Utilization of chemokine receptors, orphan receptors, and herpesvirus-encoded receptors by diverse human and simian immunodeficiency viruses

Human immunodeficiency virus type 1 (HIV-1) requires both CD4 and a coreceptor to infect cells. Macrophage-tropic (M-tropic) HIV-1 strains utilize the chemokine receptor CCR5 in conjunction with CD4 to infect cells, while T-cell-tropic (T-tropic) strains generally utilize CXCR4 as a coreceptor. Some viruses can use both CCR5 and CXCR4 for virus entry (i.e., are dual-tropic), while other chemokine receptors can be used by a subset of virus strains. Due to the genetic diversity of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) and the potential for chemokine receptors other than CCR5 or CXCR4 to influence viral pathogenesis, we tested a panel of 28 HIV-1, HIV-2, and SIV envelope (Env) proteins for the ability to utilize chemokine receptors, orphan receptors, and herpesvirus-encoded chemokine receptor homologs by membrane fusion and virus infection assays. While all Env proteins used either CCR5 or CXCR4 or both, several also used CCR3. Use of CCR3 was strongly dependent on its surface expression levels, with a larger number of viral Env proteins being able to utilize this coreceptor at the higher levels of surface expression. ChemR1, an orphan receptor recently shown to bind the CC chemokine I309 (and therefore renamed CCR8), was expressed in monocyte and lymphocyte cell populations and functioned as a coreceptor for diverse HIV-1, HIV-2, and SIV Env proteins. Use of ChemR1/CCR8 by SIV strains was dependent in part on V3 loop sequences. The orphan receptor V28 supported Env-mediated cell-cell fusion by four T- or dual-tropic HIV-1 and HIV-2 strains. Three additional orphan receptors failed to function for any of the 28 Env proteins tested. Likewise, five of six seven-transmembrane-domain receptors encoded by herpesviruses did not support Env-mediated membrane fusion. However, the chemokine receptor US28, encoded by cytomegalovirus, did support inefficient infection by two HIV-1 strains. These findings indicate that additional chemokine receptors can function as HIV and SIV coreceptors and that surface expression levels can strongly influence coreceptor use.     

A small-molecule inhibitor directed against the chemokine receptor CXCR4 prevents its use as an HIV-1 coreceptor

The chemokine receptor CXCR4 is the major coreceptor used for cellular entry by T cell- tropic human immunodeficiency virus (HIV)-1 strains, whereas CCR5 is used by macrophage (M)-tropic strains. Here we show that a small-molecule inhibitor, ALX40-4C, inhibits HIV-1 envelope (Env)-mediated membrane fusion and viral entry directly at the level of coreceptor use. ALX40-4C inhibited HIV-1 use of the coreceptor CXCR4 by T- and dual-tropic HIV-1 strains, whereas use of CCR5 by M- and dual-tropic strains was not inhibited. Dual-tropic viruses capable of using both CXCR4 and CCR5 were inhibited by ALX40-4C only when cells expressed CXCR4 alone. ALX40-4C blocked stromal-derived factor (SDF)-1alpha-mediated activation of CXCR4 and binding of the monoclonal antibody 12G5 to cells expressing CXCR4. Overlap of the ALX40-4C binding site with that of 12G5 and SDF implicates direct blocking of Env interactions, rather than downregulation of receptor, as the mechanism of inhibition. Thus, ALX40-4C represents a small-molecule inhibitor of HIV-1 infection that acts directly against a chemokine receptor at the level of Env-mediated membrane fusion.     

Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype

The biological phenotype of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according to the severity of the HIV infection. Here we show that the two previously described groups of rapid/high, syncytium-inducing (SI) and slow/low, non-syncytium-inducing (NSI) isolates are distinguished by their ability to utilize different chemokine receptors for entry into target cells. Recent studies have identified the C-X-C chemokine receptor CXCR4 (also named fusin or Lestr) and the C-C chemokine receptor CCR5 as the principal entry cofactors for T-cell-line-tropic and non-T-cell-line-tropic HIV-1, respectively. Using U87.CD4 glioma cell lines, stably expressing the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, we have tested chemokine receptor specificity for a panel of genetically diverse envelope glycoprotein genes cloned from primary HIV-1 isolates and have found that receptor usage was closely associated with the biological phenotype of the virus isolate but not the genetic subtype. We have also analyzed a panel of 36 well-characterized primary HIV-1 isolates for syncytium induction and replication in the same series of cell lines. Infection by slow/low viruses was restricted to cells expressing CCR5, whereas rapid/high viruses could use a variety of chemokine receptors. In addition to the regular use of CXCR4, many rapid/high viruses used CCR5 and some also used CCR3 and CCR2b. Progressive HIV-1 infection is characterized by the emergence of viruses resistant to inhibition by beta-chemokines, which corresponded to changes in coreceptor usage. The broadening of the host range may even enable the use of uncharacterized coreceptors, in that two isolates from immunodeficient patients infected the parental U87.CD4 cell line lacking any engineered coreceptor. Two primary isolates with multiple coreceptor usage were shown to consist of mixed populations, one with a narrow host range using CCR5 only and the other with a broad host range using CCR3, CCR5, or CXCR4, similar to the original population. The results show that all 36 primary HIV-1 isolates induce syncytia, provided that target cells carry the particular coreceptor required by the virus.     

Use of GPR1, GPR15, and STRL33 as coreceptors by diverse human immunodeficiency virus type 1 and simian immunodeficiency virus envelope proteins

Human and simian immunodeficiency viruses (HIV and SIV, respectively) use chemokine receptors as coreceptors along with CD4 to mediate viral entry. Several orphan receptors, including GPR1, GPR15, and STRL33, can also serve as coreceptors for a more limited number of HIV and SIV isolates. We investigated whether these orphan receptors could function as efficient coreceptors for a diverse group of HIV and SIV envelopes (Envs) in comparison with the principal coreceptors CCR5 and CXCR4. We found that a limited number of HIV-1 isolates could mediate inefficient cell-cell fusion with the orphan receptors relative to CCR5 and CXCR4; however, none of the orphan receptors tested could support pseudotype virus infection despite robust infection via CCR5 or CXCR4. All except one of the SIV Envs tested mediated some degree of cell-cell fusion and pseudotype infection, with target cells expressing at least one of these orphan receptors, although CCR5 proved to be the most efficient coreceptor for infection. Only one SIV Env protein, BK28, could mediate infection using GPR1 as a coreceptor, albeit much less efficiently than with CCR5. In addition, use of these coreceptors did not correlate with the published tropism of the SIV clones and was strictly CD4 dependent for both SIV and HIV. We also examined the expression of these molecules in cell lines and primary cells widely used for virus propagation and as targets for infection. All cells examined expressed STRL33, a more limited number expressed GPR15, and GPR1 was much more restricted in its expression pattern. Taken together, our results indicate that GPR15 and STRL33 are rarely used by HIV-1 but are more frequently used by SIV strains, although not in a manner that correlates with SIV tropism.     

Primary human immunodeficiency virus type 2 (HIV-2) isolates, like HIV-1 isolates, frequently use CCR5 but show promiscuity in coreceptor usage

