, which were well studied and characterized. Majority of the reported proteins were attributed with multiple functions. It is very useful to study such proteins to develop as future candidates to HIV-1 therapy.

#### **Author details**

Lokeswara S. Balakrishna and Anand K. Kondapi\*

\*Address all correspondence to: akondapi@gmail.com

Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, P O Central University, Hyderabad, Telangana, India

#### **References**


[21] Cicala, C., et al., *Induction of phosphorylation and intracellular association of CC chemokine receptor 5 and focal adhesion kinase in primary human CD4+ T cells by macrophage-tropic HIV envelope.* J Immunol, 1999. 163(1): p. 420-6.

[6] Clapham, P.R. and A. McKnight, *HIV-1 receptors and cell tropism.* Br Med Bull, 2001.

[7] Popik, W., J.E. Hesselgesser, and P.M. Pitha, *Binding of human immunodeficiency virus type 1 to CD4 and CXCR4 receptors differentially regulates expression of inflammatory genes and activates the MEK/ERK signaling pathway.* J Virol, 1998. 72(8): p. 6406-13. [8] Popik, W. and P.M. Pitha, *Binding of human immunodeficiency virus type 1 to CD4 indu‐ ces association of Lck and Raf-1 and activates Raf-1 by a Ras-independent pathway.* Mol Cell

[9] Ravichandran, K.S., T.L. Collins, and S.J. Burakoff, *CD4 and signal transduction.* Curr

[10] Power, C.A. and T.N. Wells, *Cloning and characterization of human chemokine receptors.*

[11] Baribaud, F., et al., *Antigenically distinct conformations of CXCR4.* J Virol, 2001. 75(19):

[12] Lee, B., et al., *Epitope mapping of CCR5 reveals multiple conformational states and distinct but overlapping structures involved in chemokine and coreceptor function.* J Biol Chem,

[13] Picard, L., et al., *Role of the amino-terminal extracellular domain of CXCR-4 in human im‐*

[14] Doranz, B.J., et al., *Identification of CXCR4 domains that support coreceptor and chemokine*

[15] Kwong, P.D., et al., *Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody.* Nature, 1998. 393(6686): p. 648-59. [16] Davis, C.B., et al., *Signal transduction due to HIV-1 envelope interactions with chemokine*

[17] Hesselgesser, J., et al., *CD4-independent association between HIV-1 gp120 and CXCR4: functional chemokine receptors are expressed in human neurons.* Curr Biol, 1997. 7(2): p.

[18] Weissman, D., et al., *Macrophage-tropic HIV and SIV envelope proteins induce a signal*

[19] Marchese, A., et al., *G protein-coupled receptor sorting to endosomes and lysosomes.* Annu

[20] Viola, A. and A.D. Luster, *Chemokines and their receptors: drug targets in immunity and*

*through the CCR5 chemokine receptor.* Nature, 1997. 389(6654): p. 981-5.

*inflammation.* Annu Rev Pharmacol Toxicol, 2008. 48: p. 171-97.

*munodeficiency virus type 1 entry.* Virology, 1997. 231(1): p. 105-11.

*receptors CXCR4 or CCR5.* J Exp Med, 1997. 186(10): p. 1793-8.

58: p. 43-59.

34 Advances in Molecular Retrovirology

p. 8957-67.

112-21.

Biol, 1996. 16(11): p. 6532-41.

1999. 274(14): p. 9617-26.

Top Microbiol Immunol, 1996. 205: p. 47-62.

Trends Pharmacol Sci, 1996. 17(6): p. 209-13.

*receptor functions.* J Virol, 1999. 73(4): p. 2752-61.

Rev Pharmacol Toxicol, 2008. 48: p. 601-29.


[50] Seddiki, N., et al., *A monoclonal antibody directed to sulfatide inhibits the binding of hu‐ man immunodeficiency virus type 1 (HIV-1) envelope glycoprotein to macrophages but not their infection by the virus.* Biochim Biophys Acta, 1994. 1225(3): p. 289-96.

[36] Murphy, P.M., et al., *International union of pharmacology. XXII. Nomenclature for chemo‐*

[37] Fredriksson, R., et al., *The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints.* Mol Pharmacol,

[38] Marechal, V., et al., *Opposite effects of SDF-1 on human immunodeficiency virus type 1*

[39] Roggero, R., et al., *Binding of human immunodeficiency virus type 1 gp120 to CXCR4 in‐ duces mitochondrial transmembrane depolarization and cytochrome c-mediated apoptosis in‐*

[40] Lin, G., et al., *Identification of gp120 binding sites on CXCR4 by using CD4-independent human immunodeficiency virus type 2 Env proteins.* J Virol, 2003. 77(2): p. 931-42.

[41] LaBonte, J.A., N. Madani, and J. Sodroski, *Cytolysis by CCR5-using human immunodefi‐ ciency virus type 1 envelope glycoproteins is dependent on membrane fusion and can be in‐*

[42] Sharron, M., et al., *Expression and coreceptor activity of STRL33/Bonzo on primary periph‐*

[43] Lee, S., et al., *CCR8 on human thymocytes functions as a human immunodeficiency virus*

[44] Larkin, M., et al., *Oligosaccharide-mediated interactions of the envelope glycoprotein gp120 of HIV-1 that are independent of CD4 recognition.* AIDS, 1989. 3(12): p. 793-8.

[45] Curtis, B.M., S. Scharnowske, and A.J. Watson, *Sequence and expression of a membraneassociated C-type lectin that exhibits CD4-independent binding of human immunodeficiency virus envelope glycoprotein gp120.* Proc Natl Acad Sci U S A, 1992. 89(17): p. 8356-60. [46] Geijtenbeek, T.B., et al., *DC-SIGN, a dendritic cell-specific HIV-1-binding protein that en‐*

[47] Pohlmann, S., et al., *DC-SIGNR, a DC-SIGN homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans.* Proc

[48] Fantini, J., et al., *Infection of colonic epithelial cell lines by type 1 human immunodeficiency virus is associated with cell surface expression of galactosylceramide, a potential alternative*

[49] Harouse, J.M., et al., *Inhibition of entry of HIV-1 in neural cell lines by antibodies against*

*hibited by high levels of CD4 expression.* J Virol, 2003. 77(12): p. 6645-59.

*kine receptors.* Pharmacol Rev, 2000. 52(1): p. 145-76.

*dependently of Fas signaling.* J Virol, 2001. 75(16): p. 7637-50.

*eral blood lymphocytes.* Blood, 2000. 96(1): p. 41-9.

*type 1 coreceptor.* J Virol, 2000. 74(15): p. 6946-52.

*hances trans-infection of T cells.* Cell, 2000. 100(5): p. 587-97.

*gp120 receptor.* Proc Natl Acad Sci U S A, 1993. 90(7): p. 2700-4.

*galactosyl ceramide.* Science, 1991. 253(5017): p. 320-3.

Natl Acad Sci U S A, 2001. 98(5): p. 2670-5.

*replication.* J Virol, 1999. 73(5): p. 3608-15.

2003. 63(6): p. 1256-72.

36 Advances in Molecular Retrovirology


[81] Cowan, S., et al., *Cellular inhibitors with Fv1-like activity restrict human and simian im‐ munodeficiency virus tropism.* Proc Natl Acad Sci U S A, 2002. 99(18): p. 11914-9.

[65] Miyauchi, K., et al., *HIV enters cells via endocytosis and dynamin-dependent fusion with*

[66] Marechal, V., et al., *Human immunodeficiency virus type 1 entry into macrophages mediat‐*

[67] Carter, G.C., et al., *HIV-1 infects macrophages by exploiting an endocytic route dependent*

[68] Grove, J. and M. Marsh, *The cell biology of receptor-mediated virus entry.* J Cell Biol,

[69] Marsh, M. and R. Bron, *SFV infection in CHO cells: cell-type specific restrictions to pro‐*

[70] Wang, X., et al., *Integrin alphavbeta3 is a coreceptor for human cytomegalovirus.* Nat Med,

[71] Yoder, A., et al., *HIV envelope-CXCR4 signaling activates cofilin to overcome cortical actin*

[72] Taylor, M.P., O.O. Koyuncu, and L.W. Enquist, *Subversion of the actin cytoskeleton dur‐*

[73] Birnbaumer, L., *Expansion of signal transduction by G proteins. The second 15 years or so: from 3 to 16 alpha subunits plus betagamma dimers.* Biochim Biophys Acta, 2007. 1768(4):

[74] Zhang, H., et al., *Morphologic changes in human immunodeficiency virus type 1 virions secondary to intravirion reverse transcription: evidence indicating that reverse transcription*

[75] Fassati, A. and S.P. Goff, *Characterization of intracellular reverse transcription complexes*

[76] Fassati, A. and S.P. Goff, *Characterization of intracellular reverse transcription complexes*

[77] Miller, M.D., C.M. Farnet, and F.D. Bushman, *Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition.* J Virol, 1997. 71(7): p.

[78] Forshey, B.M., et al., *Formation of a human immunodeficiency virus type 1 core of optimal*

[79] Stremlau, M., et al., *The cytoplasmic body component TRIM5alpha restricts HIV-1 infec‐*

[80] Perron, M.J., et al., *TRIM5alpha mediates the postentry block to N-tropic murine leukemia viruses in human cells.* Proc Natl Acad Sci U S A, 2004. 101(32): p. 11827-32.

*may not take place within the intact viral core.* J Hum Virol, 2000. 3(3): p. 165-72.

*of human immunodeficiency virus type 1.* J Virol, 2001. 75(8): p. 3626-35.

*of Moloney murine leukemia virus.* J Virol, 1999. 73(11): p. 8919-25.

*stability is crucial for viral replication.* J Virol, 2002. 76(11): p. 5667-77.

*tion in Old World monkeys.* Nature, 2004. 427(6977): p. 848-53.

*ductive virus entry at the cell surface.* J Cell Sci, 1997. 110 (Pt 1): p. 95-103.

*endosomes.* Cell, 2009. 137(3): p. 433-44.

2011. 195(7): p. 1071-82.

38 Advances in Molecular Retrovirology

2005. 11(5): p. 515-21.

p. 772-93.

5382-90.

*ed by macropinocytosis.* J Virol, 2001. 75(22): p. 11166-77.

*on dynamin, Rac1 and Pak1.* Virology, 2011. 409(2): p. 234-50.

*restriction in resting CD4 T cells.* Cell, 2008. 134(5): p. 782-92.

*ing viral infection.* Nat Rev Microbiol, 2011. 9(6): p. 427-39.


[112] Li, X.Y., et al., *APOBEC3G inhibits DNA strand transfer during HIV-1 reverse transcrip‐ tion.* J Biol Chem, 2007. 282(44): p. 32065-74.

[97] Rain, J.C., et al., *Yeast two-hybrid detection of integrase-host factor interactions.* Methods,

[98] Sorin, M., et al., *Recruitment of a SAP18-HDAC1 complex into HIV-1 virions and its re‐*

[99] Hamamoto, S., et al., *Identification of a novel human immunodeficiency virus type 1 inte‐ grase interactor, Gemin2, that facilitates efficient viral cDNA synthesis in vivo.* J Virol,

[100] Wang, W., et al., *Diversity and specialization of mammalian SWI/SNF complexes.* Genes

[101] Yung, E., et al., *Specificity of interaction of INI1/hSNF5 with retroviral integrases and its*

[102] Turelli, P., et al., *Cytoplasmic recruitment of INI1 and PML on incoming HIV preintegra‐ tion complexes: interference with early steps of viral replication.* Mol Cell, 2001. 7(6): p.

[103] Roy, B.B., et al., *Association of RNA helicase a with human immunodeficiency virus type 1*

[104] Pellizzoni, L., et al., *A functional interaction between the survival motor neuron complex*

[105] Fan, X.C. and J.A. Steitz, *Overexpression of HuR, a nuclear-cytoplasmic shuttling protein, increases the in vivo stability of ARE-containing mRNAs.* EMBO J, 1998. 17(12): p.

[106] Myer, V.E., X.C. Fan, and J.A. Steitz, *Identification of HuR as a protein implicated in*

[107] Vakalopoulou, E., J. Schaack, and T. Shenk, *A 32-kilodalton protein binds to AU-rich do‐ mains in the 3' untranslated regions of rapidly degraded mRNAs.* Mol Cell Biol, 1991.

[108] Lemay, J., et al., *HuR interacts with human immunodeficiency virus type 1 reverse tran‐ scriptase, and modulates reverse transcription in infected cells.* Retrovirology, 2008. 5: p.

[109] Bishop, K.N., et al., *APOBEC3G inhibits elongation of HIV-1 reverse transcripts.* PLoS

[110] Guo, F., et al., *Inhibition of tRNA(3)(Lys)-primed reverse transcription by human APO‐ BEC3G during human immunodeficiency virus type 1 replication.* J Virol, 2006. 80(23): p.

[111] Guo, F., et al., *The interaction of APOBEC3G with human immunodeficiency virus type 1 nucleocapsid inhibits tRNA3Lys annealing to viral RNA.* J Virol, 2007. 81(20): p. 11322-31.

*quirement for viral replication.* PLoS Pathog, 2009. 5(6): p. e1000463.

*functional significance.* J Virol, 2004. 78(5): p. 2222-31.

*particles.* J Biol Chem, 2006. 281(18): p. 12625-35.

*and RNA polymerase II.* J Cell Biol, 2001. 152(1): p. 75-85.

*AUUUA-mediated mRNA decay.* EMBO J, 1997. 16(8): p. 2130-9.

2009. 47(4): p. 291-7.

40 Advances in Molecular Retrovirology

2006. 80(12): p. 5670-7.

1245-54.

3448-60.

47.

11710-22.

11(6): p. 3355-64.

Pathog, 2008. 4(12): p. e1000231.

Dev, 1996. 10(17): p. 2117-30.


[141] Chen, H. and A. Engelman, *The barrier-to-autointegration protein is a host factor for HIV type 1 integration.* Proc Natl Acad Sci U S A, 1998. 95(26): p. 15270-4.

[126] Kondapi, A.K., et al., *A biochemical analysis of topoisomerase II alpha and beta kinase activ‐ ity found in HIV-1 infected cells and virus.* Arch Biochem Biophys, 2005. 441(1): p. 41-55.

[127] Ponraj, K., et al., *HIV-1 associated Topoisomerase IIbeta kinase: a potential pharmacological*

[128] Kondapi, A.K., N. Satyanarayana, and A.D. Saikrishna, *A study of the topoisomerase II activity in HIV-1 replication using the ferrocene derivatives as probes.* Arch Biochem Bio‐

[129] Filion, L.G., et al., *Inhibition of HIV-1 replication by daunorubicin.* Clin Invest Med, 1993.

[130] Bouille, P., et al., *Antisense-mediated repression of DNA topoisomerase II expression leads to an impairment of HIV-1 replicative cycle.* J Mol Biol, 1999. 285(3): p. 945-54.

[131] Farnet, C.M. and F.D. Bushman, *HIV-1 cDNA integration: requirement of HMG I(Y) pro‐ tein for function of preintegration complexes in vitro.* Cell, 1997. 88(4): p. 483-92.

[132] Strebel, K., J. Luban, and K.T. Jeang, *Human cellular restriction factors that target HIV-1*

[133] Malim, M.H., *APOBEC proteins and intrinsic resistance to HIV-1 infection.* Philos Trans

[134] Luo, K., et al., *Cytidine deaminases APOBEC3G and APOBEC3F interact with human im‐ munodeficiency virus type 1 integrase and inhibit proviral DNA formation.* J Virol, 2007.

[135] Wang, X., et al., *The cellular antiviral protein APOBEC3G interacts with HIV-1 reverse transcriptase and inhibits its function during viral replication.* J Virol, 2012. 86(7): p.

[136] Miyakawa, K., et al., *ASK1 restores the antiviral activity of APOBEC3G by disrupting*

[137] Leng, J., et al., *A cell-intrinsic inhibitor of HIV-1 reverse transcription in CD4(+) T cells*

[138] Khiytani, D.K. and N.J. Dimmock, *Characterization of a human immunodeficiency virus type 1 pre-integration complex in which the majority of the cDNA is resistant to DNase I*

[139] Lin, C.W. and A. Engelman, *The barrier-to-autointegration factor is a component of func‐ tional human immunodeficiency virus type 1 preintegration complexes.* J Virol, 2003. 77(8):

[140] Schweitzer, C.J., et al., *Proteomic analysis of early HIV-1 nucleoprotein complexes.* J Pro‐

*HIV-1 Vif-mediated counteraction.* Nat Commun, 2015. 6: p. 6945.

