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.     

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.     

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.     

The cytomegalovirus-encoded chemokine receptor US28 can enhance cell-cell fusion mediated by different viral proteins

The human cytomegalovirus (CMV) US28 gene encodes a functional CC chemokine receptor. However, this activity was observed in cells transfected to express US28 and might not correspond to the actual role of the protein in the CMV life cycle. Expression of US28 allows human immunodeficiency virus type 1 (HIV-1) entry into certain CD4(+) cells and their fusion with cells expressing HIV-1 envelope (Env) proteins. Such properties were initially reported for the cellular chemokine receptors CCR5 and CXCR4, which behave as CD4-associated HIV-1 coreceptors. We found that coexpression of US28 and either CXCR4 or CCR5 in CD4(+) cells resulted in enhanced synctium formation with HIV-1 Env+ cells. This positive effect of US28 on cell fusion seems to be distinct from its HIV-1 coreceptor activity. Indeed, enhancement of cell fusion was also observed when US28 was expressed on the HIV-1 Env+ cells instead of an CD4(+) target cells. Furthermore, US28 could enhance cell fusion mediated by other viral proteins, in particular, the G protein of vesicular stomatitis virus (VSV-G). The HIV-1 coreceptor and fusion-enhancing activities could be affected by mutations in different domains of US28. The fusion-enhancing activity of US28 seems to be cell type dependent. Indeed, cells coexpressing VSV-G and US28 fused more efficiently with human, simian, or feline target cells, while US28 had no apparent effect on fusion with the three mouse or rat cell lines tested. The positive effect of US28 on cell fusion might therefore require its interaction with a cell-specific factor. We discuss a possible role for US28 in the fusion of the CMV envelope with target cells and CMV entry.     

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.     

CD4-independent, CCR5-dependent infection of brain capillary endothelial cells by a neurovirulent simian immunodeficiency virus strain

Brain capillary endothelial cells (BCECs) are targets of CD4-independent infection by HIV-1 and simian immunodeficiency virus (SIV) strains in vitro and in vivo. Infection of BCECs may provide a portal of entry for the virus into the central nervous system and could disrupt blood-brain barrier function, contributing to the development of AIDS dementia. We found that rhesus macaque BCECs express chemokine receptors involved in HIV and SIV entry including CCR5, CCR3, CXCR4, and STRL33, but not CCR2b, GPR1, or GPR15. Infection of BCECs by the neurovirulent strain SIV/17E-Fr was completely inhibited by aminooxypentane regulation upon activation, normal T cell expression and secretion in the presence or absence of ligands, but not by eotaxin or antibodies to CD4. We found that the envelope (env) proteins from SIV/17E-Fr and several additional SIV strains mediated cell-cell fusion and virus infection with CD4-negative, CCR5-positive cells. In contrast, fusion with cells expressing the coreceptors STRL33, GPR1, and GPR15 was CD4-dependent. These results show that CCR5 can serve as a primary receptor for SIV in BCECs and suggest a possible CD4-independent mechanism for blood-brain barrier disruption and viral entry into the central nervous system.     

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.     

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.     

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.     

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.     

Dendritic cells express multiple chemokine receptors used as coreceptors for HIV entry

Cells of the dendritic lineage are thought to be among the first cells infected after mucosal exposure to HIV. In this study, we have identified the presence of multiple chemokine receptors on dendritic cells (DC) that may function as coreceptors for HIV entry. DC effectively used CCR5 for entry of macrophage (M)-tropic isolates. CCR3, the eotaxin receptor, initially identified on eosinophils, is expressed on DC and may be used as an entry coreceptor by certain dual-tropic strains. CXCR4 was not expressed on DC, although SDF-1 induced a calcium flux and DC could be infected by T cell line (T)-tropic HIV. Our findings provide evidence for the presence of a non-CXCR4 SDF-1 receptor on DC that is used mainly by T-tropic strains of HIV. DC from individuals homozygous for a 32-bp deletion of the CCR5 gene are also infectable with M-tropic strains of HIV-1, and this infection is inhibited by stromal cell-derived factor (SDF)1, suggesting that this receptor can also be used by M-tropic HIV for entry. Delineation of the spectrum of coreceptor usage on DC may offer new approaches to interfere with the initiation and propagation of HIV infection.     