Coreceptor usage of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according to biological phenotype. The chemokine receptors CCR5 and CXCR4 are the major coreceptors that, together with CD4, govern HIV-1 entry into cells. Since CXCR4 usage determines the biological phenotype for HIV-1 isolates and is more frequent in patients with immunodeficiency, it may serve as a marker for viral virulence. This possibility prompted us to study coreceptor usage by HIV-2, known to be less pathogenic than HIV-1. We tested 11 primary HIV-2 isolates for coreceptor usage in human cell lines: U87 glioma cells, stably expressing CD4 and the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, and GHOST(3) osteosarcoma cells, coexpressing CD4 and CCR5, CXCR4, or the orphan receptor Bonzo or BOB. The indicator cells were infected by cocultivation with virus-producing peripheral blood mononuclear cells and by cell-free virus. Our results show that 10 of 11 HIV-2 isolates were able to efficiently use CCR5. In contrast, only two isolates, both from patients with advanced disease, used CXCR4 efficiently. These two isolates also promptly induced syncytia in MT-2 cells, a pattern described for HIV-1 isolates that use CXCR4. Unlike HIV-1, many of the HIV-2 isolates were promiscuous in their coreceptor usage in that they were able to use, apart from CCR5, one or more of the CCR1, CCR2b, CCR3, and BOB coreceptors. Another difference between HIV-1 and HIV-2 was that the ability to replicate in MT-2 cells appeared to be a general property of HIV-2 isolates. Based on BOB mRNA expression in MT-2 cells and the ability of our panel of HIV-2 isolates to use BOB, we suggest that HIV-2 can use BOB when entering MT-2 cells. The results indicate no obvious link between viral virulence and the ability to use a multitude of coreceptors.     

Expression of chemokine receptors CXCR4 and CCR5 in HIV-1-infected and uninfected individuals

The chemokine receptors CXCR4 and CCR5 have been identified as major coreceptors for HIV-1 entry into CD4+ T cells. The majority of primary HIV-1 isolates in early disease use CCR5 as a coreceptor, whereas during disease progression with the emergence of syncytium-inducing viruses, CXCR4 is also used. We performed a cross-sectional study in which we evaluated the expression of two HIV-1 coreceptors, CCR5 and CXCR4, in whole blood samples taken from HIV-1-infected and uninfected individuals. We demonstrate that CXCR4 on CD4+ and CD8+ T cells, and CD14+ monocytes is significantly down-regulated, and CCR5 expression on CD4+ T cells is up-regulated in HIV-infected individuals compared with uninfected controls. Coreceptor expression correlated with the level of cellular activation in vivo in both HIV-infected and uninfected individuals, with CXCR4 being expressed predominantly on quiescent (HLA-DR-) T cells and CCR5 being expressed predominantly on activated (HLA-DR+) T cells. Lower expression of CXCR4 and higher expression of CCR5 on CD4+ T cells correlated with advancing disease. In addition, a tendency for greater activation of CXCR4+CD4+ T cells in patients with advanced disease was observed. Patients who harbored syncytium-inducing viruses, however, could not be distinguished from those who harbored nonsyncytium-inducing viruses based on the level of CD4+ T cell activation or chemokine receptor expression.     

Evolution of HIV-1 coreceptor usage through interactions with distinct CCR5 and CXCR4 domains

The chemokine receptor CXCR4 functions as a fusion coreceptor for T cell tropic and dual-tropic HIV-1 strains. To identify regions of CXCR4 that are important for coreceptor function, CXCR4-CXCR2 receptor chimeras were tested for the ability to support HIV-1 envelope (env) protein-mediated membrane fusion. Receptor chimeras containing the first and second extracellular loops of CXCR4 supported fusion by T tropic and dual-tropic HIV-1 and HIV-2 strains and binding of a monoclonal antibody to CXCR4, 12G5, that blocks CXCR4-dependent infection by some virus strains. The second extracellular loop of CXCR4 was sufficient to confer coreceptor function to CXCR2 for most virus strains tested but did not support binding of 12G5. Truncation of the CXCR4 cytoplasmic tail or mutation of a conserved DRY motif in the second intracellular loop did not affect coreceptor function, indicating that phosphorylation of the cytoplasmic tail and the DRY motif are not required for coreceptor function. The results implicate the involvement of multiple CXCR4 domains in HIV-1 coreceptor function, especially the second extracellular loop, though the structural requirements for coreceptor function were somewhat variable for different env proteins. Finally, a hybrid receptor in which the amino terminus of CXCR4 was replaced by that of CCR5 was active as a coreceptor for M tropic, T tropic, and dual-tropic env proteins. We propose that dual tropism may evolve in CCR5-restricted HIV-1 strains through acquisition of the ability to utilize the first and second extracellular loops of CXCR4 while retaining the ability to interact with the CCR5 amino-terminal domain.     