*from elite controllers.* Cell Host Microbe, 2014. 15(6): p. 717-28.

*digestion.* J Gen Virol, 2002. 83(Pt 10): p. 2523-32.

teome Res, 2013. 12(2): p. 559-72.

*target for viral replication.* Curr Pharm Des, 2013. 19(26): p. 4776-86.

phys, 2006. 450(2): p. 123-32.

*replication.* BMC Med, 2009. 7: p. 48.

R Soc Lond B Biol Sci, 2009. 364(1517): p. 675-87.

16(5): p. 339-47.

42 Advances in Molecular Retrovirology

81(13): p. 7238-48.

3777-86.

p. 5030-6.


## **Molecular Mechanisms Controlling HIV Transcription and Latency – Implications for Therapeutic Viral Reactivation**

Michael D. Röling, Mateusz Stoszko and Tokameh Mahmoudi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61948

#### **Abstract**

[157] Christ, F., et al., *Transportin-SR2 imports HIV into the nucleus.* Curr Biol, 2008. 18(16):

[158] Larue, R., et al., *Interaction of the HIV-1 intasome with transportin 3 protein (TNPO3 or*

[159] Woodward, C.L., et al., *Integrase interacts with nucleoporin NUP153 to mediate the nucle‐ ar import of human immunodeficiency virus type 1.* J Virol, 2009. 83(13): p. 6522-33. [160] Fouchier, R.A., et al., *Interaction of the human immunodeficiency virus type 1 Vpr protein*

[161] Le Rouzic, E., et al., *Docking of HIV-1 Vpr to the nuclear envelope is mediated by the inter‐*

[162] Matreyek, K.A. and A. Engelman, *Viral and cellular requirements for the nuclear entry of retroviral preintegration nucleoprotein complexes.* Viruses, 2013. 5(10): p. 2483-511.

*action with the nucleoporin hCG1.* J Biol Chem, 2002. 277(47): p. 45091-8.

*TRN-SR2).* J Biol Chem, 2012. 287(41): p. 34044-58.

*with the nuclear pore complex.* J Virol, 1998. 72(7): p. 6004-13.

p. 1192-202.

44 Advances in Molecular Retrovirology

Persistence of transcriptionally silent replication competent HIV-1 is a major barrier to clearance of the virus from patients; current combinatorial antiretroviral therapies are successful in abrogating active viral replication, but are unable to eradicate latent HIV-1. A "shock and kill" strategy has been proposed as a curative approach in which latent vi‐ rus is activated and infected cells are removed by immune clearance, while new rounds of infection are prevented by antiretroviral therapy. Much effort has been put toward un‐ derstanding the molecular mechanisms maintaining HIV latency and the nature of reser‐ voirs, to provide novel therapeutic targets. This has led to the development of latency reversal agents (LRAs), some of which are undergoing clinical trials. Targeting multiple mechanisms underlying HIV latency via a combination of LRAs is likely to result in more potent activation of the latent reservoir. Therefore, novel as well as synergistic combina‐ tions of therapeutic molecules are required to accomplish more potent latency reversal.

**Keywords:** HIV-1 latency, Latency reversal agents (LRAs), Combinatorial antiretroviral therapy

#### **1. Introduction**

Human immunodeficiency virus-1 (HIV-1) is a lentivirus, a subgroup of Retroviridae. Like all retroviruses, HIV-1 virions consist of an RNA genome with viral proteins encapsulated in a viral envelope. The viral proteins execute key steps to establish a productive infection by stably integrating into the host genome. Unlike most retroviruses, HIV-1 can also directly infect nondividing cells. HIV-1 preferably infects a subset of T-lymphocytes (CD4+ T-cells) that play a crucial role in the immune response. HIV-1 infection causes exhaustion and ultimately

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

depletion of the host immune system, a syndrome termed acquired immuno-deficiency syndrome (AIDS). HIV-1 came into prominence with the outbreak of the AIDS epidemic in the 1980s. Major steps have been taken toward treating this viral infection. In particular, combi‐ natorial antiretroviral therapy (cART) successfully abrogated HIV-1 replication. Thus, for compliant patients with access to c-ART, HIV infection has become a chronic rather than a lethal disease. However, cessation of antiretroviral therapy results in viral rebound in infected patients, even after years of cART. This is because in a small fraction of infected cells, HIV persists in a latent but replication-competent state. Latent HIV is unaffected by cART, but infection can rebound upon cART interruption. Therefore, HIV latency is the main challenge in developing a curative therapy for HIV.

The quest for an HIV-1 cure involves the development of either a sterilizing or a functional cure. A sterilizing cure would require complete removal of replication competent viral genetic material from the infected patient and thus the stable depletion of latently HIV-infected cells. A functional cure, on the other hand, requires the patient's immune system to suppress HIV-1 replication life-long in the absence of cART without disease progression, loss of CD4+ T cells and HIV transmission. The functional cure does not aim to eradicate the virus entirely from the patient. Both the sterilizing and functional cure strategies are currently the subject of major research efforts.

#### **2. Clinical picture of HIV**

The AIDS epidemic in the 1980s led to the identification of HIV as the causative agent. AIDS is a condition in which depletion of CD4+ T-cells overtime leads to the loss of the host immune system's ability to fight infections and cancers, eventually leading to death. As HIV was identified as the causative agent, cure efforts focused on disrupting the viral lifecycle. In the early 1990s, the first antiretroviral therapies – monotherapies – had limited success as they resulted in rebound of viremia due to the appearance of resistant viral strains. Resistant HIV required novel therapeutic strategies. Therefore, a combination of anti-retrovirals, targeting distinct steps of the viral life cycle was developed, so-called combinatorial antiretroviral therapy (cART). cART has proven to be extremely successful in lowering the amount of viral RNA in plasma below the limits of detection by standard laboratory techniques. Unfortunately, the therapy does not eradicate the virus as cessation of medication causes re-emergence of viral replication [1–3]. Thus, a fraction of the virus escapes the effects of cART. The source for this recurring viral replication is a small pool of latently infected cells that harbor integrated proviruses which, while silent, are not recognized by either the immune system nor are they subject to cART. Moreover, HIV can persist in the presence of cART in certain anatomical sites if drug penetrance is incomplete.

According to the World Health Organization (WHO), the number of HIV-infected individuals worldwide in late 2014 was estimated to be approximately 37 million [4]. The vast majority of infected people live in sub-Saharan Africa, where access to appropriate diagnostic centers and cART is limited. Estimates put new infections at 5,600 a day in 2014.

#### **2.1. HIV-1 replication cycle and state-of-the-art antiretroviral therapy**

depletion of the host immune system, a syndrome termed acquired immuno-deficiency syndrome (AIDS). HIV-1 came into prominence with the outbreak of the AIDS epidemic in the 1980s. Major steps have been taken toward treating this viral infection. In particular, combi‐ natorial antiretroviral therapy (cART) successfully abrogated HIV-1 replication. Thus, for compliant patients with access to c-ART, HIV infection has become a chronic rather than a lethal disease. However, cessation of antiretroviral therapy results in viral rebound in infected patients, even after years of cART. This is because in a small fraction of infected cells, HIV persists in a latent but replication-competent state. Latent HIV is unaffected by cART, but infection can rebound upon cART interruption. Therefore, HIV latency is the main challenge

The quest for an HIV-1 cure involves the development of either a sterilizing or a functional cure. A sterilizing cure would require complete removal of replication competent viral genetic material from the infected patient and thus the stable depletion of latently HIV-infected cells. A functional cure, on the other hand, requires the patient's immune system to suppress HIV-1 replication life-long in the absence of cART without disease progression, loss of CD4+ T cells and HIV transmission. The functional cure does not aim to eradicate the virus entirely from the patient. Both the sterilizing and functional cure strategies are currently the subject of major

The AIDS epidemic in the 1980s led to the identification of HIV as the causative agent. AIDS is a condition in which depletion of CD4+ T-cells overtime leads to the loss of the host immune system's ability to fight infections and cancers, eventually leading to death. As HIV was identified as the causative agent, cure efforts focused on disrupting the viral lifecycle. In the early 1990s, the first antiretroviral therapies – monotherapies – had limited success as they resulted in rebound of viremia due to the appearance of resistant viral strains. Resistant HIV required novel therapeutic strategies. Therefore, a combination of anti-retrovirals, targeting distinct steps of the viral life cycle was developed, so-called combinatorial antiretroviral therapy (cART). cART has proven to be extremely successful in lowering the amount of viral RNA in plasma below the limits of detection by standard laboratory techniques. Unfortunately, the therapy does not eradicate the virus as cessation of medication causes re-emergence of viral replication [1–3]. Thus, a fraction of the virus escapes the effects of cART. The source for this recurring viral replication is a small pool of latently infected cells that harbor integrated proviruses which, while silent, are not recognized by either the immune system nor are they subject to cART. Moreover, HIV can persist in the presence of cART in certain anatomical sites

According to the World Health Organization (WHO), the number of HIV-infected individuals worldwide in late 2014 was estimated to be approximately 37 million [4]. The vast majority of infected people live in sub-Saharan Africa, where access to appropriate diagnostic centers and

cART is limited. Estimates put new infections at 5,600 a day in 2014.

in developing a curative therapy for HIV.

46 Advances in Molecular Retrovirology

research efforts.

**2. Clinical picture of HIV**

if drug penetrance is incomplete.

HIV-1, as all viruses, is a parasite of the host cell and hijacks key cellular processes to establish a productive infection. To produce new virions, the virus goes through a viral replication cycle. HIV's replication cycle consists of entering the cell by docking at the cell surface receptor CD4 and co-receptors CCR5/CXCR5 and fusing to the cell, un-packaging of the genome, reverse transcription of the viral RNA genome into double-stranded DNA, which is the main compo‐ nent of the pre-integration complex, followed by integration of the double-stranded DNA genome into the host genome, transcription of the provirus, translation of viral proteins, and ultimately virion biogenesis followed by budding from host cell and maturation. Modern cART targets most steps in the HIV viral replication cycle (Figure 1). There are currently 28 approved agents for the treatment of HIV infection [5]. They fall into six mechanistic major classes, which act at different stages in the HIV replication cycle:


These antivirals comprise the various current cART regimens that are used in the clinic. cART has proven to be extremely successful in suppressing viral replication in compliant patients. In fact, it has been argued that the theoretical potential of cART has already been reached [6]. Therefore, in the developed world with access to medication, HIV has become a chronic and not a lethal disease.

#### **2.2. The burden of lifelong cART**

Implementation of cART has provided long-term suppression of viral replication, improving the life expectancy and life quality of infected patients. Unfortunately, the economic burden of cART is debilitating. According to the Centers for Disease Control and Prevention (CDC), lifetime costs of treating HIV infection is estimated to be \$379,668 per infected individual in the United States [7].

Moreover, patients on cART overtime can experience several side effects of cART such as: cardiovascular diseases (e.g., myocardial infarction); non-AIDS cancers (e.g., anal cancer, liver cancer, Hodgkin's disease); liver, kidney, and bone disease as well as neurologic complications, such as dementia [8]. Interestingly, most of these conditions are associated with the ageing process. Hence, it is thought, that HIV infection controlled by cART accelerates ageing. And importantly, HIV persists in a latent state that is not targeted by cART, rendering cART a therapeutic management of the disease as opposed to a curative treatment. Thus, there is much need to develop a curative therapy for HIV.

**Figure 1.** The viral replication cycle can be targeted pharmacologically at different stages

#### **2.3. Clinical latency**

The first step in finding a cure for HIV-1 infection is to identify the main source of cells that carry silenced, replication-competent HIV-1. Therefore, it is critical to define which cells or anatomical compartments constitute a reservoir of latent but replication-competent HIVinfected cells.

HIV-1 infects cells expressing the cell surface CD4 receptor and either of the co-receptors CCR5 or CXCR4. These cells include T helper cells, monocytes, macrophages, and dendritic cells. *In vivo,* HIV infects mostly activated CD4+ T-cells as quiescent and resting CD4+ T-cells are less permissive to infection due to low expression of CD4 and CCR5, and minimal metabolism [9– 12]. The low metabolism is characterized by low levels of available dNTPs for reverse tran‐ scription and lack of energy sources [13–17]. Additionally, the cortical actin barrier in resting cells is thought to inhibit virus entry, reverse transcription and nuclear import [18,19]. However, the biggest pool of latently infected cells comprises resting memory CD4+ T-cells. It is thought that these latent infections are predominantly generated while activated infected cells revert back to a resting memory state [20–22]. During this process, as the genome of the (partially) activated cell condenses and is silenced in transition to a memory state, so does the HIV genome [14,15]. There is also evidence for direct infection of resting cells by HIV, resulting in the generation of a latent infection [23]. Studying these cells in patients is challenging as the frequency of latently infected cells in suppressed patients is very low, estimated to be 1 latent cell per 1 million of uninfected cells [24,25]. Due to the long half-life of a latently infected resting memory CD4+ T-cells (estimated at 44 months), cART would take over 70 years in order to eradicate HIV from the infected patient [6,26,27].

Naive T-cells are also found to be latently infected; however, the frequency of such cells is even smaller than resting memory cells [28]. Interestingly, the naive T-cell reservoir may increase over time in suppressed individuals due to high proliferation of these cells compared to resting memory cells [29].

HIV is found also in cells of monocyte/macrophage lineage such as macrophages in brain and lung sections of infected individuals on anti-retroviral therapy [30,31]. However, proviral transcription occurs in these cells at low levels; therefore, it is debatable whether these cells are part of the latent reservoir [32,33].

Among the anatomical compartments affected by HIV-1, the central nervous system (CNS) and gut-associated lymphoid tissues (GALT) are two major sites [34–36]. The source of infection in the CNS is most likely infected monocytes, which are able to cross the blood– brain barrier as the virus itself cannot [37–39]. Approximately 5-10 times more HIV-1 RNA can be obtained from GALT than from blood cells in patients receiving cART [40,41], potentially indicative of lower penetrance of cART in cells within this anatomical site. However, the contribution of these compartments to rebound of viremia after cART cessation remains controversial [42,43].

#### **2.4. Clinical proof-of-concepts for HIV-1 eradication**

Moreover, patients on cART overtime can experience several side effects of cART such as: cardiovascular diseases (e.g., myocardial infarction); non-AIDS cancers (e.g., anal cancer, liver cancer, Hodgkin's disease); liver, kidney, and bone disease as well as neurologic complications, such as dementia [8]. Interestingly, most of these conditions are associated with the ageing process. Hence, it is thought, that HIV infection controlled by cART accelerates ageing. And importantly, HIV persists in a latent state that is not targeted by cART, rendering cART a therapeutic management of the disease as opposed to a curative treatment. Thus, there is much

need to develop a curative therapy for HIV.

48 Advances in Molecular Retrovirology

**2.3. Clinical latency**

infected cells.

**Figure 1.** The viral replication cycle can be targeted pharmacologically at different stages

The first step in finding a cure for HIV-1 infection is to identify the main source of cells that carry silenced, replication-competent HIV-1. Therefore, it is critical to define which cells or anatomical compartments constitute a reservoir of latent but replication-competent HIV-

HIV-1 infects cells expressing the cell surface CD4 receptor and either of the co-receptors CCR5 or CXCR4. These cells include T helper cells, monocytes, macrophages, and dendritic cells. *In vivo,* HIV infects mostly activated CD4+ T-cells as quiescent and resting CD4+ T-cells are less permissive to infection due to low expression of CD4 and CCR5, and minimal metabolism [9– 12]. The low metabolism is characterized by low levels of available dNTPs for reverse tran‐ scription and lack of energy sources [13–17]. Additionally, the cortical actin barrier in resting cells is thought to inhibit virus entry, reverse transcription and nuclear import [18,19]. However, the biggest pool of latently infected cells comprises resting memory CD4+ T-cells. Thus far, only one patient, the so-called Berlin patient, was cured from HIV-1 after receiving treatment for acute myeloid leukemia [44,45]. HIV eradication in this patient was accomplished after several rounds of radio- and chemotherapy, total body irradiation, and two hematopoietic stem cell (HSC) transplantations from a donor bearing homozygous thirty-two base pair deletion in the CCR5 co-receptor gene (CCR5Δ32) were performed. The mutant CCR5 impedes viral entry of R5 tropic viruses in the first phase of the infection [46–49]. It is estimated that between 1% and 15% of the European Caucasian population harbor this mutation, while it occurs less frequently in African and Asian populations [47, 48]. In this patient, cART was ceased a day before the first transplant and after 7 years, no viremia or other indications of viral replication have been detectable [52].

