**Coreceptor Usage in HIV Infection**

#### Kerina Duri

234 Immunodeficiency

100(6): 372-7.

[37] Bhaijee F, Wainwright H, Meintjes G et al. Warfarin-induced skin necrosis in HIV-1 infected patients with tuberculosis and venous thrombosis. S Afr Med J. 2010 Jun 1;

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52060

#### **1. Introduction**

Chemokines are small low molecular weight proteins or cytokines secreted by cells that function as chemical messengers. They were originally found to attract leukocytes to site(s) of inflammation. As ligands they activate and signal through their respective chemokine receptors triggering an influx of intracellular calcium (Ca2+) ions causing a process known as chemotaxis. Chemokine receptors are integral membrane proteins that specifically bind and respond to chemokines. They are members of the class A subfamily of G-protein coupled receptor (GPCRs) superfamily, a name derived from the characteristic cysteine motif of the group of chemokines they interact with. Despite their pivotal roles in the immune system and angiogenesis, chemokines as well as their receptors have been associated with a number of pathologies including autoimmune disorders, pulmonary diseases, transplant rejection, cancers, vascular diseases and human immunodeficiency virus (HIV) infection. Scientists have noted that while the CD4 receptor is necessary for the successful infection of host immune cells by all naturally occurring HIV-1 strains it is not sufficient. Thus, another specific cell surface molecule called chemokine receptor is required. The recent introduction of entry inhibitors in the clinic as components of antiretroviral treatment has increased the research interest of coreceptor usage in HIV infection. Chemokine receptors are subjects of significant medical importance which not only provide new insights into the mechanisms of viral entry, tropism and pathogenesis, but have also culminated into new control strategies from the host's perspective influencing HIV transmission along with disease progression. Identification of the different phenotypes of HIV-1 strains with different prevalence during various stages of disease progression and the role of these phenotypes in treatment outcome has further revolutionalised research in this field. This chapter seeks to depict a simple and clear understanding of the basics of HIV phenotypes or genotypes and the respective current as well as prospective diagnostic tools. Milestones and challenges in this relatively new class of antiretroviral therapy of coreceptor antagonists will also be highlighted.

© 2012 Duri, licensee InTech. This is an open access chapter 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. © 2012 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.

#### **2. Structure of chemokine receptors**

Chemokine receptors consist of about 350 amino acids that are divided into a short and acidic N-terminal end, seven helical transmembrane domains since they span the cell membrane seven times with three intracellular and three extracellular hydrophilic loops, and an intracellular C-terminus containing serine and threonine residues that act as phosphorylation sites during receptor regulation as shown in **Figure 1** below. Chemokine receptors are usually linked to a G-protein through which they signal. The N-terminus of the chemokine receptor is an extracellular domain that binds the chemokine(s) and has been shown to influence infection tropism. The first two extracellular loops of chemokine receptors are linked together by disulphide bonding between two conserved cysteine residues. The disulfide bonds keep the extracellular loops in place thereby maintaining the structural integrity necessary for ligand binding and signal transduction. In spite of the high amino acid similarity of their primary sequences, chemokine receptors bind a limited number of ligands.

Coreceptor Usage in HIV Infection 237

similarly an identifying number. Thus, for instance one of the receptors of CCL5 is called CCR5. Despite the numerous chemokine receptors that are prospective coreceptors for HIV *in vitro*, only CCR5 and CXCR4 have attracted substantial interest because they form portals of cellular entry for both HIV-1 and HIV-2 inclusive of other related simian or feline retroviruses. The lack of crystallography for these two highly hydrophobic coreceptors