Identification of determinants on a dualtropic human immunodeficiency virus type 1 envelope glycoprotein that confer usage of CXCR4

The chemokine receptors CCR5 and CXCR4, in combination with CD4, mediate cellular entry of macrophage-tropic (M-tropic) and T-cell-tropic strains of human immunodeficiency virus type 1 (HIV-1), respectively, while dualtropic viruses can use either receptor. We have constructed a panel of chimeric viruses and envelope glycoproteins in which various domains of the dualtropic HIV-1(DH12) gp160 were introduced into the genetic background of an M-tropic HIV-1 isolate, HIV-1(AD8). These constructs were employed in cell fusion and virus infectivity assays using peripheral blood mononuclear cells, MT4 T cells, primary monocyte-derived macrophages, or HOS-CD4 cell lines, expressing various chemokine receptors, to assess the contributions of different gp120 subdomains in coreceptor usage and cellular tropism. As expected, the dualtropic HIV-1(DH12) gp120 utilized either CCR3, CCR5, or CXCR4, whereas HIV-1(AD8) gp120 was able to use only CCR3 or CCR5. We found that either the V1/V2 or the V3 region of HIV-1(DH12) gp120 individually conferred on HIV-1(AD8) the ability to use CXCR4, while the combination of both the V1/V2 and V3 regions increased the efficiency of CXCR4 use. In addition, while the V4 or the V5 region of HIV-1(DH12) gp120 failed to confer the capacity to utilize CXCR4 on HIV-1(AD8), these regions were required in conjunction with regions V1 to V3 of HIV-1(DH12) gp120 for efficient utilization of CXCR4. Comparison of virus infectivity analyses with various cell types and cell fusion assays revealed assay-dependent discrepancies and indicated that events occurring at the cell surface during infection are complex and cannot always be predicted by any one assay.     

STRL33, A novel chemokine receptor-like protein, functions as a fusion cofactor for both macrophage-tropic and T cell line-tropic HIV-1

The chemokine receptors CXCR4, CCR2B, CCR3, and CCR5 have recently been shown to serve along with CD4 as coreceptors for HIV-1. The tropisms of HIV-1 strains for subgroups of CD4(+) cells can be explained, at least partly, by the selective use of G protein-coupled receptors (GPCRs). We have identified a novel human gene, STRL33, located on chromosome 3 that encodes a GPCR with sequence similarity to chemokine receptors and to chemokine receptor-like orphan receptors. STRL33 is expressed in lymphoid tissues and activated T cells, and is induced in activated peripheral blood lymphocytes. When transfected into nonhuman NIH 3T3 cells expressing human CD4, the STRL33 cDNA rendered these cells competent to fuse with cells expressing HIV-1 envelope glycoproteins (Envs). Of greatest interest, STRL33, in contrast with CXCR4 or CCR5, was able to function as a cofactor for fusion mediated by Envs from both T cell line-tropic and macrophage-tropic HIV-1 strains. STRL33-transfected Jurkat cell lines also supported enhanced productive infection with HIV-1 compared with control Jurkat cells. Despite the sequence similarities between STRL33 and chemokine receptors, STRL33-transfected cell lines did not respond to any in a panel of chemokines. Based on the pattern of tissue expression of the STRL33 mRNA, and given the ability of STRL33 to function with Envs of differing tropisms, STRL33 may play a role in the establishment and/or progression of HIV-1 infection.     