Following the success of the case of the "Berlin patient", two HIV-1-positive patients, the "Boston patients", received HSCs transplants after developing Hodgkin's lymphoma [53]. Both patients carried heterozygous CCR5Δ32 mutation. While still under cART regimen, no viral production was observed which led to cessation of therapy. Unfortunately, after several months, strong viral rebound occurred in these patients. Follow-up analysis pointed to the likely presence of a small refractory source of cells, which is thought to have seeded the viral rebound; phylogenic studies revealed that only a few latent proviruses contributed to the viral rebound [53]. Several other similar studies have been conducted with infected patients suffering from either leukemias or lymphomas who received autologous or allogenic HSC transplantation alongside cART as a strategy to deplete the latent pool of cells. However, in most of these studies, viral rebound was detected following therapy interruption [54].

In another case, the Mississippi baby, an infant presumably infected *in utero*, received cART 30 h after birth. As newborns do not have resting memory CD4+ T-cells, it was reasoned that cART will prevent establishment of the latent reservoir – the main impediment in eradication strategies. One month after therapy, viremia reached undetectable levels and cART was stopped after 18 months. Unfortunately, 2 years post therapy interruption, rebound of viremia was detected (52, http://www.niaid.nih.gov/news/newsreleases/2014/pages/mississippibaby‐ hiv.aspx).

The immune system of rare "elite controllers" maintains low HIV-1 plasma levels, without the need of medication for many years. Although the capability of these patient to control viral replication is not completely understood, their circulating myeloid dendritic cells and CD8+ T-cells are more effective in depletion of infected CD4 T-cells [56–61]. Interestingly, the ARNS VISCONTI cohort showed that cessation of long-term cART, started during the acute phase of HIV-1 infection, resulted in post-treatment control (PST) of infection. Fourteen of the studied individuals were able to keep or even further reduce the viral reservoir. Furthermore, these individuals were able to maintain long-lasting, low level of viremia [62]. Recently, a perinatally infected baby displayed more than 11 years of HIV-1 remission. At 3 months of age, plasma HIV-RNA reached 2.1 x 106 copies/ml, and cART was administered for about 5–6 years. At 6.8 years of age, no HIV-1 RNA was detectable and cART was discontinued. After more than 12 years, plasma viremia still remains undetectable [63]. Therefore, this case provides the first evidence that early initiated, long-term cART can result in stable and durable HIV-1 remission.

Data from the Berlin and Boston patients provided a rationale for the creation of HIV-resistant cells. Since the CCR5Δ32 homozygous mutation is not lethal and not associated with abnormal immune functions [52], many approaches to silence the CCR5 gene have been or are under investigation [64–67]. These studies all employ genome editing technologies such as tran‐ scription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats (CRISPRs) or zinc-finger nucleases (ZNFs), which target the genome with high specificity and introduce deletions in the sequence of interest, in this case in the DNA sequence of CCR5 or/and CXCR4 co-receptors [64,65,68]. The rationale for this approach is based on the notion that cells bearing mutated CCR5 protein are not permissive to infection with R5 HIV-1 viruses, while cells with a mutated CXCR4 are resistant to C4 viruses. The double knock-out of both CCR5 and CXCR4 would allow resistance to infection regardless of viral tropism. However, the safety of such an approach remains to be elucidated. Uninfected HSCs isolated from infected individuals are engineered with either technology and then transfused back into patients. The ZNF approach targeting CCR5 has shown some promising results, although the sizes of cohorts used have been small. Gene-modified cells persisted in patients over 9 months, and cells seemed to expand and undergo trafficking to other tissues [66]. An increase in CD4+ T-cell counts was observed in all individuals. Importantly HIV-1 DNA in the blood decreased. The encouraging outcome of this study has resulted in phase II clinical trials.

Another gene therapy-based approach is the introduction of HIV-1 expression-dependent suicide genes encoding either toxic or pro-apoptotic proteins such as members of the Bcl-2 protein family. Constructs that are responsive to Tat and Rev viral proteins were tested [69]. While obtaining encouraging results, activity of such suicide genes only affects cells that are actively producing viruses, thus the latent pool of cells would still be unaffected.

Despite many attempts at HIV-1 cure, thus far only two cases, the "Berlin patient" and the early treated infant have resulted in eradication [44,45,63]. Due to safety and economic issues associated with transplantation and gene therapy approaches, broad use of such a therapeutic approach is not feasible for HIV cure. Moreover, the gene therapy approach provides a functional rather than sterilizing cure. Nevertheless, all these studies provided valuable insights into the biology of the latent reservoirs. They constitute a proof-of-concept for HIV-1 cure. Moreover, it seems that immediate initiation of cART contributes to restricting the establishment of the latent pool.

These studies highlight the need for more robust, cheaper, and feasible treatments in order to achieve HIV-1 eradication among all infected individuals. In 2004, the concept of so-called "shock and kill" or "kick and kill" therapy was proposed [70–72]. The aim is to specifically reactivate proviruses in latently infected cells ("shock") and eliminate the infected cells via viral cytophatic effects or/and render the cells susceptible to immune clearance ("kill"). New rounds of infection would be prevented by cART. "Shock and kill" therapy relies on the identification of potent and specific latency reversal agents (LRAs) alongside induction of an effective immune response against the reactivated latent pool of cells. The LRAs currently under investigation do not result in sufficient reactivation of latent HIV *in vivo*. Therefore, novel molecules that specifically reactivate latent HIV-1 are urgently needed.

#### **3. Model systems and assays to detect and study HIV-1**

To study the complex nature of HIV-1 latency, reliable model systems are required that recapitulate the nature and dynamics of the latent reservoir in vivo. Several cell lines of lymphocytic or monocytic lineage, primary-cell models, as well as animal models, are used to study HIV latency [73].

#### **3.1. Cell lines**

months, strong viral rebound occurred in these patients. Follow-up analysis pointed to the likely presence of a small refractory source of cells, which is thought to have seeded the viral rebound; phylogenic studies revealed that only a few latent proviruses contributed to the viral rebound [53]. Several other similar studies have been conducted with infected patients suffering from either leukemias or lymphomas who received autologous or allogenic HSC transplantation alongside cART as a strategy to deplete the latent pool of cells. However, in most of these studies, viral rebound was detected following therapy interruption [54].

In another case, the Mississippi baby, an infant presumably infected *in utero*, received cART 30 h after birth. As newborns do not have resting memory CD4+ T-cells, it was reasoned that cART will prevent establishment of the latent reservoir – the main impediment in eradication strategies. One month after therapy, viremia reached undetectable levels and cART was stopped after 18 months. Unfortunately, 2 years post therapy interruption, rebound of viremia was detected (52, http://www.niaid.nih.gov/news/newsreleases/2014/pages/mississippibaby‐

The immune system of rare "elite controllers" maintains low HIV-1 plasma levels, without the need of medication for many years. Although the capability of these patient to control viral replication is not completely understood, their circulating myeloid dendritic cells and CD8+ T-cells are more effective in depletion of infected CD4 T-cells [56–61]. Interestingly, the ARNS VISCONTI cohort showed that cessation of long-term cART, started during the acute phase of HIV-1 infection, resulted in post-treatment control (PST) of infection. Fourteen of the studied individuals were able to keep or even further reduce the viral reservoir. Furthermore, these individuals were able to maintain long-lasting, low level of viremia [62]. Recently, a perinatally infected baby displayed more than 11 years of HIV-1 remission. At 3 months of age, plasma HIV-RNA reached 2.1 x 106 copies/ml, and cART was administered for about 5–6 years. At 6.8 years of age, no HIV-1 RNA was detectable and cART was discontinued. After more than 12 years, plasma viremia still remains undetectable [63]. Therefore, this case provides the first evidence that early initiated, long-term cART can result in stable and durable HIV-1 remission.

Data from the Berlin and Boston patients provided a rationale for the creation of HIV-resistant cells. Since the CCR5Δ32 homozygous mutation is not lethal and not associated with abnormal immune functions [52], many approaches to silence the CCR5 gene have been or are under investigation [64–67]. These studies all employ genome editing technologies such as tran‐ scription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats (CRISPRs) or zinc-finger nucleases (ZNFs), which target the genome with high specificity and introduce deletions in the sequence of interest, in this case in the DNA sequence of CCR5 or/and CXCR4 co-receptors [64,65,68]. The rationale for this approach is based on the notion that cells bearing mutated CCR5 protein are not permissive to infection with R5 HIV-1 viruses, while cells with a mutated CXCR4 are resistant to C4 viruses. The double knock-out of both CCR5 and CXCR4 would allow resistance to infection regardless of viral tropism. However, the safety of such an approach remains to be elucidated. Uninfected HSCs isolated from infected individuals are engineered with either technology and then transfused back into patients. The ZNF approach targeting CCR5 has shown some promising results, although the sizes of cohorts used have been small. Gene-modified cells persisted in

hiv.aspx).

50 Advances in Molecular Retrovirology

Immortalized cell lines of T-cell and monocytic origin are cost-effective and easy to use in the study of latent HIV. They allow fast read-outs in large scale for mechanistic molecular characterization of HIV gene expression. Therefore, cell lines are an attractive platform for screening and mechanistic characterization of LRAs. To generate a latent cell line, cells must first be latently infected with a HIV derived virus. Several different HIV derived viruses are used ranging from full length to minimal virus and can make use of a wide range of reporter constructs (e.g. GFP or luciferase). The viral Tat/TAR axis is of vital importance for the transcriptional regulation of HIV and can be included or excluded from the viral construct used. Latent infection of relevant cell lines derived from T-cells or monocytic lineage, depend‐ ing on reservoir of interest generate cell lines that can be used to study the molecular mecha‐ nisms of HIV latency [74–78].

Ach-2 and U1 cells are characterized by low expression of HIV-1, which can be strongly upregulated upon TNFα or mitogens stimulation [74,79]. However, in these cell lines, latency results from mutations in Tat protein (U1 cell-line) or in RNA stem loop TAR (Ach-2) [76,77]. Therefore, these cell lines do not represent complexity of latency found *in vivo*, however, they do allow Tat/TAR-independent HIV-1 reactivation investigation.

A more appropriate system to study latency are J-Lat cell lines derived from Jurkat cells of Tlymphocytic origin [78, 80,81]. These cells have integrated replication-competent full-length or minimal proviral constructs with an intact promoter and Tat-TAR axis, a *GFP* reporter gene replaces the *Nef* sequence in full-length proviruses or is located downstream of *Tat* in minimal proviruses [78].

These cell lines have been extremely useful to delineate the molecular requirements of HIV transcription activation and silencing. Although useful for molecular analysis and screening platforms, the cell line model systems of HIV latency also present some limitations; first, clonal cell lines are derived from a single integration event, and therefore do not reflect the diverse distribution of integration sites in the host chromatin [82,83]. Consistently, results vary depending on the cell lines used, indicating possible clonal cell line effects [84]. Due to the above mentioned limitations and the considerable difference between cell line models and primary cells in terms of proliferative capacity, genomic stability and mechanisms involved in establishing and maintaining latency, generally latency models based on primary cells are preferable.

#### **3.2. Primary cells**

To more closely resemble infection *in vivo* and validate putative LRAs more accurately, several primary cell models have been developed. Depending on the cell status at infection, these models can be divided into two groups.

The first group relies on purification of CD4+ T-cells from healthy donors, that are then activated and subsequently infected. Depending on the method, CD4+T-cells are purified and stimulated with a-CD3/IL-2 [85], a-CD3/aCD-28 [86], a-CD3/aCD-28/IL-2 [87], or Ag-MDDC (antigen-loaded monocyte-derived dendritic cells; [88]), and infected with virus. Productively infected cells die due to virus-induced apoptosis or become latent by reverting back to a resting state. To limit infection to only one replication cycle, replication-defective viruses or antire‐ troviral drugs are also used. The rationale for these systems rely on the notion that a portion of activated, infected CD4+ T-cells transition to a quiescent state, shutting down general transcription and slowing down metabolism, resulting in latency [6,25,28,89–91]. Depending on the method used, different populations of latently infected cells are generated for use in reactivation studies. In the methods suggested by Sahu and Marini central memory T (TCM) cells remain in culture, in Yang's protocol mainly effector memory T (TEM) cells are produced, in Bosque and Planelles's method cells phenotype resembles central memory-like (TCM). The main disadvantage of these methods is the time needed to obtain results, which varies from 1 to 4 months. Furthermore, they are labor-intensive and technically challenging.

The second group uses direct infection of resting memory CD4+ T-cells, which immediately after integration become latent. Cells are infected after purification and can be used after several days for reactivation studies [90, 91]. Stimulation of CCR7, CXCR3, or CCR6 receptors increases the susceptibility of resting memory CD4+ T-cells to infection without T-cell activa‐ tion. In the methods of Swiggard and Lassen, central memory T (TCM) and effector memory T (TEM) cells are the source of latent HIV-1; in Saleh's method naïve resting memory T-cells, in addition to TCM and TEM cells, constitute the latent pool. The main advantage of these methods is the time needed to evaluate the potency of putative LRA, as results can be obtained within one week.

Depending on the protocol used, the amounts of cells that become latent differ from as little as 1% to up to 40%. In models where cells are activated, on average more latently infected cells are generated. Using these models, we can quantify the level of reactivation of HIV-1 in a reliable manner by measuring the production of the viral protein p24 by enzyme-linked immunosorbent assay (ELISA) or quantification of viral transcription by quantitative RT-PCR, or by detection of GFP/luciferase in case of reporter-based constructs.

A novel detection method distinguishes uninfected, productively infected, and latently infected cells using a dual reporter system. A modified HIV-1 derived genome containing GFP as a reporter of viral transcriptional activity and mCherry under an EF1a promoter as a reporter of infection (latent or productive) allows easy isolation of the different cell populations [23].

Ultimately, the golden standard for testing activity of LRAs are primary cells from infected individuals under cART obtained by leukophoresis, a process in which white blood cells are specifically isolated while other blood components are reverted back to the patients' circula‐ tory system. The isolated cells are uninfected, latently infected, and infected with defective viruses. Large amounts of CD4+ T-cells are required and isolated from patients for testing LRAs.

The development of primary cell models greatly improved the quest for LRAs, yet results differ between each model system [84]. No *in vitro* models completely recapitulate the full range of latent cells *in vivo*; instead, only a small sub-fraction of latently infected cells is represented. Hence, the validation process of putative LRAs requires testing on cells derived from infected individuals [93].

#### **3.3. Animal models of HIV-1 infection**

first be latently infected with a HIV derived virus. Several different HIV derived viruses are used ranging from full length to minimal virus and can make use of a wide range of reporter constructs (e.g. GFP or luciferase). The viral Tat/TAR axis is of vital importance for the transcriptional regulation of HIV and can be included or excluded from the viral construct used. Latent infection of relevant cell lines derived from T-cells or monocytic lineage, depend‐ ing on reservoir of interest generate cell lines that can be used to study the molecular mecha‐

Ach-2 and U1 cells are characterized by low expression of HIV-1, which can be strongly upregulated upon TNFα or mitogens stimulation [74,79]. However, in these cell lines, latency results from mutations in Tat protein (U1 cell-line) or in RNA stem loop TAR (Ach-2) [76,77]. Therefore, these cell lines do not represent complexity of latency found *in vivo*, however, they

A more appropriate system to study latency are J-Lat cell lines derived from Jurkat cells of Tlymphocytic origin [78, 80,81]. These cells have integrated replication-competent full-length or minimal proviral constructs with an intact promoter and Tat-TAR axis, a *GFP* reporter gene replaces the *Nef* sequence in full-length proviruses or is located downstream of *Tat* in minimal

These cell lines have been extremely useful to delineate the molecular requirements of HIV transcription activation and silencing. Although useful for molecular analysis and screening platforms, the cell line model systems of HIV latency also present some limitations; first, clonal cell lines are derived from a single integration event, and therefore do not reflect the diverse distribution of integration sites in the host chromatin [82,83]. Consistently, results vary depending on the cell lines used, indicating possible clonal cell line effects [84]. Due to the above mentioned limitations and the considerable difference between cell line models and primary cells in terms of proliferative capacity, genomic stability and mechanisms involved in establishing and maintaining latency, generally latency models based on primary cells are

To more closely resemble infection *in vivo* and validate putative LRAs more accurately, several primary cell models have been developed. Depending on the cell status at infection, these

The first group relies on purification of CD4+ T-cells from healthy donors, that are then activated and subsequently infected. Depending on the method, CD4+T-cells are purified and stimulated with a-CD3/IL-2 [85], a-CD3/aCD-28 [86], a-CD3/aCD-28/IL-2 [87], or Ag-MDDC (antigen-loaded monocyte-derived dendritic cells; [88]), and infected with virus. Productively infected cells die due to virus-induced apoptosis or become latent by reverting back to a resting state. To limit infection to only one replication cycle, replication-defective viruses or antire‐ troviral drugs are also used. The rationale for these systems rely on the notion that a portion of activated, infected CD4+ T-cells transition to a quiescent state, shutting down general transcription and slowing down metabolism, resulting in latency [6,25,28,89–91]. Depending

do allow Tat/TAR-independent HIV-1 reactivation investigation.

nisms of HIV latency [74–78].