The first HIV coreceptor to be identified was CXCR4 which was originally an orphan receptor called leukocyte-derived seven-transmembrane domain receptor (LESTR). By then it did not receive much attention until it was recognized as a coreceptor for HIV-1. Thus, in the mid 1990s the second receptor, CXCR4, needed for successful entry of HIV into cells was discovered. At that time this coreceptor was termed "fusin" as it facilitated certain HIV strains to fuse with and enter immune cells called T cells. A detailed analysis of the structure of fusin revealed that it was a receptor for chemokines which were previously shown to suppress HIV activity. CXCR4 has a wide cellular distribution. It is commonly expressed on most immature and mature hematopoietic cell types, cells of the central nervous system, neutrophils, monocytes, T and B cells, dendritic cells (DCs), Langerhans cells and macrophages. To date there are seven CXC chemokine receptors in mammals, named CXCR1 through CXCR7. The CXC chemokines include stromal cell-derived factor-1 alpha and beta now officially designated CXCL12a and CXCL12b respectively. CXCL12 is often induced by pro-inflammatory stimuli such as lipopolysaccharide, interleukin-1 (IL-1) or tumour necrosis factor-alpha (TNF-α) and has been shown to be strongly chemotactic for lymphocytes. Its high level of expression in the genital mucosa may help to explain the

Another chemokine receptor necessary for the entry of HIV into macrophages called CCR5 that was subsequently identified. CCR5 receptors have been shown to be involved in leukocyte activation and mobilization. CCR5 is expressed on several cell types including peripheral blood-derived DCs, CD34+ hematopoietic progenitor cells and some activated/memory Th1 lymphocyte. Chemokines that bind these receptors are RANTES (regulated-upon-activation normal T expressed and secreted); MIP-1α and MIP-1β (macrophage inflammatory protein-1 alpha and beta) previously observed to suppress HIV infection but now officially called CCL5, CCL3 and CCL4 respectively. I*n vitro* studies have demonstrated several CC chemokines bind CCR5. However, CCL3, CCL4, CCL5, and CCL8 have shown the most suppressive effects in HIV-1 infection assays. To date ten members of the CC chemokine receptor subfamily have been described, namely CCR1 through to CCR10 according to the IUIS/WHO Subcommittee on Chemokine Nomenclature. CCR5 is one of the major coreceptor implicated in susceptibility to HIV-1 infection and disease progression. The lack of CCR5 gene expression has been associated with resistant to HIV-1 infection as

makes them difficult to isolate.

**4. CXC chemokine receptors** 

inefficient transmission of CXCR4-tropic HIV isolates.

**5. CC chemokine receptors** 

will be discussed later on.

**Figure 1.** Typical structure of a chemokine receptor

#### **3. Classification of chemokine receptors**

To date, approximately 50 human chemokines and 20 receptors have been discovered. Basically chemokines and their receptors are divided into four subfamilies; CXC, CC, CX3C and XC depending on the position of the two pairs of the highly conserved cysteine residues on the ligands, where C denotes the cysteine amino acid residue whilst X represents noncysteine amino acids. For the main subfamilies, the first two cysteines are either contiguous (CC) or intercalated by one non cysteine amino acid residue (CXC). A system of nomenclature has been introduced where each ligand and receptor is identified by its subfamily and an identifying number. For example, CCL2 refers to a chemokine ligand of the CC subfamily, number 2. Similarly, the **r**eceptors are referred to by subfamily **R** and similarly an identifying number. Thus, for instance one of the receptors of CCL5 is called CCR5. Despite the numerous chemokine receptors that are prospective coreceptors for HIV *in vitro*, only CCR5 and CXCR4 have attracted substantial interest because they form portals of cellular entry for both HIV-1 and HIV-2 inclusive of other related simian or feline retroviruses. The lack of crystallography for these two highly hydrophobic coreceptors makes them difficult to isolate.

### **4. CXC chemokine receptors**

236 Immunodeficiency

number of ligands.