CXCR4 utilization is sufficient to trigger CD4+ T cell depletion in HIV-1-infected human lymphoid tissue

The human chemokine receptors CCR5 and CXCR4 have emerged as the predominant cofactors, along with CD4, for cellular entry of HIV-1 in vivo whereas the contribution of other chemokine receptors to HIV disease has not been yet determined. CCR5-specific (R5) viruses predominate during primary HIV-1 infection whereas viruses with specificity for CXCR4 (R5/X4 or X4 viruses) often emerge in late stages of HIV disease. The evolution of X4 viruses is associated with a rapid decline in CD4+ T cells, although a causative relationship between viral tropism and CD4+ T cell depletion has not yet been proven. To rigorously test this relationship, we assessed CD4+ T cell depletion in suspensions of human peripheral blood mononuclear cells and in explants of human lymphoid tissue on exposure to paired viruses that are genetically identical (isogenic) except for select envelope determinants specifying reciprocal tropism for CXCR4 or CCR5. In both systems, X4 HIV-1 massively depleted CD4+ lymphocytes whereas matched R5 viruses depleted such cells only mildly despite comparable viral replication kinetics. These findings demonstrate that the coreceptor specificities of HIV-1 are a causal factor in CD4+ T cell depletion ex vivo and strongly support the hypothesis that the evolution of viral envelope leading to usage of CXCR4 in vivo accelerates loss of CD4+ T cells, causing immunodeficiency.     

A small molecule CXCR4 inhibitor that blocks T cell line-tropic HIV-1 infection

Several members of the chemokine receptor family have been shown to function in association with CD4 to permit human immunodeficiency virus type 1 (HIV-1) entry and infection. The CXC chemokine receptor CXCR4/fusin is a receptor for pre-B cell growth stimulating factor (PBSF)/stromal cell-derived factor 1 (SDF-1) and serves as a coreceptor for the entry of T cell line-tropic HIV-1 strains. Thus, the development of CXCR4 antagonists or agonists may be useful in the treatment of HIV-1 infection. T22 ([Tyr5,12,Lys7]-polyphemusin II) is a synthesized peptide that consists of 18 amino acid residues and an analogue of polyphemusin II isolated from the hemocyte debris of American horseshoe crabs (Limulus polyphemus). T22 was found to specifically inhibit the ability of T cell line-tropic HIV-1 to induce cell fusion and infect the cell lines transfected with CXCR4 and CD4 or peripheral blood mononuclear cells. In addition, T22 inhibited Ca2+ mobilization induced by pre-B cell growth stimulating factor (PBSF)/SDF-1 stimulation through CXCR4. Thus, T22 is a small molecule CXCR4 inhibitor that blocks T cell line-tropic HIV-1 entry into target cells.     

Promonocytic U937 subclones expressing CD4 and CXCR4 are resistant to infection with and cell-to-cell fusion by T-cell-tropic human immunodeficiency virus type 1

Different strains of human immunodeficiency virus type 1 (HIV-1) vary markedly in the ability to infect cells of the monocyte/macrophage (M/M) lineage. M/M are generally resistant to infection with T-cell-tropic (T-tropic) strains of HIV-1. Recently, the chemokine receptors CCR5 and CXCR4 were identified as cofactors for fusion/entry of macrophage- and T-tropic strains of HIV-1, respectively. To investigate the mechanisms of resistance of M/M to T-tropic HIV-1 infection, we examined a number of subclones of the U937 promonocytic cell line. We found that certain subclones of U937 (plus clones) could, while others (minus clones) could not, support replication of T-tropic strains of HIV-1. We demonstrate that (i) both minus and plus clones support HIV-1 replication when transfected with an infectious molecular cDNA clone of a T-tropic HIV-1; (ii) minus clones do not, but plus clones do, efficiently support fusion with cells expressing HIV-1 IIIB Env; (iii) both plus and minus clones (with the exception of one clone) express physiologically functional CXCR4 protein as well as CD4 on the cell surface; (iv) introduction of CXCR4 into the CXCR4-negative clone does not restore fusogenicity with or susceptibility to T-tropic HIV-1; and (v) a ligand (stromal cell-derived factor 1) for or a monoclonal antibody (12G5) to CXCR4 does not effectively inhibit HIV-mediated cell-to-cell fusion of U937 cells. These data indicate that resistance to T-tropic HIV-1 infection of U937 minus clones occurs at fusion/ entry events and that expression of functional CXCR4 and CD4 is not a sole determinant for susceptibility to T-tropic HIV-1 infection; furthermore, they suggest that other factors are positively or negatively involved in HIV-mediated cell-to-cell fusion in U937 promonocytic cells.     