52 Advances in Molecular Retrovirology

proviruses [78].

preferable.

**3.2. Primary cells**

models can be divided into two groups.

The number of animal models available to study latency is limited. The toxicity of putative LRAs can be assessed with use of mouse and non-human primate (NHP) models [94]. Two mouse models have been developed and used in HIV latency studies: the humanized SCID (SCID-hu) mouse, transplanted with human thymus and liver fragments, and the humanized blood, liver, and thymus (BLT) mouse which has a human immune system with full mucosal immunity [95–97]. Unfortunately, SCID-hu mice do not express human proteins involved in the viral replication cycle; therefore, the study of HIV-1 in these mice is restricted to events taking place within organs of human origin in this model. In addition, HIV-1 is not responsive to cART in these animals. BLT mice are a better model of HIV-1 infection, as they produce resting memory CD4+ T-cells of human origin. However, some components of cART do not repress replication in BLT mice [34].

NHP models employ the Simian immunodeficiency virus (SIV) infection in rhesus and pig tailed macaques to recapitulate HIV-1 infection in humans [98,99]. NHP models allow the monitoring of the spread of infection. Moreover, infection in this model can be controlled by antiretroviral therapy. NHP models are helpful in studying the first stages of latency estab‐ lishment, as investigating this part of HIV-1 infection is extremely challenging in patients, as the pool of latently infected cells is established early during infection [100]. One caveat to the use of SIV-based NHP models of HIV latency is that the viral 5′LTR or promoter of SIV is considerably different in sequence from HIV-1 [101] and therefore latent SIV response to LRAs, which is a direct consequence of promoter-mediated transcription activation may vary substantially from latent HIV-1. In addition, animal models are far more expensive than cellbased systems. Nor do they fully reflect human infection or metabolism. Finally, ethical concerns are inherent to the use of NHP models of HIV latency.

#### **3.4. Detection of the latent reservoir**

The study of latent HIV infection requires accurate measurement of the size of the latent reservoir and the extent of reactivation following LRA treatment. Depending on the experi‐ mental aim, different detection methods can be employed. These methods generally rely on PCR, protein quantification, or reporter detection.

The quantitative viral outgrowth assay (QVOA) is a well-established method to estimate the latent pool. The assay relies on the use of serial dilutions of cells obtained from an infected individual in co-culture with uninfected cells that are permissive to infection. Viral proteins are detected by ELISA. Unfortunately, QVOA is time-consuming, costly, and might generate false-negative results as not all replication-competent proviruses are reactivated, and thus not detected [83].

The HIV reservoir can be approximated by detecting the number of viral DNA copies present in the cells. The recently introduced digital droplet PCR (ddPCR) improves on classic and nested qRT-PCR by simultaneously amplifying thousands of nanoliter reactions in combina‐ tion with very sensitive detection system based on flow cytometry [94,102,103]. ddPCR is therefore superior to nested qRT-PCR in its ability to resolve rare events such as latent HIV-1. Although PCR based methods provide increased sensitivity for the detection of viral genetic material, these approaches also detect defective proviruses, which results in false-positive results.

Another recent PCR-based method for reservoir detection evades false positive results from defective proviruses. The *Tat/rev* induced limiting diluting assay (TILDA) relies on PCR amplification of multiply spliced RNA (msRNA) of *tat*/*rev* transcripts that are present in productively infected cells and absent in latent infection [104]. Small amounts of cells isolated from patients are divided into two equal parts and distributed in limiting dilution. One half is left unstimulated while the other is activated with PMA/Ionomycin. After 12 hours, cells are lysed and subjected to ultrasensitive nested RT-PCR. By employing statistical modeling, the frequency of cells that are expressing msRNA in both groups is estimated and based on the unstimulated group a threshold of activation can be set. Using the TILDA assay, the size of the reservoir is estimated at 24 cells per million, which is more than measured by QVOA but less than measured by PCR methods [24,83,104]. The assay more accurately estimates the true size of the latent reservoir, is highly sensitive, reproducible, fast, relatively inexpensive, and requires only 10 mL of patients' blood. However, a limitation on the TILDA assay is that it detects the presence of viral transcripts but not the production or release of infectious viral particles; therefore, it may still overestimate the true size of the reservoir, yet to a smaller extent than other PCR-based methods. Additionally, signal detection relies on amplification of highly variable region of the HIV-1 DNA; therefore, detection of all subspecies of HIV-1 might be challenging and require extra optimization steps.

Unfortunately, all current methods to detect latent HIV-1 have limitations. First, the pool of latently infected cells in patients is extremely low, resulting in a high noise-to-signal ratio. Furthermore, defective or hyper-mutated proviruses are detectable by PCR-based techniques, yet irrelevant for eradication strategies. Moreover, not all replication-competent proviruses are inducible in the first round of treatment, yet get reactivated upon subsequent rounds of stimulation [83]. Thus, assays to measure latency reversal are overestimating – in the case of PCR-based methods – or underestimating – in the case of QVOA – the latent pool. This poses a main problem in measuring efficiency of the reactivation of HIV-1. A captivating approach employing the use of a biomarker (e.g., gene), which responds to treatment in the same way as HIV-1, would allow more easily quantifiable assessments as to whether latent HIV in patient cells would be responsive to a particular treatment.

#### **4. Molecular mechanisms of latency**

(SCID-hu) mouse, transplanted with human thymus and liver fragments, and the humanized blood, liver, and thymus (BLT) mouse which has a human immune system with full mucosal immunity [95–97]. Unfortunately, SCID-hu mice do not express human proteins involved in the viral replication cycle; therefore, the study of HIV-1 in these mice is restricted to events taking place within organs of human origin in this model. In addition, HIV-1 is not responsive to cART in these animals. BLT mice are a better model of HIV-1 infection, as they produce resting memory CD4+ T-cells of human origin. However, some components of cART do not

NHP models employ the Simian immunodeficiency virus (SIV) infection in rhesus and pig tailed macaques to recapitulate HIV-1 infection in humans [98,99]. NHP models allow the monitoring of the spread of infection. Moreover, infection in this model can be controlled by antiretroviral therapy. NHP models are helpful in studying the first stages of latency estab‐ lishment, as investigating this part of HIV-1 infection is extremely challenging in patients, as the pool of latently infected cells is established early during infection [100]. One caveat to the use of SIV-based NHP models of HIV latency is that the viral 5′LTR or promoter of SIV is considerably different in sequence from HIV-1 [101] and therefore latent SIV response to LRAs, which is a direct consequence of promoter-mediated transcription activation may vary substantially from latent HIV-1. In addition, animal models are far more expensive than cellbased systems. Nor do they fully reflect human infection or metabolism. Finally, ethical

The study of latent HIV infection requires accurate measurement of the size of the latent reservoir and the extent of reactivation following LRA treatment. Depending on the experi‐ mental aim, different detection methods can be employed. These methods generally rely on

The quantitative viral outgrowth assay (QVOA) is a well-established method to estimate the latent pool. The assay relies on the use of serial dilutions of cells obtained from an infected individual in co-culture with uninfected cells that are permissive to infection. Viral proteins are detected by ELISA. Unfortunately, QVOA is time-consuming, costly, and might generate false-negative results as not all replication-competent proviruses are reactivated, and thus not

The HIV reservoir can be approximated by detecting the number of viral DNA copies present in the cells. The recently introduced digital droplet PCR (ddPCR) improves on classic and nested qRT-PCR by simultaneously amplifying thousands of nanoliter reactions in combina‐ tion with very sensitive detection system based on flow cytometry [94,102,103]. ddPCR is therefore superior to nested qRT-PCR in its ability to resolve rare events such as latent HIV-1. Although PCR based methods provide increased sensitivity for the detection of viral genetic material, these approaches also detect defective proviruses, which results in false-positive

concerns are inherent to the use of NHP models of HIV latency.

repress replication in BLT mice [34].

54 Advances in Molecular Retrovirology

**3.4. Detection of the latent reservoir**

detected [83].

results.

PCR, protein quantification, or reporter detection.

Although replication-competent, latent HIV is transcriptionally silenced but susceptible to reactivation upon certain stimuli. Following integration into the host genome, transcription from the HIV genome is controlled by key cellular host factors, and subject to host cell gene regulation similar to endogenous genes. Since viral transcription initiation, elongation, and termination are tightly regulated by host proteins, HIV is also widely used as a model system to study gene regulation.

#### **4.1. Host antiretroviral mechanisms thwart infection**

Host defense mechanisms impede HIV-1 infection. Upon entering the cell, HIV's RNA genome is reverse transcribed into double-stranded DNA (dsDNA). This process requires freely available deoxynucleotide triphosphates (dNTPs). By limiting the pool of freely available dNTPs, the nucleotide scavenger SAMHD1 restricts viral replication in non-cycling myeloid cells and quiescent CD4+ T-cells [105–108]. Additionally, SAMHD1 has 3′–5′ exoribonucleases (RNAse) activity that specifically cleaves single-stranded RNA [109,110]. Interestingly, Vpx, encoded by HIV-2 and Simian immunodeficiency virus, is an accessory protein packaged into the virion, which induces SAMHD1 degradation [111].

Additionally, APOBEC3G limits viral replication by catalyzing the deamination of cytidine to uridine in the viral single-stranded DNA (ssDNA) genome during reverse transcription [112]. Interestingly, APOBEC3G is inactive in memory CD4+T-cells, which helps to explain why this cell type is more permissive to HIV-1 infection. Therefore, activated CD4+ T-cells are the main target cell type of HIV infection and of the main source of the latent reservoir.

#### **4.2. Integration of HIV into the host genome required by host factors**

The reverse-transcribed viral DNA genome is incorporated in the pre-integration complex (PIC). The PIC is imported into the nucleus. Host factors identified so far that affect viral integration are lens epithelium-derived growth factor (LEDGF/p75/PSIP1) and hepatomaderived growth factor related protein 2 (HRP- 2/ HDGFRP2), through an integrase binding domain. In the absence of LEDGF, provirus integration is decreased 10-fold and HIV's pattern of integration is altered [113–115]. Simultaneous LEDGF and HRP-2 knockdown further decreases viral replication [116]. Nevertheless, knockdown of both factors does not completely abolish HIV-1 integration, indicating that IN alone and/or in cooperation with other host factors can still integrate the viral genome [117]. PIC nuclear import stimulates export to the cytoplasm of INI-1 and PML, disrupting this effect greatly improves integration efficiency [118–120]. Upon knockdown of transportin-3/TNPO3 and nuclear pore protein RanBP2/Nup35 HIV-1 integrates randomly [121]. Therefore, nuclear import affects the site of integration with a preference for open chromatin.

#### **4.3. Pre-integration vs post-integration latency**

Two states of latency can be defined based on the integration state of HIV: pre-integration latency and post-integration latency. Defects in integration or in a prior phase of the viral replication cycle (e.g., incomplete reverse transcription) might result in unintegrated viral DNA. The half-life of the linear pre-integration complex is approximately 1 day [122]. The linear unintegrated viral DNA can also be circularized, resulting in slightly extended half-life of the virus [123]. In quiescent cells, the pre-integrated virus can reside near the centromere for weeks [124]. Unintegrated virus can replicate, albeit very inefficiently [125]. The half-life of both forms of unintegrated virus is too short and replication inefficient to serve as the source required for the long-term persistence of latent HIV making pre-integration latency less clinically relevant.

Post-integration latency occurs when the HIV virus is stably integrated into the host genome, but a productive infection is not achieved. The site of integration and the abundance of transcription factors are crucial for determining whether an infection will be latent or produc‐ tive. The site of integration will determine the chromatin environment (such as histone modifications), relative position to other genes (intronic insertion vs gene desert) and position within the nucleus of the provirus.

#### **4.4. Integration biases**

available deoxynucleotide triphosphates (dNTPs). By limiting the pool of freely available dNTPs, the nucleotide scavenger SAMHD1 restricts viral replication in non-cycling myeloid cells and quiescent CD4+ T-cells [105–108]. Additionally, SAMHD1 has 3′–5′ exoribonucleases (RNAse) activity that specifically cleaves single-stranded RNA [109,110]. Interestingly, Vpx, encoded by HIV-2 and Simian immunodeficiency virus, is an accessory protein packaged into

Additionally, APOBEC3G limits viral replication by catalyzing the deamination of cytidine to uridine in the viral single-stranded DNA (ssDNA) genome during reverse transcription [112]. Interestingly, APOBEC3G is inactive in memory CD4+T-cells, which helps to explain why this cell type is more permissive to HIV-1 infection. Therefore, activated CD4+ T-cells are the main

The reverse-transcribed viral DNA genome is incorporated in the pre-integration complex (PIC). The PIC is imported into the nucleus. Host factors identified so far that affect viral integration are lens epithelium-derived growth factor (LEDGF/p75/PSIP1) and hepatomaderived growth factor related protein 2 (HRP- 2/ HDGFRP2), through an integrase binding domain. In the absence of LEDGF, provirus integration is decreased 10-fold and HIV's pattern of integration is altered [113–115]. Simultaneous LEDGF and HRP-2 knockdown further decreases viral replication [116]. Nevertheless, knockdown of both factors does not completely abolish HIV-1 integration, indicating that IN alone and/or in cooperation with other host factors can still integrate the viral genome [117]. PIC nuclear import stimulates export to the cytoplasm of INI-1 and PML, disrupting this effect greatly improves integration efficiency [118–120]. Upon knockdown of transportin-3/TNPO3 and nuclear pore protein RanBP2/Nup35 HIV-1 integrates randomly [121]. Therefore, nuclear import affects the site of integration with

Two states of latency can be defined based on the integration state of HIV: pre-integration latency and post-integration latency. Defects in integration or in a prior phase of the viral replication cycle (e.g., incomplete reverse transcription) might result in unintegrated viral DNA. The half-life of the linear pre-integration complex is approximately 1 day [122]. The linear unintegrated viral DNA can also be circularized, resulting in slightly extended half-life of the virus [123]. In quiescent cells, the pre-integrated virus can reside near the centromere for weeks [124]. Unintegrated virus can replicate, albeit very inefficiently [125]. The half-life of both forms of unintegrated virus is too short and replication inefficient to serve as the source required for the long-term persistence of latent HIV making pre-integration latency less

Post-integration latency occurs when the HIV virus is stably integrated into the host genome, but a productive infection is not achieved. The site of integration and the abundance of transcription factors are crucial for determining whether an infection will be latent or produc‐

target cell type of HIV infection and of the main source of the latent reservoir.

**4.2. Integration of HIV into the host genome required by host factors**

the virion, which induces SAMHD1 degradation [111].

56 Advances in Molecular Retrovirology

a preference for open chromatin.

clinically relevant.

**4.3. Pre-integration vs post-integration latency**

The site of integration greatly determines the transcriptional activity of the provirus. HIV preferentially integrates into active genes both in patient material and transformed cell lines [82,126–128]. Moreover, HIV-1 integrates in regions of genome that are in close proximity to nuclear envelope [129]. Latent integrations are in or close to alphoid repeat elements in heterochromatin, whereas productive integrations avoid insertion in or near heterochromatin [78]. Integration is associated with transcription-inducing histone modifications (i.e., H3 & H4 acetylation and H3K4 methylation) but not transcription-inhibiting modifications (i.e., H3K27 trimethylation and DNA CpG methylation) [130]. A comparison of integration sites in resting and activated CD4+ T-cells showed that in both cell types HIV integrates in active genes. However, in activated cells, insertions were enriched for gene dense, CpG island-rich and high G/C-content regions [131]. Latency in infected Jurkat cell lines correlated with integrations in gene deserts, centromeric heterochromatin, and highly expressed cellular genes [128]. Within the nucleus, HIV-1 is located mostly in decondensed chromatin at the nuclear periphery, while it disfavors heterochromatic regions [132]. Interestingly, latent proviruses were found to interact with a pericentromeric region of chromosome 12 in quiescent cells [133]. In a study of viremic progressors and viremic controllers, integration was enriched into, or in close prox‐ imity to, Alu repeats, local hotspots, and silent regions of the genome [134]. In addition, close proximity of the provirus to PML bodies is associated with latency, an association that is lost upon reactivation [135].

