**8. Tropism**

HIV-1 was initially isolated from peripheral blood cells and consequently characterised as a virus that infects the CD4+ T-lymphocyte population, T tropic isolates. However, subsequent isolation of HIV-1 from non-lymphoid organs demonstrated that HIV-1 could also infect cells of the monocyte-macrophage lineage; macrophage tropic isolates. Studies have shown that T-cell and macrophage isolates display significant different biological properties with respect to cellular tropism, genetic diversity and relative replication rates including their inherent ability to induce syncytia.

### **9. MT-2 cell tropism**

Biological differences of HIV-1 isolates and depletion of CD4 positive lymphocytes have been shown to correlate with the pathogenesis of AIDS. Direct cytopathic effects of HIV can be studied *in vitro* in T cell lines. An MT-2 tumour cell line assay is generally used for the phenotypic characterisation of HIV-1 isolates. The ability of HIV-1 isolates to replicate in MT-2 cell lines is a prototype where viruses that do not infect MT-2 cells are designated nonsyncytium inducing (NSI), while those that infect cells are termed syncytium inducing (SI). Studies have shown that HIV-1 isolates from patients with low CD4 counts have been shown to replicate rapidly to high titres in peripheral blood mononuclear cells (PBMCs) with the infected cells forming syncytia and such isolates are called rapid/high replicating or syncytium inducing (SI). HIV-1 isolates from asymptomatic individuals replicate much slowly with low titres and such isolates are termed slow/low or NSI. Consequently, HIV-1 isolates can be classified into two main groups; those that replicate in T–cell lines, grow 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 serious AIDS related neurological complications.

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

238 Immunodeficiency

**7. XCR1** 

**8. Tropism** 

**9. MT-2 cell tropism** 

**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

The "C" sub-family of chemokine receptors contains only one member: XCR1, also known as GPR5. It is the receptor for chemokines XCL1 and XCL2 formerly, lymphotactin-1 and-2, respectively. This receptor is closely related to the MIP-1 α and RANTES yet to date its expression and the function of its ligand XCL1 remain elusive. However, at least in murine models, XCR1 has been shown to be expressed on CD8+ DCs and that its ligand XCL1 has

HIV-1 was initially isolated from peripheral blood cells and consequently characterised as a virus that infects the CD4+ T-lymphocyte population, T tropic isolates. However, subsequent isolation of HIV-1 from non-lymphoid organs demonstrated that HIV-1 could also infect cells of the monocyte-macrophage lineage; macrophage tropic isolates. Studies have shown that T-cell and macrophage isolates display significant different biological properties with respect to cellular tropism, genetic diversity and relative replication rates

Biological differences of HIV-1 isolates and depletion of CD4 positive lymphocytes have been shown to correlate with the pathogenesis of AIDS. Direct cytopathic effects of HIV can be studied *in vitro* in T cell lines. An MT-2 tumour cell line assay is generally used for the phenotypic characterisation of HIV-1 isolates. The ability of HIV-1 isolates to replicate in MT-2 cell lines is a prototype where viruses that do not infect MT-2 cells are designated nonsyncytium inducing (NSI), while those that infect cells are termed syncytium inducing (SI). Studies have shown that HIV-1 isolates from patients with low CD4 counts have been shown to replicate rapidly to high titres in peripheral blood mononuclear cells (PBMCs) with the infected cells forming syncytia and such isolates are called rapid/high replicating or syncytium inducing (SI). HIV-1 isolates from asymptomatic individuals replicate much slowly with low titres and such isolates are termed slow/low or NSI. Consequently, HIV-1 isolates can be classified into two main groups; those that replicate in T–cell lines, grow

shown to be a potent and highly specific chemo-attractant for this subset of DCs.

susceptibility to HIV-1 infection and rapid progression to AIDS.

including their inherent ability to induce syncytia.

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 emergence of CXCR4-using variants.

### **11. Evolution of X4 viruses**

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 ancestor incorporated a chemokine gene into its *env* gene such that this captured chemokine gene rapidly diverge by frequent mutations thereby attaining the ability to effectively interact with various chemokine receptors in a short period of time. Another possible explanation is based on cell division rate where it has been observed that X4 and R5 viruses show preferential tropism for naive and memory T cells, respectively. Since memory T cells divide 10 times more frequently than naive cells, it would be an advantage to have a tropism for CCR5 coreceptor during the first stages of infection.

Coreceptor Usage in HIV Infection 241

tropic/NSI whilst T-tropic/ SI are associated with disease progression. The emergence of CXCR4-using HIV-1 variants in a patient is almost invariably associated with a subsequent increase in the rate of decline of circulating CD4+ T cells, an accelerated disease progression and a poor prognosis for survival. While CXCR4-using variants can emerge at any stage of infection, untreated individuals who develop such variants progress to AIDS within an average of two years after their first detection. However, the presence of CXCR4-using variants is not an obligatory prerequisite for disease progression as a significant proportion of individuals' progress to AIDS whilst harbouring exclusive R5 HIV-1 variants. The emergence of SI HIV-1 in a sero-positive individual is now generally regarded as a negative prognostic indicator and thus considerable interest has been focused on the HIV-1 genetic determinants of the SI phenotype. Tropism assays are important for determining HIV phenotype before administering coreceptor antagonists in combination antiretroviral therapy. In view of this, tropism assays must taken by HIV infected people with drug resistance who are considering taking coreceptor antagonists. This new class of antiretroviral targets CCR5-specific HIV during entry into the cell. Thus, knowing the patient's viral tropism can assist and guide the clinician make an informed and effective HIV treatment plan. Consequently, the importance of tropism screening to detect the presence of

With the recent introduction of HIV-1 chemokine receptor antagonists on the market as components of antiretroviral therapy, it is increasingly important to screen HIV patients' coreceptor phenotype or genotype prior to therapy. Hence simple and efficient methods for routinely characterising and monitoring HIV-1 coreceptor phenotypes or genotypes are of paramount importance. Studies have demonstrated that the ability to induce syncytia is determined by regions of env outside the V3 loop that encompass residues that contribute to the binding of CD4 by gp120. Such observations suggest that areas of the HIV-1 *env* gene contributing to the CD4 binding site may also contribute to the determination of SI and NSI genotypes. HIV-1 tropism can be determined by phenotypic

HIV-1 tropism can be assessed using phenotypic assays which are currently the most accurate method based on recombinant viruses. Determination of coreceptor use of HIV-1 isolates is done in cell lines such as U87 and GHOST transfected with CCR5 or CXCR4. Patients' plasma is used to generate pseudoviruses or infectious recombinant viruses of fulllength or partial viral envelopes derived from the patient's viral population. The recombinants are subsequently tested on indicator cell lines expressing CD4 and either CCR5 or CXCR4. The first commercially available tropism assay became available on the market almost at the same time of approval of the CCR5 antagonist, maraviroc. Its brand name of phenotypic assay is Trofile® manufactured by Monogram Biosciences South San

R5-using HIV may not be over emphasized.

or genotypic based methods.

**15. Phenotypic methods** 

**14. Methods for determination of coreceptor usage** 

**Figure 2.** HIV classification adopted from Moyle G, 2008.