CXCR4 and CD4 mediate a rapid CD95-independent cell death in CD4(+) T cells

AIDS is characterized by a progressive decrease of CD4(+) helper T lymphocytes. Destruction of these cells may involve programmed cell death, apoptosis. It has previously been reported that apoptosis can be induced even in noninfected cells by HIV-1 gp120 and anti-gp120 antibodies. HIV-1 gp120 binds to T cells via CD4 and the chemokine coreceptor CXCR4 (fusin/LESTR). Therefore, we investigated whether CD4 and CXCR4 mediate gp120-induced apoptosis. We used human peripheral blood lymphocytes, malignant T cells, and CD4/CXCR4 transfectants, and found cell death induced by both cell surface receptors, CD4 and CXCR4. The induced cell death was rapid, independent of known caspases, and lacking oligonucleosomal DNA fragmentation. In addition, the death signals were not propagated via p56(lck) and Gialpha. However, the cells showed chromatin condensation, morphological shrinkage, membrane inversion, and reduced mitochondrial transmembrane potential indicative of apoptosis. Significantly, apoptosis was exclusively observed in CD4(+) but not in CD8(+) T cells, and apoptosis triggered via CXCR4 was inhibited by stromal cell-derived factor-1, the natural CXCR4 ligand. Thus, this mechanism of apoptosis might contribute to T cell depletion in AIDS and might have major implications for therapeutic intervention.     

Selective employment of chemokine receptors as human immunodeficiency virus type 1 coreceptors determined by individual amino acids within the envelope V3 loop

The chemokine receptor CCR5 acts as an essential cofactor for cell entry by macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains, whereas CXCR4 acts as an essential cofactor for T-cell-line-adapted strains. We demonstrated that the specific amino acids in the V3 loop of the HIV-1 envelope protein that determine cellular tropism also regulate chemokine coreceptor preference for cell entry by the virus. Further, a strong correlation was found between HIV-1 strains classified as syncytium inducing in standard assays and those using CXCR4 as a coreceptor. These data support the hypothesis that progressive adaptation to additional coreceptors is a key molecular basis for HIV-1 phenotypic evolution in vivo.     

Determinant in human immunodeficiency virus type 1 for efficient replication under cytokine-induced CD4(+) T-helper 1 (Th1)- and Th2-type conditions

Cytokines are potent stimuli for CD4(+)-T-cell differentiation. Among them, interleukin-12 (IL-12) and IL-4 induce naive CD4(+) T cells to become T-helper 1 (Th1) or Th2 cells, respectively. In this study we found that macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains replicated more efficiently in IL-12-induced Th1-type cultures derived from normal CD4(+) T cells than did T-cell-line-tropic (T-tropic) strains. In contrast, T-tropic strains preferentially infected IL-4-induced Th2-type cultures derived from the same donor CD4(+) T cells. Additional studies using chimeric viruses demonstrated that the V3 region of HIV-1 gp120 was the principal determinant for efficiency of replication. Cell fusion analysis showed that cells expressing envelope protein from a T-tropic strain effectively fused with IL-4-induced Th2-type culture cells. Flow cytometric analysis showed that the level of CCR5 expression was higher on IL-12-induced Th1-type culture cells, whereas CXCR4 was highly expressed on IL-4-induced Th2-type culture cells, although a low level of CXCR4 expression was observed on IL-12-induced Th1-type culture cells. These results indicate that HIV-1 isolates exhibit differences in the ability to infect CD4(+)-T-cell subsets such as Th1 or Th2 cells and that this difference may partly correlate with the expression of particular chemokine receptors on these cells. The findings suggest that immunological conditions are one of the factors responsible for inducing selection of HIV-1 strains.     