#### **4.5. Integration relative to host genes affects transcriptional state of the provirus**

Sense and antisense integration relative to host genes can greatly affect the transcriptional state of HIV. Integration in sense orientation can lead to promoter occlusion, whereas integration in antisense orientation can lead to collision of the transcriptional machinery. Promoter occlusion occurs when the transcriptional machinery is depleted from the viral promoter by a dominant host promoter that is transcribed and negatively affects proviral expression.

Indeed, chimeric transcripts of the host gene and in sense viral integrations were observed [136,137]. Additionally, Han et al. compared the effect of sense and antisense insertions of HIV relative to the active HPRT gene [138]. In this setting, sense integration enhanced viral expression whereas antisense integration (transcriptional collision) led to suppression. Sense integrations were shown to be modestly preferred in latent cells, a preference that was not present in productively infected cells [139]. Transcriptional interference and transcriptional collision are examples of host genes interference with viral expression. On the other hand, reactivation of HIV may lead to suppression of host gene expression [136]. Indeed, in a cell model with a latent integration into the HMBOX1 gene, the host gene was repressed upon viral reactivation [140].

#### **4.6. Viral transcription starts at the 5'LTR**

The provirus is flanked by a 5′ and 3′ long terminal repeats (LTRs). While transcription can be initiated from both LTRs, the 5′ LTR is dominant and serves as the HIV promoter, although 3′ transcription is activated when the 5′ LTR is defective [141]. Transcriptional interference has been proposed as the mechanism by which the 5′ LTR exerts its dominance over the 3′ [142]. Interestingly, low-level antisense transcription takes place at the 3′ LTR, a mechanism by which latency can be maintained [143–147]. Sense transcription results in at least 40 coding transcripts due to alternative splicing of the HIV-1 genome [148]. Finally, both LTRs also act as a source of negative sense transcription, which could potentially affect the expression of neighboring genes [149,150].

#### **4.7. The 5′ LTR contains numerous putative transcription factor binding sites**

HIV-1 encodes a potent trans-activating protein – Tat – that drives viral expression during productive infection. However, initially, before sufficient levels of Tat are expressed, the provirus relies on host factors to initiate transcription. The 5′ LTR contains three regions – U3, R, and U5 (Figure 2) [151]. The R region, immediately next to the transcription start site (TSS), contains the trans-activation response (TAR) element, an important regulator of HIV expres‐ sion. The U3 region contains the core promoter (nucleotides –78 to –1 upstream of TSS), a core enhancer (nucleotides –105 to –79), and a modulator region (nucleotides –454 to –104) [152,153]. The core promoter contains three Sp1 binding sites in tandem, a TATA box, and an initiator element at the transcription start site. The core enhancer contains two NF-kB-binding sites. The modulator region – so-called because early experiments with deletion upstream of the LTR caused increased activity of the LTR – was shown by later experiments to contain binding sites for both repressive and activating factors including nuclear factor of activated Tcells NFAT, STAT5, NF-kB p65/p50 heterodimers, lymphocyte enhancer factor (LEF-1), CCAAT/enhancer binding protein (C/EBP) factors, AP-1, and activating transcription factor/ cyclic AMP response element binding (ATF/CREB) factors (Figure 2) [152,154–162]. It is well established that these transcription factors have binding sites within HIV-1 sequence. More‐ over, they are strong activators of HIV-1 transcription of which NF-κB is considered the most critical [163–166]. In addition to the presence of these sites, bioinformatic tools indicate that this region of the HIV LTR contains a tightly clustered distribution of multiple transcription factor consensus binding elements [167].

#### **4.8. Positive host factors bind to the 5′ LTR**

Initial transcription of HIV-1 is entirely dependent on host factors. Nuclear factor kappa-lightchain-enhancer of activated B cells (NF-kB) is a hetero dimer comprised of p50 and p65 subunits involved in T-cell activation. NF-kB acts as a transcription factor and is a potent activator of HIV-1 transcription initiation and elongation. It interacts and functions coopera‐ tively with numerous proteins. Independent of Tat, NF-kB can reactivate HIV to high expres‐ sion levels [168]. Mutated NF-kB-binding sites on the LTR inhibit basal transcription and Tat transactivation [169]. NF-kB, Sp1, and other factors (LEF-1, Ets1, and TFE-3) bind to sites near NF-kB sites and synergistically activate HIV transcription, even in the presence of repressive Molecular Mechanisms Controlling HIV Transcription and Latency – Implications for Therapeutic Viral Reactivation http://dx.doi.org/10.5772/61948 59

**Figure 2.** The genome of HIV-1

**4.6. Viral transcription starts at the 5'LTR**

58 Advances in Molecular Retrovirology

factor consensus binding elements [167].

**4.8. Positive host factors bind to the 5′ LTR**

genes [149,150].

The provirus is flanked by a 5′ and 3′ long terminal repeats (LTRs). While transcription can be initiated from both LTRs, the 5′ LTR is dominant and serves as the HIV promoter, although 3′ transcription is activated when the 5′ LTR is defective [141]. Transcriptional interference has been proposed as the mechanism by which the 5′ LTR exerts its dominance over the 3′ [142]. Interestingly, low-level antisense transcription takes place at the 3′ LTR, a mechanism by which latency can be maintained [143–147]. Sense transcription results in at least 40 coding transcripts due to alternative splicing of the HIV-1 genome [148]. Finally, both LTRs also act as a source of negative sense transcription, which could potentially affect the expression of neighboring

HIV-1 encodes a potent trans-activating protein – Tat – that drives viral expression during productive infection. However, initially, before sufficient levels of Tat are expressed, the provirus relies on host factors to initiate transcription. The 5′ LTR contains three regions – U3, R, and U5 (Figure 2) [151]. The R region, immediately next to the transcription start site (TSS), contains the trans-activation response (TAR) element, an important regulator of HIV expres‐ sion. The U3 region contains the core promoter (nucleotides –78 to –1 upstream of TSS), a core enhancer (nucleotides –105 to –79), and a modulator region (nucleotides –454 to –104) [152,153]. The core promoter contains three Sp1 binding sites in tandem, a TATA box, and an initiator element at the transcription start site. The core enhancer contains two NF-kB-binding sites. The modulator region – so-called because early experiments with deletion upstream of the LTR caused increased activity of the LTR – was shown by later experiments to contain binding sites for both repressive and activating factors including nuclear factor of activated Tcells NFAT, STAT5, NF-kB p65/p50 heterodimers, lymphocyte enhancer factor (LEF-1), CCAAT/enhancer binding protein (C/EBP) factors, AP-1, and activating transcription factor/ cyclic AMP response element binding (ATF/CREB) factors (Figure 2) [152,154–162]. It is well established that these transcription factors have binding sites within HIV-1 sequence. More‐ over, they are strong activators of HIV-1 transcription of which NF-κB is considered the most critical [163–166]. In addition to the presence of these sites, bioinformatic tools indicate that this region of the HIV LTR contains a tightly clustered distribution of multiple transcription

Initial transcription of HIV-1 is entirely dependent on host factors. Nuclear factor kappa-lightchain-enhancer of activated B cells (NF-kB) is a hetero dimer comprised of p50 and p65 subunits involved in T-cell activation. NF-kB acts as a transcription factor and is a potent activator of HIV-1 transcription initiation and elongation. It interacts and functions coopera‐ tively with numerous proteins. Independent of Tat, NF-kB can reactivate HIV to high expres‐ sion levels [168]. Mutated NF-kB-binding sites on the LTR inhibit basal transcription and Tat transactivation [169]. NF-kB, Sp1, and other factors (LEF-1, Ets1, and TFE-3) bind to sites near NF-kB sites and synergistically activate HIV transcription, even in the presence of repressive

**4.7. The 5′ LTR contains numerous putative transcription factor binding sites**

chromatin structures [170,171]. NF-kB and AP-1, a heterodimer of proteins from the c-Fos, c-Jun, ATF, and JDP families, cooperatively trans-activated LTR activity through the ERK1/ERK2 mitogen-activated protein kinase (MAPK) pathway [161]. Acetylation of Lys310 in NF-kB p65 subunit is an activating mark that is removed by NAD+-dependent protein deacetylases SIRT1 and SIRT2 [172]. Tat positively affects NF-kB by inhibiting SIRT1 and stimulating degradation of IkB, a protein that sequesters NF-kB in the cytoplasm [169,173]. The viral nucleocapsid (NC) protein enhances NF-kB-mediated activity by interacting with the LTR [174]. p65 recruits THIIH which is part of the preinitiation complex and its subunit CDK7 with kinase activity activates CDK9, resulting in increased HIV transcription [175,176]. The cell surface receptor OX40, bound by its ligand gp34, activates transcription from 5′ LTR, in a manner dependent on the presence of NF-kB-binding sites on the LTR [177]. The transcription factor E2F-1, a regulator of S-phase gene expression, inhibits LTR transcription through the recruitment of p50 at the NF-kB-binding sites on the LTR [178].

Members of the SV40-promoter (Sp) specific transcription factor family regulate LTR activity. Sp1 and Sp4 are activators of HIV-1 [179]. Expression of Sp transcription factors changes during monocytic maturation, suggesting differences in susceptibility to LTR activation during differentiation [180].

Nuclear factor of activated T-cells (NFAT) can induce LTR activity in T-cells [155]. NFAT recruits HATs through CBP/p300, which results in reactivation of HIV-1 transcription [181]. The Janus kinase (JAK)/signal transducers and activators of transcription (STAT5) can stimulate or inhibit HIV transcription. STAT5 binds to its binding sites in the U3 enhancer region on the LTR where it promotes transcription [156]. In response to a broad range of cytokines (e.g., IL-2, IL-7, IL-15) and granulocyte-macrophage colony-stimulating factor (GM- CSF) JAK-mediated phosphorylation of a C-terminal tyrosine residue activates STAT5A and STAT5B. Homodimers or heterodimers of activated STAT5A and STAT5B translocate to the nucleus to stimulate HIV expression [182,183]. Interestingly, STAT5Δ, an isoform of STAT5 truncated on the C-terminus, acts as a repressor of LTR activity [184]. Indeed, in the promon‐ ocytic cell line U1 high levels of STAT5Δ are present. Upon stimulation with GM-CSF, STAT5Δ blocks RNAPII from binding to LTR U3 region, inhibiting activity of HIV promoter [185]. STAT5Δ promotes p50 homodimers binding to the LTR, contributing to latency main‐ tenance [186].

In monocytes and macrophages, CCAAT/enhancer binding protein (C/EBP) factors are crucial for activation of HIV-1 [160,187–189]. C/EBP, a member of the bZIP superfamily, contains a DNA-binding domain and a leucine zipper for homo- and heterodimerizations. Similar to Sp-1, levels of C/EBP vary during myeloid development [190]. Interestingly, the HIV-1 LTR contains several C/EBP binding sites [159].

Some studies employing mutagenesis of binding sites for activator protein-1 (AP-1) within proviral genome showed that AP-1 transcription factor is the crucial activator of proviral transcription, as proviruses with altered AP-1-binding sites were less prone to reactivation even if treated with strong activator such as phorbol 12-myristate 13-acetate – PMA [191]. Furthermore, the latent pool was bigger in cells infected with a virus carrying a deletion in AP-1 sites, implicating that the AP-1 protein is necessary for successful provirus transcription [192]. Heterodimeric protein AP-1 is formed upon phosphorylation od c-Jun N-terminal kinase (JNK) in JNK/MAPK pathway [193]. It is well established that activation of TLR signaling induces nuclear localization of NF-kB and AP-1 mediated via JNK pathway [194–196].

In addition to the already mentioned host factors, the potent viral trans-activating protein Tat and to a lesser extent the multifunctional viral protein, viral protein R (Vpr), positive‐ ly affect viral transcription. Productive infection requires the presence of Tat. Exogenous expression of Tat rescues HIV from latency [197]. A defective Tat mutant (C22G) is incapable of full-length viral expression [198]. Additionally, the Tat mutant (H13L) is more prone to establish latency [197]. Tat recruits the positive transcription elongation factor b (P-TEFb), which shifts RNAPII promoter proximal pausing to transcriptional elongation leading to a productive infection [199,200]. P-TEFb consists of CDK9, a serine/threonine kinase, and CyclinT1. The N-terminal cystein-rich region of Tat (Cy22-Cy37) binds to CycT1 through Zn2+-mediated interactions [201–203].

Vpr is a multifunctional HIV-1 protein that plays a role in nuclear import of the PIC and cell cycle arrest in proliferating cells. Vpr also activates LTR activity through multiple mechanisms. Vpr recruits p300 to the 5′ LTR increasing acetylation, resulting in HIV-1 transcription [204]. Moreover, Vpr interacts with Sp1 and TFIIB, part of the transcription initiation complex, stimulating proviral transcription [204–206].

#### **4.9. Repressive host factors at the 5′ LTR**

Not all host transcription factors have an activating effect on LTR activity (Figure 3). YY1 and LSF recognize binding sequences in the LTR and repress transcription through epigenetic modification [207]. C-promoter binding factor-1 (CBF-1) also represses HIV through epigenetic silencing [208,209]. c-Myc recruits an epigenetic silencing factor to repress HIV-1 [210].

CSF) JAK-mediated phosphorylation of a C-terminal tyrosine residue activates STAT5A and STAT5B. Homodimers or heterodimers of activated STAT5A and STAT5B translocate to the nucleus to stimulate HIV expression [182,183]. Interestingly, STAT5Δ, an isoform of STAT5 truncated on the C-terminus, acts as a repressor of LTR activity [184]. Indeed, in the promon‐ ocytic cell line U1 high levels of STAT5Δ are present. Upon stimulation with GM-CSF, STAT5Δ blocks RNAPII from binding to LTR U3 region, inhibiting activity of HIV promoter [185]. STAT5Δ promotes p50 homodimers binding to the LTR, contributing to latency main‐

In monocytes and macrophages, CCAAT/enhancer binding protein (C/EBP) factors are crucial for activation of HIV-1 [160,187–189]. C/EBP, a member of the bZIP superfamily, contains a DNA-binding domain and a leucine zipper for homo- and heterodimerizations. Similar to Sp-1, levels of C/EBP vary during myeloid development [190]. Interestingly, the HIV-1 LTR contains

Some studies employing mutagenesis of binding sites for activator protein-1 (AP-1) within proviral genome showed that AP-1 transcription factor is the crucial activator of proviral transcription, as proviruses with altered AP-1-binding sites were less prone to reactivation even if treated with strong activator such as phorbol 12-myristate 13-acetate – PMA [191]. Furthermore, the latent pool was bigger in cells infected with a virus carrying a deletion in AP-1 sites, implicating that the AP-1 protein is necessary for successful provirus transcription [192]. Heterodimeric protein AP-1 is formed upon phosphorylation od c-Jun N-terminal kinase (JNK) in JNK/MAPK pathway [193]. It is well established that activation of TLR signaling induces nuclear localization of NF-kB and AP-1 mediated via JNK pathway [194–196].

In addition to the already mentioned host factors, the potent viral trans-activating protein Tat and to a lesser extent the multifunctional viral protein, viral protein R (Vpr), positive‐ ly affect viral transcription. Productive infection requires the presence of Tat. Exogenous expression of Tat rescues HIV from latency [197]. A defective Tat mutant (C22G) is incapable of full-length viral expression [198]. Additionally, the Tat mutant (H13L) is more prone to establish latency [197]. Tat recruits the positive transcription elongation factor b (P-TEFb), which shifts RNAPII promoter proximal pausing to transcriptional elongation leading to a productive infection [199,200]. P-TEFb consists of CDK9, a serine/threonine kinase, and CyclinT1. The N-terminal cystein-rich region of Tat (Cy22-Cy37) binds to CycT1 through

Vpr is a multifunctional HIV-1 protein that plays a role in nuclear import of the PIC and cell cycle arrest in proliferating cells. Vpr also activates LTR activity through multiple mechanisms. Vpr recruits p300 to the 5′ LTR increasing acetylation, resulting in HIV-1 transcription [204]. Moreover, Vpr interacts with Sp1 and TFIIB, part of the transcription initiation complex,

Not all host transcription factors have an activating effect on LTR activity (Figure 3). YY1 and LSF recognize binding sequences in the LTR and repress transcription through epigenetic

tenance [186].