**2. Structure of chemokine receptors** 

**Figure 1.** Typical structure of a chemokine receptor

**3. Classification of chemokine receptors** 

Chemokine receptors consist of about 350 amino acids that are divided into a short and acidic N-terminal end, seven helical transmembrane domains since they span the cell membrane seven times with three intracellular and three extracellular hydrophilic loops, and an intracellular C-terminus containing serine and threonine residues that act as phosphorylation sites during receptor regulation as shown in **Figure 1** below. Chemokine receptors are usually linked to a G-protein through which they signal. The N-terminus of the chemokine receptor is an extracellular domain that binds the chemokine(s) and has been shown to influence infection tropism. The first two extracellular loops of chemokine receptors are linked together by disulphide bonding between two conserved cysteine residues. The disulfide bonds keep the extracellular loops in place thereby maintaining the structural integrity necessary for ligand binding and signal transduction. In spite of the high amino acid similarity of their primary sequences, chemokine receptors bind a limited

To date, approximately 50 human chemokines and 20 receptors have been discovered. Basically chemokines and their receptors are divided into four subfamilies; CXC, CC, CX3C and XC depending on the position of the two pairs of the highly conserved cysteine residues on the ligands, where C denotes the cysteine amino acid residue whilst X represents noncysteine amino acids. For the main subfamilies, the first two cysteines are either contiguous (CC) or intercalated by one non cysteine amino acid residue (CXC). A system of nomenclature has been introduced where each ligand and receptor is identified by its subfamily and an identifying number. For example, CCL2 refers to a chemokine ligand of the CC subfamily, number 2. Similarly, the **r**eceptors are referred to by subfamily **R** and The first HIV coreceptor to be identified was CXCR4 which was originally an orphan receptor called leukocyte-derived seven-transmembrane domain receptor (LESTR). By then it did not receive much attention until it was recognized as a coreceptor for HIV-1. Thus, in the mid 1990s the second receptor, CXCR4, needed for successful entry of HIV into cells was discovered. At that time this coreceptor was termed "fusin" as it facilitated certain HIV strains to fuse with and enter immune cells called T cells. A detailed analysis of the structure of fusin revealed that it was a receptor for chemokines which were previously shown to suppress HIV activity. CXCR4 has a wide cellular distribution. It is commonly expressed on most immature and mature hematopoietic cell types, cells of the central nervous system, neutrophils, monocytes, T and B cells, dendritic cells (DCs), Langerhans cells and macrophages. To date there are seven CXC chemokine receptors in mammals, named CXCR1 through CXCR7. The CXC chemokines include stromal cell-derived factor-1 alpha and beta now officially designated CXCL12a and CXCL12b respectively. CXCL12 is often induced by pro-inflammatory stimuli such as lipopolysaccharide, interleukin-1 (IL-1) or tumour necrosis factor-alpha (TNF-α) and has been shown to be strongly chemotactic for lymphocytes. Its high level of expression in the genital mucosa may help to explain the inefficient transmission of CXCR4-tropic HIV isolates.

#### **5. CC chemokine receptors**

Another chemokine receptor necessary for the entry of HIV into macrophages called CCR5 that was subsequently identified. CCR5 receptors have been shown to be involved in leukocyte activation and mobilization. CCR5 is expressed on several cell types including peripheral blood-derived DCs, CD34+ hematopoietic progenitor cells and some activated/memory Th1 lymphocyte. Chemokines that bind these receptors are RANTES (regulated-upon-activation normal T expressed and secreted); MIP-1α and MIP-1β (macrophage inflammatory protein-1 alpha and beta) previously observed to suppress HIV infection but now officially called CCL5, CCL3 and CCL4 respectively. I*n vitro* studies have demonstrated several CC chemokines bind CCR5. However, CCL3, CCL4, CCL5, and CCL8 have shown the most suppressive effects in HIV-1 infection assays. To date ten members of the CC chemokine receptor subfamily have been described, namely CCR1 through to CCR10 according to the IUIS/WHO Subcommittee on Chemokine Nomenclature. CCR5 is one of the major coreceptor implicated in susceptibility to HIV-1 infection and disease progression. The lack of CCR5 gene expression has been associated with resistant to HIV-1 infection as will be discussed later on.