Microglia express CCR5, CXCR4, and CCR3, but of these, CCR5 is the principal coreceptor for human immunodeficiency virus type 1 dementia isolates

Microglia are the main human immunodeficiency virus (HIV) reservoir in the central nervous system and most likely play a major role in the development of HIV dementia (HIVD). To characterize human adult microglial chemokine receptors, we analyzed the expression and calcium signaling of CCR5, CCR3, and CXCR4 and their roles in HIV entry. Microglia expressed higher levels of CCR5 than of either CCR3 or CXCR4. Of these three chemokine receptors, only CCR5 and CXCR4 were able to transduce a signal in microglia in response to their respective ligands, MIP-1beta and SDF-1alpha, as recorded by single-cell calcium flux experiments. We also found that CCR5 is the predominant coreceptor used for infection of human adult microglia by the HIV type 1 dementia isolates HIV-1DS-br, HIV-1RC-br, and HIV-1YU-2, since the anti-CCR5 antibody 2D7 was able to dramatically inhibit microglial infection by both wild-type and single-round luciferase pseudotype reporter viruses. Anti-CCR3 (7B11) and anti-CXCR4 (12G5) antibodies had little or no effect on infection. Last, we found that virus pseudotyped with the DS-br and RC-br envelopes can infect cells transfected with CD4 in conjunction with the G-protein-coupled receptors APJ, CCR8, and GPR15, which have been previously implicated in HIV entry.     

HIV type 1 subtypes, coreceptor usage, and CCR5 polymorphism

Identification of the chemokine receptors CCR5 and CXCR4 as the major coreceptors for HIV-1 entry has greatly assisted our understanding of HIV-1 pathogenesis, transmission, and tropism. However, most of our current knowledge on coreceptor usage comes from studies using HIV-1 strains or env genes derived from the genetic subtype B predominant in North America and western Europe. In this report, the coreceptor usage of 20 primary viral isolates representative of genetic subtypes A, B, C, D, E, and group O was examined. Thirty-nine full-length CCR5 sequences from individuals of diverse geographic origins were also obtained to examine the possible effect of CCR5 polymorphism on HIV-1 subtype distribution. Our results indicate that (1) CCR5 and CXCR4 serve as the two major coreceptors for viruses belonging to HIV-1 subtypes A, B, C, D, E, and group O, whereas other chemokine receptors such as CCR2b and CCR3 play only a minor role in facilitating viral entry into stimulated PBMCs; (2) the coreceptor usage is determined by the viral phenotype rather than its genotype because all NSI strains, irrespective of their subtype classification, utilize CCR5, whereas all SI strains are able to use CXCR4; and (3) there is no geographic clustering of CCR5 polymorphism in different ethnic populations, suggesting that CCR5 diversity is not the underlying explanation for differences in the spread of different HIV-1 subtypes. Therefore, the uneven worldwide distribution of HIV-1 subtypes is more likely the result of stochastic dissemination.     

Spontaneous mutations in the env gene of the human immunodeficiency virus type 1 NDK isolate are associated with a CD4-independent entry phenotype

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is a multistep process initiated by envelope protein gp120 binding to cell surface CD4. The conformational changes induced by this interaction likely favor a second-step interaction between gp120 and a coreceptor such as CXCR4 or CCR5. Here, we report a spontaneous and stable CD4-independent entry phenotype for the HIV-1 NDK isolate. This mutant strain, which emerged from a population of chronically infected CD4-positive CEM cells, can replicate in CD4-negative human cell lines. The presence of CXCR4 alone renders cells susceptible to infection by the mutant NDK, and infection can be blocked by the CXCR4 natural ligand SDF-1. Furthermore, we have correlated the CD4-independent phenotype with seven mutations in the C2 and C3 regions and the V3 loop. We propose that the mutant gp120 spontaneously acquires a conformation allowing it to interact directly with CXCR4. This virus provides us with a powerful tool to study directly gp120-CXCR4 interactions.