60 Advances in Molecular Retrovirology

several C/EBP binding sites [159].

Zn2+-mediated interactions [201–203].

stimulating proviral transcription [204–206].

**4.9. Repressive host factors at the 5′ LTR**

Transcription factors initiate LTR activity, but full-length transcripts are not produced because transcription elongation is inhibited. DRB sensitivity-inducing factor (DSIF), a heterodimer composed of hSpt4 and hSpt5 proteins, induces capping of RNA from newly initiated transcription complexes [211]. The subunit hSpt5 interacts directly with nascent RNA as it appears from the RNAPII exit site and recruits negative elongation factor (NELF) (Figure 3) [212–214]. Escape of transcripts from the promoter proximal pause site is prevented by NELF, which induces termination of transcription over several hundred bases [215]. Moreover, the binding sequence of NELF subunit E recognizes a homologous sequence on TAR, increasing association of NELF with the LTR, which results in transcription silencing. Indeed, experi‐ ments where NELF is knocked down show higher basal HIV transcription and reactivation from latency [216–218].

A novel, RNA interference independent, mechanism mediated by microprocessor and termination factors causes transcriptional silencing and chromatin remodeling at the HIV-1 promoter [219]. Microprocessor binds to TAR, which is then cleaved by Drosha into two RNAs, a 5′-end and 3′-end product. The 5′ is further processed in an Rrp6-dependent manner into a transcription repressing RNA species. The 3′ RNA recruits termination factor Xrn2 and Setx, which induces RNAPII pausing and premature termination of transcription [219].

**Figure 3.** Molecular mechanisms in latent and productive HIV-1 infection

#### **4.10. Host factors induce transcriptional initiation, but not elongation**

While some host transcription factors recruit RNAPII, in the absence of Tat, transcription elongation does not occur resulting in the generation of short abortive transcripts by promoter proximal pausing [220,221]. These ~60nt transcripts include TAR, which has a stem-loop structure and binds near the HIV 5′LTR, inhibiting RNA-polymerase. TAR directly binds Tat, which recruits transcriptional elongation complex to the LTR [222].

#### **4.11. Tat-dependent transcription leads to productive infection**

If cells become activated or due to leaky transcription, Tat can be produced. Tat binding to P-TEFb induces significant conformational changes in P-TEFb, allowing Tat and CycT1 to cooperatively recognize and stably bind TAR [200,223].

Tat-P-TEFb phosphorylates NELF-E resulting in the dissociation of NELF from TAR and the paused RNAPII complex [214,216,218,224]. CDK9 phosphorylates RNAPII at the carboxyl terminal domain (CTD) at Ser2 and Ser5 residues of the 52 heptad repeats, which regulates progression to the elongation phase of transcription [225–227]. The phosphorylation status determines regular and alternative RNA splicing and the 3′ end recruitment of polyadenyla‐ tion factors [228,229]. Ser2 phosphorylation of the RNAPII CTD recruits splicing-associated c-Ski-interacting protein, SKIP, and stimulates elongation transcription and alternative splicing of the Tat-specific splice site through interactions with U5snRNP proteins and tri-snRNP110K [230].

Phosphorylation of hSpt5, a subunit of DSIF, by CDK9 converts it into a positive elongation factor that prevents nascent RNA from breaking of from the transcription complex prema‐ turely and inhibits pausing of RNAPII at arrest sites [231,232]. By removing several blocks Tat-P-TEFb induces transcriptional elongation as well as co-transcriptional processing. During active transcription elongation, increased recruitment of RNAPII to TSS maintains a stable level of RNAPII at the promoter proximal region [218]. Throughout transcription, Tat-P-TEFb remains associated with the elongating transcription machinery [231,233,234].

#### **4.12. P-TEFb can be recruited in active and inactive form in the nucleus by Tat**

In activated T-cells, inactive P-TEFb predominantly resides in the 7SK small nuclear ribonu‐ cleoprotein (snRNP) complex (Figure 3) [235–237]. The 7SK snRNP complex consists of 7SK snRNA, HEXIM1 (or its homolog HEXIM2), the La-related protein 7 (LARP7), and the 7SKspecific 5′ methylphosphate capping enzyme (MePCE). The snRNA functions as a scaffold: it binds two units of P-TEFb and one HEXIM1/2 homo-/heterodimers [238,239]. MePCE and LARP7 protect the 7SK RNA from nuclease degradation, MePCE binds the 5′ end, LARP7 the polyuridine 3′ end [240–242]. Tat disrupts the interaction between pTEFb and HEXIM1/7SK snRNA and recruits P-TEFb to 5′ LTR, resulting in active transcription [226].

BRD4 can also recruit P-TEFb from 7SK snRNP [241,243], to promote transcription. Due to similarities in their C-terminal P-TEFb interacting domains [244], Tat and BRD4 compete for P-TEFb [245,246]. In a latent model, knockdown of BRD4 results in Tat-dependent reactivation of HIV-1 [247].

Bromodomain and extra-terminal domain family of proteins (BET) play an important role in repression of the HIV-1 transcription. BET proteins are responsible for the recruitment of P- TEFb to transcribed genes [246,248]. BRD4 competes with viral protein Tat for binding site on pTEFb, and it represses HIV-1 transcription [245,246]. Knockdown of BRD2 indicates this protein contributes to the maintenance of latency. These results are consistent with the notion that BRD2 is binding to remodeling factors such as HDACs [249,250].

P-TEFb can be recruited to transcription complexes by other factors. CTIP2 recruits P-TEFb by binding HEXIM1 and negatively regulates the complex by repressing the CDK9 kinase activity of P-TEFb [251]. Phosphorylation of HEXIM1 at Tyr271 and Tyr 274 decreases retention of P-TEFb in the 7SK RNP [252]. Additionally, through the binding of nascent RNA, SRSF2 and P-TEFb are released from the 7SK complex and induce transcription elongation in a manner similar to TAR/Tat-mediated recruitment of P-TEFb [253].

#### **4.13. P-TEFb is a subunit of the super elongation complex**

proximal pausing [220,221]. These ~60nt transcripts include TAR, which has a stem-loop structure and binds near the HIV 5′LTR, inhibiting RNA-polymerase. TAR directly binds Tat,

If cells become activated or due to leaky transcription, Tat can be produced. Tat binding to P-TEFb induces significant conformational changes in P-TEFb, allowing Tat and CycT1 to

Tat-P-TEFb phosphorylates NELF-E resulting in the dissociation of NELF from TAR and the paused RNAPII complex [214,216,218,224]. CDK9 phosphorylates RNAPII at the carboxyl terminal domain (CTD) at Ser2 and Ser5 residues of the 52 heptad repeats, which regulates progression to the elongation phase of transcription [225–227]. The phosphorylation status determines regular and alternative RNA splicing and the 3′ end recruitment of polyadenyla‐ tion factors [228,229]. Ser2 phosphorylation of the RNAPII CTD recruits splicing-associated c-Ski-interacting protein, SKIP, and stimulates elongation transcription and alternative splicing of the Tat-specific splice site through interactions with U5snRNP proteins and tri-snRNP110K

Phosphorylation of hSpt5, a subunit of DSIF, by CDK9 converts it into a positive elongation factor that prevents nascent RNA from breaking of from the transcription complex prema‐ turely and inhibits pausing of RNAPII at arrest sites [231,232]. By removing several blocks Tat-P-TEFb induces transcriptional elongation as well as co-transcriptional processing. During active transcription elongation, increased recruitment of RNAPII to TSS maintains a stable level of RNAPII at the promoter proximal region [218]. Throughout transcription, Tat-P-TEFb

In activated T-cells, inactive P-TEFb predominantly resides in the 7SK small nuclear ribonu‐ cleoprotein (snRNP) complex (Figure 3) [235–237]. The 7SK snRNP complex consists of 7SK snRNA, HEXIM1 (or its homolog HEXIM2), the La-related protein 7 (LARP7), and the 7SKspecific 5′ methylphosphate capping enzyme (MePCE). The snRNA functions as a scaffold: it binds two units of P-TEFb and one HEXIM1/2 homo-/heterodimers [238,239]. MePCE and LARP7 protect the 7SK RNA from nuclease degradation, MePCE binds the 5′ end, LARP7 the polyuridine 3′ end [240–242]. Tat disrupts the interaction between pTEFb and HEXIM1/7SK

BRD4 can also recruit P-TEFb from 7SK snRNP [241,243], to promote transcription. Due to similarities in their C-terminal P-TEFb interacting domains [244], Tat and BRD4 compete for P-TEFb [245,246]. In a latent model, knockdown of BRD4 results in Tat-dependent reactivation

Bromodomain and extra-terminal domain family of proteins (BET) play an important role in repression of the HIV-1 transcription. BET proteins are responsible for the recruitment of P-

remains associated with the elongating transcription machinery [231,233,234].

snRNA and recruits P-TEFb to 5′ LTR, resulting in active transcription [226].

**4.12. P-TEFb can be recruited in active and inactive form in the nucleus by Tat**

which recruits transcriptional elongation complex to the LTR [222].

**4.11. Tat-dependent transcription leads to productive infection**

cooperatively recognize and stably bind TAR [200,223].

62 Advances in Molecular Retrovirology

[230].

of HIV-1 [247].

P-TEFb is required for activation of HIV transcription but does not explain the maximum observed viral expression; therefore, additional factors are necessary [254,255]. P-TEFb is an integral part of the super elongation complex (SEC) (Figure 3), which is a potent activator of transcriptional elongation of host genes [234,256]. It is composed of one of two scaffold proteins, AF4/FMR2 proteins AFF1 or AFF4. Translocations of AFF1 and AFF4 resulting in fusion proteins are commonly found in mixed lineage leukemia (MLL) [257–259]. The resultant fusion proteins cause aberrant recruitment of SEC to MML-specific genes [260]. AFF1 and AFF4 recruit many other proteins to the SEC [261], such as ELL family of elongation stimulatory factors ELL1 and ELL2, which inhibit RNAPII pausing and synergistically improve Tattransactivation with P-TEFb [256]. Moreover, knockdown of ELL2 strongly suppresses viral expression. [203,210,230,252]. Tat and AFF4 inhibit the polyubiquitination-mediated degra‐ dation of ELL2, increasing available levels of SEC. [256,262].

#### **4.14. Tat can be extensively post-translationally modified – "Tat code"**

Modifications on numerous amino residues of Tat regulate the interaction with a wide variety of host proteins. In comparison to the histone code which is used to explain the multiple modification on histone tails and their function, a "Tat-code" has been proposed [34]. Tat is phosphorylated on Ser16 and Ser 46 by CDK2, modifications which result in transcription inhibition [263]. Acetylation of Lys28 increases affinity for P-TEFb binding and is removed by HDAC6 [264–266]. Tat dissociates from TAR and binds acetyltransferase PCAF which acetylates Tat at Lys50 and Lys51 [264,265,267–270]. Acetylated Lys50 allows recruitment of the PBAF (SWI/SNF B) chromatin remodeling complex to the LTR [267,271–273]. SIRT1 deacetylates Tat at Lys50 as part of a late phase of transcriptional regulation, striping Tat of acetyl groups allowing its reuse in subsequent rounds of transcriptional cycles [274]. Mono‐ methyl-transferase Set7/9 and LSD1, respectively, methylate and demethylate Lys51. Deme‐ thylated Lys51 of Tat enhances HIV-1 transcription [275,276]. Hdm2 polyubiquitinates Lys71, activating Tat [277].

#### **4.15. Nucleosome positioning at the 5′ LTR controls viral expression**

Regardless of integration position, the latent 5′ LTR typically contains two nucleosomes, Nuc-0 and Nuc-1, at fixed positions [278]. Nuc-1 blocks transcription elongation as it is positioned just downstream of the TSS. Nuc-1 is displaced upon virus reactivation [278–280]. Nucleo‐ somes can be altered by chromatin remodeling complexes. A third unstable or loosely positioned nucleosome is located in between nuc-0 and nuc-1 [281] (Figures 2 and 3A).

BCL11B, together with the chromatin remodeling complex NuRD, strongly represses HIV-1 transcription [282]. BCL11B is specifically expressed in T-cells and neurons. Interestingly, the NuRD complex consists of several proteins with histone deacetylase activities – i.e., HDAC1 and HDAC2 [283,284].

The ATP-dependent chromatin remodeler BAF (SWI/SNF-A) was discovered by our group to be essential to both the establishment and maintenance of HIV latency (Figure 3). The BAF complex utilizes energy from ATP to push Nuc-1 from an energetically favorable position upstream of the TSS to a suboptimal region, downstream of TSS, resulting in a transcriptional block [281]. siRNA depletion of the BAF complex de-repressed proviral transcription. Fur‐ thermore, in siRNA-mediated BAF knockdown, latency establishment occurred less frequent‐ ly than in the presence of the functional complex. The PIC through LEDGF interacts with INI-1 a subunit of BAF, allowing nucleosomes to be deposited at the provirus, contributing to latency establishment [118].

#### **4.16. Epigenetic modifications regulate latency**

Epigenetic modifications of nucleosomes such as histone-acetylation and -methylation and of DNA such as DNA-methylation play an important role in regulating the proviral transcription. Nucleosomes are the basic units of organization of chromatin and consist of a combination of histone subunits. Histones have an amino acids tail that can be extensively modified. Two broadly studied modifications that regulate expression effects are histone-acetylation and histone-methylation

Histone-acetylation by histone acetyl transferases (HATs) induces chromatin loosening, while histone deacetylases (HDACs) reverse the effect by removing the acetyl group (Figure 3). HATs such as p300/CREB-binding protein (p300/CBP) and p300/CBP-associated factor (P/CAF) can be recruited to activate the HIV LTR [158,285]. HDAC1, HDAC2, HDAC3, and HDAC6 repress HIV [286–289]. Numerous host factors recruit HDACs to the LTR. A negative regulator of P-TEFb, CTIP2 in cooperation with COUP-TF and Sp1 also recruits HDAC1 and HDAC2 to the HIV LTR in microglial cells [290,291]. Host factors LSF and YY1 co-operatively bind to the LTR, where YY1 recruits HDAC1 to deacetylate Nuc-1[207]. CBF-1 and c-Myc also repress HIV through the recruitment of HDAC1 [208–210].

Methylation of histones by histone methyltransferases (HMT) can act as an activating or repressing mark depending on the histone tail residue modified (e.g., methylation of lysine 4 on histone 3 (H3K4) is activating whereas H3K9, H3K27, and H4K20 methylation is repressive). HMTs modify specific histone residues, e.g., EZH2 (H3K27me3), SUV39H1 (H3K9me3), G9a (H3K9me2), and G9a like protein, GLP (H3K9me2). The repressive methyl groups deposited by these HMTs contribute to the maintenance of latency [292–295]. Moreover, EZH2 is suspected to recruit additional repressive proteins such as HDACs and other HMTs [294].

DNA methylation at CpG dinucleotides represses transcription by disrupting the binding of transcription activators to their binding sites or indirectly through the binding methyl-CpG binding proteins (MeCPs). In cell line models of latency, the HIV-1 LTR contains two CpG islands that are hypermethylated (Figure 3) [296]. Methyl-CpG binding domain protein 2 (MDB2) and HDAC-2 bind to the second CpG island on the HIV LTR and are displaced from there when cells are treated with cytosine-methylation inihibitor 5-aza-2′deoxycytidine [296]. In memory CD4+ T-cells from long-term aviremic and viremic patients, an increase in HIV LTR DNA methylation was observed in the aviremic patients [297]. The methylation of the HIV LTR in long-term non-progressors and elite controllers is increased compared to the LTR of aviremic patients on cART [298]. In contrast, this difference was not found in the first CpG island of resting memory CD4+ T-cells from aviremic patients, indicating that the mechanism by which DNA-methylation regulates latency deserves further exploration.

#### **4.17. Viral and host non-coding RNAs regulate viral expression**

**4.15. Nucleosome positioning at the 5′ LTR controls viral expression**

and HDAC2 [283,284].

64 Advances in Molecular Retrovirology

establishment [118].

histone-methylation

**4.16. Epigenetic modifications regulate latency**

through the recruitment of HDAC1 [208–210].