#### **6. CX3CR1**

CX3C chemokine receptor 1 (CX3CR1) also known as the fractalkine receptor or G-protein coupled receptor 13 (GPR13) has been shown to bind chemokine CX3CL1, also called fractalkine. Fractalkine is a transmembrane chemokine involved in the adhesion and migration of leukocytes. CX3CR1 is expressed on monocytes and plays a major role in the survival of monocytes. It has been shown to interact with human respiratory syncytial virus protein G consequently, modulating the host immune response. It also interacts with HIV-1 envelope (env) polyprotein glycoprotein (gp) 160. Thus CX3CR1 is also a minor coreceptor for HIV-1. Certain variations in the expression this gene has been associated with increased susceptibility to HIV-1 infection and rapid progression to AIDS.

Coreceptor Usage in HIV Infection 239

rapidly in cultures forming syncytia in target cells and the other group that replicate in macrophages, grow relatively slowly in culture but are not able to induce syncytia, SI and NSI, respectively. The formation of syncytia does not always happen in HIV infected people. However, autopsies have found syncytia in the spleens of some patients. More frequently CD4 syncytia have been observed in the brains of patients who would have died from

Following the identification of the coreceptors, HIV-1 isolates have also been characterized based on their ability to infect and induce syncytia in CD4+ T-cell lines that express CXCR4 but not CCR5.While all the HIV-1 strains require CD4 to enter and infect cells, some isolates utilize the chemokine receptors X4 or R5 while other variants use both receptors, dual tropic (R5X4) strains for binding and entry. Coreceptor usages correspond to the phenotypes previously defined by the MT-2 assay with SI and NSI viruses using CXCR4 and CCR5, respectively. Dual tropic isolates exhibit both the M and T tropic characteristics. They are further classified as dual-R; R5X4 variants with more efficient use of CCR5 than of CXCR4 or dual-X; R5X4 with more efficient use of CXCR4 than of CCR5. *In vitro* studies have shown that some HIV-1 strains can use a variety of other chemokine receptors. Interestingly, this does not appear to have major relevance to HIV infection nor pathogenesis *in vivo*. MT-2 positive variants are defined as either X4 or R5X4. Absence of viral growth in this assay may

be either due to the exclusive presence of R5 variants or failure to isolate HIV.

Frequencies of R5 HIV-1 variants vary among different populations, being 80% and 50% in drug naïve individuals and patients receiving antiretroviral therapy, respectively. Tropism shifts have been associated with long term use of antiretroviral therapy. Some studies have shown that drug selection pressure may gradually select for pre-existing X4 virus from cellular reservoirs during sustained highly active antiretroviral therapy. Thus, the use of CCR5 coreceptor antagonists is associated with a selective pressure that promotes the

There are different theories regarding the origin of X4 viruses. One theory postulates that X4 viruses emerge directly from the pre-existing R5 pool as a result of mutations within the HIV V3 loop *env* gene. At least for HIV-1 subtype C, X4 variants have been associated with an amino acid substitution mutation from the conserved GPGQ crown motif to a GPGR. Still some authors argue that X4 viruses are already in the existing pool of viruses but somehow the X4 viruses and R5/X4 viruses remain suppressed by varied host mechanisms and only to be detected in the late phases of infection suggestive that the immune system exerts a selective pressure that hinders the emergence of CXCR4-using variants. However, when the host immune competence begins to deteriorate during the HIV disease progression, it paves way for the emergence of CXCR4-using variants. Another hypothesis proposes that chemokines and the C2-V3-C3 region of HIV gp120 have a common origin such that the HIV

serious AIDS related neurological complications.

emergence of CXCR4-using variants.

**11. Evolution of X4 viruses** 

**10. HIV strains classification based on tropism** 