Chemokine coreceptor usage by diverse primary isolates of human immunodeficiency virus type 1

We tested chemokine receptor subset usage by diverse, well-characterized primary viruses isolated from peripheral blood by monitoring viral replication with CCR1, CCR2b, CCR3, CCR5, and CXCR4 U87MG.CD4 transformed cell lines and STRL33/BONZO/TYMSTR and GPR15/BOB HOS.CD4 transformed cell lines. Primary viruses were isolated from 79 men with confirmed human immunodeficiency virus type 1 (HIV-1) infection from the Chicago component of the Multicenter AIDS Cohort Study at interval time points. Thirty-five additional well-characterized primary viruses representing HIV-1 group M subtypes A, B, C, D, and E and group O and three primary simian immunodeficiency virus (SIV) isolates were also used for these studies. The restricted use of the CCR5 chemokine receptor for viral entry was associated with infection by a virus having a non-syncytium-inducing phenotype and correlated with a reduced rate of disease progression and a prolonged disease-free interval. Conversely, broadening chemokine receptor usage from CCR5 to both CCR5 and CXCR4 was associated with infection by a virus having a syncytium-inducing phenotype and correlated with a faster rate of CD4 T-cell decline and progression of disease. We also observed a greater tendency for infection with a virus having a syncytium-inducing phenotype in men heterozygous for the defective CCR5 Delta32 allele (25%) than in those men homozygous for the wild-type CCR5 allele (6%) (P = 0.03). The propensity for infection with a virus having a syncytium-inducing phenotype provides a partial explanation for the rapid disease progression among some men heterozygous for the defective CCR5 Delta32 allele. Furthermore, we did not identify any primary viruses that used CCR3 as an entry cofactor, despite this CC chemokine receptor being expressed on the cell surface at a level commensurate with or higher than that observed for primary peripheral blood mononuclear cells. Whereas isolates of primary viruses of SIV also used STRL33/BONZO/TYMSTR and GPR15/BOB, no primary isolates of HIV-1 used these particular chemokine receptor-like orphan molecules as entry cofactors, suggesting a limited contribution of these other chemokine receptors to viral evolution. Thus, despite the number of chemokine receptors implicated in viral entry, CCR5 and CXCR4 are likely to be the physiologically relevant chemokine receptors used as entry cofactors in vivo by diverse strains of primary viruses isolated from blood.     

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 is required by a nonprimate lentivirus: heterologous expression of feline immunodeficiency virus in human, rodent, and feline cells

A heterologous feline immunodeficiency virus (FIV) expression system permitted high-level expression of FIV proteins and efficient production of infectious FIV in human cells. These results identify the FIV U3 element as the sole restriction to the productive phase of replication in nonfeline cells. Heterologous FIV expression in a variety of human cell lines resulted in profuse syncytial lysis that was FIV env specific, CD4 independent, and restricted to cells that express CXCR4, the coreceptor for T-cell-line-adapted strains of human immunodeficiency virus. Stable expression of human CXCR4 in CXCR4-negative human and rodent cell lines resulted in extensive FIV Env-mediated, CXCR4-dependent cell fusion and infection. In feline cells, stable overexpression of human CXCR4 resulted in increased FIV infectivity and marked syncytium formation during FIV replication or after infection with FIV Env-expressing vectors. The use of CXCR4 is a fundamental feature of lentivirus biology independent of CD4 and a shared cellular link to infection and cytopathicity for distantly related lentiviruses that cause AIDS. Their conserved use implicates chemokine receptors as primordial lentivirus receptors.     

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.     

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.     

Continued utilization of CCR5 coreceptor by a newly derived T-cell line-adapted isolate of human immunodeficiency virus type 1

The differential use of CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) may be intimately involved in the transmission and progression of human immunodeficiency virus infection. Changes in coreceptor utilization have also been noted upon adaptation of primary isolates (PI) to growth in established T-cell lines. All of the T-cell line-adapted (TCLA) viruses studied to date utilize CXCR4 but not CCR5. This observation had been suggested as an explanation for the sensitivity of TCLA, but not PI, viruses to neutralization by recombinant gp120 antisera and V3-directed monoclonal antibodies, but recent studies have shown coreceptor utilization to be independent of neutralization sensitivity. Here we describe a newly isolated TCLA virus that is sensitive to neutralization but continues to utilize both CXCR4 and CCR5 for infection. This finding further divorces coreceptor specificity from neutralization sensitivity and from certain changes in cell tropism. That the TCLA virus can continue to utilize CCR5 despite the changes that occur upon adaptation and in the apparent absence of CCR5 expression in the FDA/H9 T-cell line suggests that the interaction between envelope protein and coreceptor may be mediated by multiple weak interactions along a diffuse surface.