Regardless of integration position, the latent 5′ LTR typically contains two nucleosomes, Nuc-0 and Nuc-1, at fixed positions [278]. Nuc-1 blocks transcription elongation as it is positioned just downstream of the TSS. Nuc-1 is displaced upon virus reactivation [278–280]. Nucleo‐ somes can be altered by chromatin remodeling complexes. A third unstable or loosely positioned nucleosome is located in between nuc-0 and nuc-1 [281] (Figures 2 and 3A).

BCL11B, together with the chromatin remodeling complex NuRD, strongly represses HIV-1 transcription [282]. BCL11B is specifically expressed in T-cells and neurons. Interestingly, the NuRD complex consists of several proteins with histone deacetylase activities – i.e., HDAC1

The ATP-dependent chromatin remodeler BAF (SWI/SNF-A) was discovered by our group to be essential to both the establishment and maintenance of HIV latency (Figure 3). The BAF complex utilizes energy from ATP to push Nuc-1 from an energetically favorable position upstream of the TSS to a suboptimal region, downstream of TSS, resulting in a transcriptional block [281]. siRNA depletion of the BAF complex de-repressed proviral transcription. Fur‐ thermore, in siRNA-mediated BAF knockdown, latency establishment occurred less frequent‐ ly than in the presence of the functional complex. The PIC through LEDGF interacts with INI-1 a subunit of BAF, allowing nucleosomes to be deposited at the provirus, contributing to latency

Epigenetic modifications of nucleosomes such as histone-acetylation and -methylation and of DNA such as DNA-methylation play an important role in regulating the proviral transcription. Nucleosomes are the basic units of organization of chromatin and consist of a combination of histone subunits. Histones have an amino acids tail that can be extensively modified. Two broadly studied modifications that regulate expression effects are histone-acetylation and

Histone-acetylation by histone acetyl transferases (HATs) induces chromatin loosening, while histone deacetylases (HDACs) reverse the effect by removing the acetyl group (Figure 3). HATs such as p300/CREB-binding protein (p300/CBP) and p300/CBP-associated factor (P/CAF) can be recruited to activate the HIV LTR [158,285]. HDAC1, HDAC2, HDAC3, and HDAC6 repress HIV [286–289]. Numerous host factors recruit HDACs to the LTR. A negative regulator of P-TEFb, CTIP2 in cooperation with COUP-TF and Sp1 also recruits HDAC1 and HDAC2 to the HIV LTR in microglial cells [290,291]. Host factors LSF and YY1 co-operatively bind to the LTR, where YY1 recruits HDAC1 to deacetylate Nuc-1[207]. CBF-1 and c-Myc also repress HIV

Methylation of histones by histone methyltransferases (HMT) can act as an activating or repressing mark depending on the histone tail residue modified (e.g., methylation of lysine 4 on histone 3 (H3K4) is activating whereas H3K9, H3K27, and H4K20 methylation is repressive). HMTs modify specific histone residues, e.g., EZH2 (H3K27me3), SUV39H1 (H3K9me3), G9a (H3K9me2), and G9a like protein, GLP (H3K9me2). The repressive methyl groups deposited

Non-coding RNAs exert post transcriptional control on gene expression. Small non-coding RNAs (<200 nt) and in particular microRNAs (miRNAs) are well established to have regulatory function. The study of long non-coding RNAs (lncRNA, >200 nt) is an emerging field because of their epigenetic regulatory potential. Both viral and host miRNAs and lncRNAs affect replication of HIV-1 [146,299–301].

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism. miRNAs posttranscriptionally suppress or silence gene expression as part of the RNA-induced silencing complex (RISC) forming a protein–RNA complex. Pri-miRNAs are generated by RNAPII and are subsequently processed by microprocessor into pre-miRNAs in the nucleus. Following export to the cytoplasm, they are cleaved by Dicer and incorporated into RISC. RISC generally binds in the 3′-untranslated region (3'UTR) of a target mRNA. The bound transcript is degraded or transcription is impeded depending on the level of homology, resulting in translational repression. The RNAi affects the infectivity of monocytes and macrophages [302]. Comparisons of productively infected, suppressed, and uninfected patients found difference in miRNA profiles, but it is very unlikely that the observed effects are due to viral activity because the number of infected cells is low in elite controllers or under cART [303–305]. Knockdown of Dicer or Drosha, a component of microprocessor, stimulates HIV-1 replication, indicating that miRNA generally are responsible for suppression of proviral transcription [299,300]. However, phenotypic effects are hard to interpret due to the pleiotropic side effects of microprocessor depletion. RNAi affects infectivity by targeting transcripts of key host factors and viral proteins involved in HIV-1 repression. In resting T-cells, the polycistronic miRNA cluster miR-17/92 is suppressed by HIV, resulting in PCAF upregulation [299]. Additionally, CycT1 is negatively regulated by miR27b [306]. Moreover, during differentiation from monocytes to macrophages, expression of miRNA198 and miR27b decreases relieving suppression of CycT1 [307,308]. In infected cells Tat, and possibly Vpr, inhibit RNAi [309– 311]. In resting, but not activated, CD4+ T-cells a cluster of five miRNAs (miR-28, miR-125b, miR-150, miR-223, and miR-382) were found to be upregulated. They all target viral mRNAs for degradation; therefore, these miRNAs are contributing to latency maintenance [312]. However, further studies are required as results thus far are inconsistent [313–319].

The viral protein Nef is targeted by miR29a which interferes with HIV replication [300,320]. TRIM32 activates HIV-1 expression through the NF-kB pathway and is downregulated by miRNA-155 [321]. Tat-induced upregulation of miR34a and miR217 inhibits SIRT1 expression, which in turn results in high abundance of NF-kB, enhancing proviral transcription [322,323]. miRNA-182 has a positive effect on LTR activation by Tat [324]. miR-1236 restricts viral replication by repressing Vpr (HIV-1)-binding protein expression, VprBP [325].

HIV-1-derived miRNAs (vmiRNAs) were predicted *in silico* [326]. Applying deep sequenc‐ ing technologies vmiRNAs were observed in cell line model systems of latency [327,328]. The TAR-derived miRNA-TAR5p and miR-TAR3p are asymmetrically processed and both repress LTR activity [329]. The *Nef*-derived miR-N367 inhibits viral promotor activity [330]. Nevertheless, relevance of vmiRNAs is debatable as no vmiRNAs were detected in PBMCs or macrophages of infected patients [331].

lncRNAs can modulate gene expression through different proposed mechanisms: (1) affecting mRNAs through sequence recognition, (2) recruiting proteins to DNA, (3) blocking host factors by assuming a secondary structure, (4) functioning as a scaffold for protein complexes. An anti-sense lncRNA of HIV-1 inhibits viral replication[146]. The non-coding repressor of NFAT (NRON) inhibits LTR activity in a NFAT-dependent manner [301].

#### **4.18. Stochastic gene expression**

The current model of HIV latency proposes that resting memory CD4+ T-cells are deprived of host factors that are necessary for viral expression. An alternative model proposes that expression is highly stochastic. Due to fluctuations in chromatin state and availability of the transcription factors, the latent and productive state co-exist [332]. In support, clonal lines (containing the same integration) showed binominal distributions of viral expression [333]. Transcriptional bursts of 2–10 mRNA transcripts were estimated to be the source of HIV-1 gene expression [334]. Tat-controlled positive feedback extends the expression reactivation [335]. The sensitivity to reactivation is also stochastic, as cells derived from patients remained latent during a first round of activation and were reactivatable in the next round of activation [83]. Moreover, molecules that increase gene expression fluctuations synergistically enhance HIV-1 reactivation [336].

#### **5. HIV cure**

Mechanistic insight into the complex nature of latent HIV-1 infection provides a rationale for eradication strategies. Therefore, identification of molecules that inhibit activity of repressors or potentiate HIV-1 activators alongside with immune system boosting are important objec‐ tives in eradication strategies.

#### **5.1. Shocking the virus: screening for Latency Reversal Agents (LRAs)**

The initial step of LRA discovery is screening drug libraries with cell-line-based models. Positive hits are evaluated further using primary-cell-based models as they better recapitulate the nature of latent reservoirs. If effective and not toxic, putative LRAs should undergo reactivation studies using primary cells derived from HIV-1-positive individuals that are on cART as well as toxicology studies in animal models, in case of novel molecules. It is advan‐ tageous to include molecules that are already approved drugs in such putative LRAs libraries, employing them into clinical practice would be time and resources effective. Moreover, in order to easily diffuse through cell membranes, ideal LRAs are small molecules, with molec‐ ular weight below 900 daltons, although clinical practice shows that most effective compounds do not exceed 500 daltons [337,338].

The first attempts to reactivate proviral DNA failed, due to the use of agents (e.g., IL-2 or a monoclonal antibody against CD3 receptor) which resulted in global T-cell activation. Indeed, viral p24 and plasma HIV-1 RNA levels increased, but the toxicity of such treatment left this approach useless [339–341]. Therefore, there is a need for more specific agents, which are able to reactivate proviral transcription without T-cell activation.

#### **5.2. HDAC inhibitors (HDACis)**

miR-150, miR-223, and miR-382) were found to be upregulated. They all target viral mRNAs for degradation; therefore, these miRNAs are contributing to latency maintenance [312].

The viral protein Nef is targeted by miR29a which interferes with HIV replication [300,320]. TRIM32 activates HIV-1 expression through the NF-kB pathway and is downregulated by miRNA-155 [321]. Tat-induced upregulation of miR34a and miR217 inhibits SIRT1 expression, which in turn results in high abundance of NF-kB, enhancing proviral transcription [322,323]. miRNA-182 has a positive effect on LTR activation by Tat [324]. miR-1236 restricts viral

HIV-1-derived miRNAs (vmiRNAs) were predicted *in silico* [326]. Applying deep sequenc‐ ing technologies vmiRNAs were observed in cell line model systems of latency [327,328]. The TAR-derived miRNA-TAR5p and miR-TAR3p are asymmetrically processed and both repress LTR activity [329]. The *Nef*-derived miR-N367 inhibits viral promotor activity [330]. Nevertheless, relevance of vmiRNAs is debatable as no vmiRNAs were detected in PBMCs

lncRNAs can modulate gene expression through different proposed mechanisms: (1) affecting mRNAs through sequence recognition, (2) recruiting proteins to DNA, (3) blocking host factors by assuming a secondary structure, (4) functioning as a scaffold for protein complexes. An anti-sense lncRNA of HIV-1 inhibits viral replication[146]. The non-coding repressor of NFAT

The current model of HIV latency proposes that resting memory CD4+ T-cells are deprived of host factors that are necessary for viral expression. An alternative model proposes that expression is highly stochastic. Due to fluctuations in chromatin state and availability of the transcription factors, the latent and productive state co-exist [332]. In support, clonal lines (containing the same integration) showed binominal distributions of viral expression [333]. Transcriptional bursts of 2–10 mRNA transcripts were estimated to be the source of HIV-1 gene expression [334]. Tat-controlled positive feedback extends the expression reactivation [335]. The sensitivity to reactivation is also stochastic, as cells derived from patients remained latent during a first round of activation and were reactivatable in the next round of activation [83]. Moreover, molecules that increase gene expression fluctuations synergistically enhance

Mechanistic insight into the complex nature of latent HIV-1 infection provides a rationale for eradication strategies. Therefore, identification of molecules that inhibit activity of repressors or potentiate HIV-1 activators alongside with immune system boosting are important objec‐

However, further studies are required as results thus far are inconsistent [313–319].

replication by repressing Vpr (HIV-1)-binding protein expression, VprBP [325].

(NRON) inhibits LTR activity in a NFAT-dependent manner [301].

or macrophages of infected patients [331].

**4.18. Stochastic gene expression**

66 Advances in Molecular Retrovirology

HIV-1 reactivation [336].

tives in eradication strategies.

**5. HIV cure**

Histone deacetylase inhibitors (HDACis) are a very promising class of LRAs which include valporic acid (VPA), Vorinostat (SAHA), Romidepsin, Panobinostat, Givinostat, Droxinostat, or Entinostat. Some (Vorinostat (SAHA), Romidepsin, Panobinostat) are undergoing clinical trials [94,342–344].

The focus on HDACis is due to their ability to loosen up the compact chromatin structure at the latent proviral promoter. Inhibition of HDACs results in an increase of histone acetylation level by HATs. HDACs 1, 2, and 3 are of particular interest as they considerably contribute to HIV-1 repression [287]. Fortunately, HDACis are already used in clinical therapies, e.g., VPA is used in epilepsy and bipolar disorders, Vorinostat and Romidepsin are used to treat cutaneous T-cell lymphoma (CTCL) while Panabinostat is used in patients with multiple myeloma. In a very promising study by Archin et al., a single treatment with Vorinostat resulted in an increase in proviral RNA [345]. Unfortunately, the follow-up study with additional, multiple-dose rounds of treatment showed that increase on HIV-1 transcription is neither sustained nor elevated [346]. It is possible that other mechanisms maintaining latency compensate histone acetylation, in order to restrain proviral transcription. Alternatively such low concentrations of Vorinostat result in activation of pTEF-b instead of HDAC inhibition [347]. Since HDACs are involved in general regulation of gene expression; they have pleio‐ tropic effects causing toxicities. Therefore, their use must be strictly controlled and monitored in order to provide maximal safety [348]. Nevertheless, HDACis are still under much interest. Especially, finding more specific HDAC inhibitors is very appealing, as current drugs are inhibiting a wide range of different HDACs, contributing to high toxicity [349].

#### **5.3. BET inhibitors (BETi's)**

Since BET proteins repress the HIV-1 promoter, it is worth to use their inhibitors in latency reversal strategies. Treatment with BET protein inhibitor JQ1 reactivates HIV-1 transcription in Tat-independent fashion [247]. Furthermore, BET inhibitor activity was positively tested in more relevant primary model system of latency [249]. Unfortunately, JQ-1 is not clinically available, due to its short half-life.

#### **5.4. HMT inhibitors (HMTis)**

Several histone methyltransferases (HMTs) such as EZH2, SUV39H1, and G9a interact with 5′ LTR contributing to maintenance of latency by deposition of repressive methyl groups on nucleosomal proteins [292–295]. Moreover, EZH2 recruits additional repressive proteins such as HDACs and other HMTs [294]. Several inhibitors of these proteins were tested in cell lines or primary cells from HIV-1 positive patients. Among which, Chaetocin (SUV39H1 inhibitor) and BIX-01294 (G9a inhibitor) were most potent [292,350]. However, high toxicity, due to pleiotropic effects, makes them unsuitable for clinical practice. Therefore, identification of novel compounds that are able to inhibit the activity of HMTs is needed.

#### **5.5. DNMT inhibitors (DNMTis)**

Inhibition of DNA methyltransferases (DNMTs) with 5-aza-2′ deoxycytidine (aza-CdR or Decitabine) leads to modest reactivation of latent HIV-1. This activity can be further enhanced with PKC agonists [351]. However, 5′ LTR methylation in patients material remains contro‐ versial [352]. Thus, further investigation of provirus methylation *in vivo* is needed.

#### **5.6. Toll-like receptors (TLRs) stimulation**

TLRs recently gained more attention, as theirs agonists are strong reactivators of HIV-1 [353– 357]. The main role of these receptors is to activate an immune response against bacterial or viral infections [358]. Stimulating TLRs (as adjuvants in immunization) as well as opportunistic bacterial infections elevate plasma HIV-RNA and improve immune function [359–363].

Vaccine adjuvant – CPG 7909 (TLR 9 agonist) is able to decrease plasma HIV-1 RNA via activation of HIV-specific CD8+ T-cells in peripheral blood [359]. More recently, in SIV-positive rhesus monkeys undergoing cART were treated with GS-9620, a TLR7 agonist, reversible CD8 cytotoxic T-cells activation alongside with modest CD4 T-cell activation were observed. Moreover, elevated plasma viremia was observed as well as decrease in HIV-1 DNA in blood, colon, and lymph nodes. Interestingly, viral load returned back to undetectable levels when GS-9620 was no longer administrated. More strikingly, when cART was stopped, GS-9620 treated monkeys had 0.5 log lower viral set-point than untreated, infected animals. Addition‐ ally, in cells isolated from HIV-positive individuals transcription of HIV-1 was observed. However, some variability between samples was noticed. Clinical trials with the use of this compound are planned [364,365].

#### **5.7. Super elongation complex stimulation**

**5.3. BET inhibitors (BETi's)**

68 Advances in Molecular Retrovirology

available, due to its short half-life.

**5.5. DNMT inhibitors (DNMTis)**

compound are planned [364,365].

**5.6. Toll-like receptors (TLRs) stimulation**

**5.4. HMT inhibitors (HMTis)**

Since BET proteins repress the HIV-1 promoter, it is worth to use their inhibitors in latency reversal strategies. Treatment with BET protein inhibitor JQ1 reactivates HIV-1 transcription in Tat-independent fashion [247]. Furthermore, BET inhibitor activity was positively tested in more relevant primary model system of latency [249]. Unfortunately, JQ-1 is not clinically

Several histone methyltransferases (HMTs) such as EZH2, SUV39H1, and G9a interact with 5′ LTR contributing to maintenance of latency by deposition of repressive methyl groups on nucleosomal proteins [292–295]. Moreover, EZH2 recruits additional repressive proteins such as HDACs and other HMTs [294]. Several inhibitors of these proteins were tested in cell lines or primary cells from HIV-1 positive patients. Among which, Chaetocin (SUV39H1 inhibitor) and BIX-01294 (G9a inhibitor) were most potent [292,350]. However, high toxicity, due to pleiotropic effects, makes them unsuitable for clinical practice. Therefore, identification of

Inhibition of DNA methyltransferases (DNMTs) with 5-aza-2′ deoxycytidine (aza-CdR or Decitabine) leads to modest reactivation of latent HIV-1. This activity can be further enhanced with PKC agonists [351]. However, 5′ LTR methylation in patients material remains contro‐

TLRs recently gained more attention, as theirs agonists are strong reactivators of HIV-1 [353– 357]. The main role of these receptors is to activate an immune response against bacterial or viral infections [358]. Stimulating TLRs (as adjuvants in immunization) as well as opportunistic bacterial infections elevate plasma HIV-RNA and improve immune function [359–363].

Vaccine adjuvant – CPG 7909 (TLR 9 agonist) is able to decrease plasma HIV-1 RNA via activation of HIV-specific CD8+ T-cells in peripheral blood [359]. More recently, in SIV-positive rhesus monkeys undergoing cART were treated with GS-9620, a TLR7 agonist, reversible CD8 cytotoxic T-cells activation alongside with modest CD4 T-cell activation were observed. Moreover, elevated plasma viremia was observed as well as decrease in HIV-1 DNA in blood, colon, and lymph nodes. Interestingly, viral load returned back to undetectable levels when GS-9620 was no longer administrated. More strikingly, when cART was stopped, GS-9620 treated monkeys had 0.5 log lower viral set-point than untreated, infected animals. Addition‐ ally, in cells isolated from HIV-positive individuals transcription of HIV-1 was observed. However, some variability between samples was noticed. Clinical trials with the use of this

versial [352]. Thus, further investigation of provirus methylation *in vivo* is needed.

novel compounds that are able to inhibit the activity of HMTs is needed.

Treatment of cell lines and cells isolated from aviremic patients on cART with hexamethylene bisacetamide (HMBA), an anticancer drug that transiently activates PI3K/Akt pathway, results in phosphorylation of HEXIM1. P-TEFb is subsequently released and interacts with RNAP II, resulting in latency reversal [366–368]. Moreover, HMBA provides CDK9 recruitment to the viral promoter by interaction with SP1, which enhances transcription from proviral DNA. Furthermore, Klichko et al. showed that treatment with HMBA resulted in a decrease of CD4 receptor expression without affecting transcription of CCR5 and CXCR4 co-receptors [369]. Moreover, HMBA does not trigger activation of T-cells. Studies on P-TEFb's role in HIV-1 latency indicate that this heterocomplex might be an interesting target for inclusion in "shock and kill" therapies.

#### **5.8. PKC pathway activation**

Another interesting approach is the use of molecules that are able to selectively activate the protein kinase C (PKC) pathway. PKC pathway agonists trigger nuclear localization of NF-kB, NFAT, and AP-1 transcription factors. Therefore, PKC agonists are one of the most potent activators of HIV-1 transcription. Currently, two PKC agonists are being scrutinized clinically: prostratin and bryostatin, due to their safety and specificity toward HIV-1 reactivation. The latter is a clinically available drug [370]. Moreover, these two compounds prevent *de novo* infections, as they downregulate viral receptor and co-receptors CD4, CCR5 and CXCR4 in PBMCs [371]. A rather controversial molecule that reactivates HIV-1 transcription via NF-κB pathway is arsenic trioxide (As2O3). In the Jurkat model system of latency, As2O3 activates NFκB leading to HIV-1 replication. Moreover, it synergizes with prostratin, tumor necrosis factor alpha (TNFα), and VPA [372]. Interestingly, arsenic is already used in clinical practice to treat acute promyelocytic leukemia (APL). Therefore, it would be interesting to test this compound in more relevant models of HIV-1 latency such as primary cells infected *ex vivo* and in cells derived from aviremic patients.

The use of PKC agonists raises concerns about their safety in a clinical setting. The protein kinase enzyme family consists of several isoenzymes that play important roles in signal transduction cascades [373]. As activation of latent HIV-1 is mediated via PKCα and PKCθ, the identification of more specific agonists of PKCα and PKCθ is needed. Alternatively, lowering the concentration of a specific agonists might decrease toxicity and contribute to latency reversal [374].

#### **5.9. JNK/MAPK pathway activation**

Studies employing mutagenesis of binding sites for activator protein-1 (AP-1) within the proviral genome showed that the AP-1 transcription factor is a crucial activator of proviral transcription, as proviruses with altered AP-1 binding sites were less prone to reactivation even if treated with a strong activator such as phorbol 12-myristate 13-acetate – PMA [191]. Furthermore, the latent pool of cells infected by virus with deletion in AP-1 sites was bigger, implicating that AP-1 is necessary for provirus transcription [192]. Heterodimeric protein AP-1 is formed upon phosphorylation of c-Jun N-terminal kinase (JNK) in JNK/MAPK pathway [193]. It is well established that activation of TLR signaling induces nuclear localization of NFkB and AP-1 mediated via JNK pathway [194,196,376,377].

Virtual screening followed by validation of positive hits in cell line model systems for HIV-1 latency discovered 8-methoxy-6-methylquinolin-4-ol (MMQO) as a specific activator of the JNK-AP-1 pathway, which is able to reactivate HIV-1 from its latent state. Interestingly, MMQO inhibits IL-2 and TNFa expression, contributing to maintenance of resting state of CD4+ T-cells [378]. The recently synthetized panel of inhibitors of farnesyl transferase (FTase) are able to moderately reactivate HIV-1 transcription via JNK pathway. Interestingly, strong synergy with other LRAs, such as Vorinostat or TNF-a, was observed for these molecules in latency reversal [379,380].

#### **5.10. Canonical Wnt signaling pathway activation**

Recently, our group showed that treatment with Wnt3A/Rsp (natural stimulators of Wnt pathway) and lithium (inhibitor of Wnt repressor protein GSK3) leads to latency reversal in latent cell lines and enhances the latency reversal potential of HDAC inhibitors in CD4+ T primary cells obtained from patient volunteers when co-treated [381]. This observation shows a functional role for three LEF1 binding sites in the 5′ LTR contains, which are downstream targets of the classical Wnt pathway [381,382]. It would be very interesting to find more potent and selective inducers of Wnt pathway, as lithium exhibits many pleiotropic, toxic effects [383,384].

#### **5.11. Chromatin loosening**

It was discovered by our group that a main player in the establishment and maintenance of latency is the BAF complex (SWI/SNF-A), which belongs to ATP-dependent chromatin remodelers' family. Interestingly, Dykhuizen et al. [378] screened a library of compounds that would be able to mimic BRG-1 knock out. In their study, they found 20 compounds that were transcriptionally mimicking BAF complex disruption. We showed that several of those molecules were able to decrease the frequency of latency establishment and reactivate HIV-1 in cell line and primary cells models of latency [386, in press]. Moreover, they synergize with other LRAs – SAHA and prostratin. Two most potent inhibitors – caffeic acid phenethyl ester (CAPE) and pyrimethamine (PYR) did not activate T-cells derived from healthy donors and cells obtained from aviremic patients. Moreover, PYR is a registered drug used in malaria treatment. Therefore, these inhibitors are promising molecules to include in eradication strategies.

#### **5.12. Multifunctional LRAs**

*In vitro* treatment with cocaine leads to increase in HIV- replication in PBMCs as well as increased viral load in mouse models of HIV infection [387–389]. Interestingly, in *ex vivo* infected primary CD4+ cocaine treatment resulted in downregulation of miR125-b expression, which led to enhanced replication of HIV-1 [314]. In primary human macrophages and myeloid cell systems of latency, cocaine increased replication of HIV-1. Cocaine treatment activates NFκB and leads to phosphorylation of mitogen- and stress-activated kinase 1 (MSK1). Further‐ more, pMSK1 phosphorylates RELA (p65), a subunit of NF-κB promoting the interaction of NF-κB with p300 and recruitment of P-TEFb to the proviral 5′ LTR [390]. Moreover, treatment with cocaine results in histone H3 phosphorylation, thus increasing accessibility of HIV-1 promoter for transcription factors [390]. Therefore, cocaine not only reverses latency via NFκB pathway but also causes epigenetic changes on 5′ LTR as well as blocks repressive miRNA.

Oral bacteria secrete short-chain fatty acids (SCFAs) including butyric acid, propionic acid, isovaleric acid, and isobutyric acid that are capable of HIV-1 and herpesviruses latency reversal [384,385]. Some of these molecules are known HDACis (e.g. Butyric acid) [393]. Moreover, SCFAs not only promotes histone acetylation, but also inhibit repressive histone formation and DNA methylation. Furthermore, they activate P-TEFb resulting in increased elongation of transcription from 5' LTR. [345,385,386].

#### **5.13. Immune clearance of reactivated cells – "Kill"**

is formed upon phosphorylation of c-Jun N-terminal kinase (JNK) in JNK/MAPK pathway [193]. It is well established that activation of TLR signaling induces nuclear localization of NF-

Virtual screening followed by validation of positive hits in cell line model systems for HIV-1 latency discovered 8-methoxy-6-methylquinolin-4-ol (MMQO) as a specific activator of the JNK-AP-1 pathway, which is able to reactivate HIV-1 from its latent state. Interestingly, MMQO inhibits IL-2 and TNFa expression, contributing to maintenance of resting state of CD4+ T-cells [378]. The recently synthetized panel of inhibitors of farnesyl transferase (FTase) are able to moderately reactivate HIV-1 transcription via JNK pathway. Interestingly, strong synergy with other LRAs, such as Vorinostat or TNF-a, was observed for these molecules in

Recently, our group showed that treatment with Wnt3A/Rsp (natural stimulators of Wnt pathway) and lithium (inhibitor of Wnt repressor protein GSK3) leads to latency reversal in latent cell lines and enhances the latency reversal potential of HDAC inhibitors in CD4+ T primary cells obtained from patient volunteers when co-treated [381]. This observation shows a functional role for three LEF1 binding sites in the 5′ LTR contains, which are downstream targets of the classical Wnt pathway [381,382]. It would be very interesting to find more potent and selective inducers of Wnt pathway, as lithium exhibits many pleiotropic, toxic effects

It was discovered by our group that a main player in the establishment and maintenance of latency is the BAF complex (SWI/SNF-A), which belongs to ATP-dependent chromatin remodelers' family. Interestingly, Dykhuizen et al. [378] screened a library of compounds that would be able to mimic BRG-1 knock out. In their study, they found 20 compounds that were transcriptionally mimicking BAF complex disruption. We showed that several of those molecules were able to decrease the frequency of latency establishment and reactivate HIV-1 in cell line and primary cells models of latency [386, in press]. Moreover, they synergize with other LRAs – SAHA and prostratin. Two most potent inhibitors – caffeic acid phenethyl ester (CAPE) and pyrimethamine (PYR) did not activate T-cells derived from healthy donors and cells obtained from aviremic patients. Moreover, PYR is a registered drug used in malaria treatment. Therefore, these inhibitors are promising molecules to include in eradication

*In vitro* treatment with cocaine leads to increase in HIV- replication in PBMCs as well as increased viral load in mouse models of HIV infection [387–389]. Interestingly, in *ex vivo* infected primary CD4+ cocaine treatment resulted in downregulation of miR125-b expression, which led to enhanced replication of HIV-1 [314]. In primary human macrophages and myeloid

kB and AP-1 mediated via JNK pathway [194,196,376,377].

**5.10. Canonical Wnt signaling pathway activation**

latency reversal [379,380].

70 Advances in Molecular Retrovirology

**5.11. Chromatin loosening**

**5.12. Multifunctional LRAs**

[383,384].

strategies.

The majority of chronic patients are facing immune exhaustion, characterized by low cytokine secretion, smaller proliferative capacity, and low cytopathic potential of CD8+ T-cells [394,395]. Therefore, the first line of action would be reviving normal immune activity. Indeed, inhibition of programmed cell death protein 1 (PD-1) leads to restoration of immune functions in mouse models of HIV-1 infection [396]. However, these results were obtained in viremic animals. Nevertheless, an IgG4 antibody targeting PD-1 receptor is undergoing clinical trials to assess safety, immunotherapeutic activity, and the ability of treatment to reduce pool of latently infected cells [397].

In so-called "elite controllers", CD8+ T-cells effectively restrain infection without intervention of cART, by killing CD4+ T-cells that are actively producing HIV-1 particles [398,399]. The immune system can be boosted by specific amplification of HIV-1-specific CD8+ T-cells. These observations again aroused the idea of developing a vaccine. Indeed, rhesus monkeys vaccinated with CMV vectors resulted in broad cellular immune response to SIV [400–402]. However, safety issues related to the use of such vectors remain to be elucidated. Another platform being investigated to increase immune response against HIV-1 are Ad26 vectors, as it was shown that vaccinated rhesus monkeys were protected against infection with SIV as well as viral loads were lowered after vaccination [403,404].

A very interesting group of immunoglobulins to include in eradication strategies are broadly neutralizing monoclonal antibodies (mAbs or bNAbs) isolated from chronically infected patients. New generations of bNAbs exert higher potency and wider range of activity against many HIV-1 subtypes. It was shown that a combination of bNAbs is potent enough to transiently suppress viremia in rhesus monkeys as well as to reduce the amount of HIV-1 DNA in the blood, lymph nodes, and gastrointestinal mucosa [403,405,406].

#### **6. Future perspectives and challenges**

A reservoir of latent HIV is the main obstacle in finding a functional and sterilizing cure. Several challenges need to be addressed in order to overcome this obstacle. Defining the latent reservoir is impeded by the rare occurrence of a latent infection in a high background of defective proviral integration. Although HIV prefers integration in or near transcriptionally active genes which leaves ample room for variation in chromatin environment and available host transcription factors. This puts considerable demands on LRAs. LRAs should be effective, yet specific, without being toxic. As LRAs act via pathways involved in distinct cellular processes, pleiotropic effects are to be expected. Furthermore, recent studies on material obtained from HIV-1-positive suppressed patients revealed that currently available LRAs are not strong enough to reactivate the whole pool of latent proviruses, even after multiple rounds of stimulation. One of the concerns arising from "shock and kill" therapy is whether putative LRAs are strong enough to drive virus production to a level at which the immune system will be able to recognize and destroy HIV-1-producing cells. Indeed, trials aiming at testing HDAC inhibitors are inconsistent in showing depletion of latently infected cells while showing increased proviral transcription [407–412]. A complementary strategy would be to use multiple LRAs in combination to broadly and potentially synergistically reactivate the diversely integrated latent proviruses. Synergism between LRAs was already identified, e.g., Vorinostat and Prostratin [84]. Therefore, the quest for identification and characterization of novel compounds which are able to reactivate HIV-1 transcription as well as identifying combina‐ tions of drugs that can synergize to reverse latency is needed. Currently, no cell model is able to recapitulate the complexities of latency *in vivo*. A better system that more closely resembles the *in vivo* situation would greatly aid the understanding of molecular mechanisms underlying latency and the screening of new LRA. Moreover, as HIV-1 persists in a silent state, it contrib‐ utes to a low level of inflammation, which over time leads to immune exhaustion. Furthermore, depletion of cells harboring latent provirus requires antigen-specific CTLs stimulation [399]. Most likely successful eradication therapies will be based on the combination of LRAs coupled with boosting HIV-1-specific immune response. A "shock and kill" approach in combination with immune therapies provides hope for reversing HIV-1 infection.

#### **Author details**

Michael D. Röling# , Mateusz Stoszko# , Tokameh Mahmoudi\*

\*Address all correspondence to: t.mahmoudi@erasmusmc.nl

Department of Biochemistry, Erasmus Medical Centre, Rottrerdam, The Netherlands
