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.     

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.     

Coreceptor Utilization by Human Immunodeficiency Virus Type 1 Is Not a Primary Determinant of Neutralization Sensitivity

We have examined the relationship between coreceptor utilization and sensitivity to neutralization in a primary isolate of human immunodeficiency virus type 1 and its T-cell line-adapted (TCLA) derivative. We determined that adaptation of the primary-isolate (PI) virus 168P results in the loss of the unique capacity of PI viruses to utilize the CCR5 coreceptor and in the acquisition by the TCLA 168C virus of sensitivity to neutralization by V3-directed monoclonal antibodies (MAbs). In experiments wherein infection by 168P is directed via either the CCR5 or the CXCR4 pathway, we demonstrate that the virus, as well as pseudotyped virions bearing a molecularly cloned 168P envelope protein, remains refractory to neutralization by MAbs 257-D, 268-D, and 50.1 regardless of the coreceptor utilized. This study suggests that coreceptor utilization is not a primary determinant of differential neutralization sensitivity in PI and TCLA viruses.Although CD4 had long been recognized as the cellular receptor to which the human immunodeficiency virus type 1 (HIV) envelope protein binds (9, 21, 22), it had also been recognized that expression of CD4 alone is insufficient to render nonhuman cells susceptible to HIV infection (4, 5, 22). Similarly, different HIV isolates display different abilities to infect CD4-positive human macrophages, T lymphocytes, and established T-cell lines (31, 32, 35), suggesting that additional molecules may be responsible for cell tropism specificity. During the past year, cellular molecules that act in conjunction with CD4 have been identified as required cofactors for HIV envelope protein-mediated binding and entry (1, 6, 10–12, 14). These HIV coreceptors are members of the superfamily of seven-transmembrane segment G-protein-coupled receptors and act primarily as cellular receptors for chemokines.The discovery of cellular coreceptors for HIV has provided new perspectives for understanding these early events in HIV infection (see review in reference 2). Thus, phenotypically distinct isolates of HIV utilize as coreceptors different chemokine receptor molecules. Although all primary isolates of HIV infect primary T lymphocytes, some also infect cells of the macrophage lineage (31, 32). These monocyteropic isolates utilize the CCR5 chemokine receptor, whose natural ligands include the chemokines RANTES, MIP-1α, and MIP-1β (1, 6, 10–12). Monocytropic isolates do not induce syncytia in primary lymphocyte culture and do not infect established T-cell lines (31). During the late course of HIV infection, syncytium-inducing (SI) primary viruses often arise from the population of monocytropic viruses (31, 32). These SI primary isolates no longer infect macrophages, and they utilize both CCR5 and another chemokine receptor, CXCR4 (7, 33, 38). CXCR4, whose natural chemokine ligand is SDF-1 (3, 27), was originally identified by Feng et al. as the cofactor used by laboratory-adapted viruses (14). In fact, the common laboratory viruses (IIIb/LAI, LAV, and RF) are unable to utilize CCR5 coreceptor (1, 6, 10–12), presumably reflecting the lack of CCR5 expression in most established T-cell lines (1, 13). Although some primary isolates utilize additional chemokine receptor molecules, notably CCR3 and CCR2b (6, 11, 18), the relationship between these coreceptors and viral phenotypes is less clear. The ability to utilize CCR5 coreceptor, however, is unique to primary-isolate (PI) viruses.Paralleling these differences in coreceptor utilization and cell tropism are differences in sensitivity to virus neutralization. Although laboratory-adapted isolates of HIV can be potently neutralized by sera elicited by recombinant gp120 (rgp120) protein, primary isolates are largely refractory to neutralization by rgp120 vaccine sera (23, 37). Similarly, PI viruses are significantly more resistant than T-cell line-adapted (TCLA) viruses to neutralization by gp120-directed monoclonal antibodies (MAbs) (25, 37) and to inhibition by soluble forms of CD4 (8). We and others have demonstrated that neutralization sensitivity develops concomitantly with adaptation of primary isolates to persistent growth in established T-cell lines (24, 37). By studying pedigreed PI and TCLA viruses (168P and 168C, respectively), we have shown that adaptation renders the TCLA virus sensitive not only to rgp120 vaccine sera and CD4 immunoadhesin but also to MAbs directed to the V3 loop of gp120 (37). However, the basis for this increase in neutralization sensitivity remains unclear.In this report, we explore the relationship between neutralization sensitivity and coreceptor utilization, especially with regard to changes that accompany adaptation. We examined neutralization sensitivity of the well-characterized SI primary isolate 168P under experimental conditions where infection can be directed via either the CXCR4 or the CCR5 pathway. The pedigreed TCLA derivative 168C utilizes only CXCR4 and was sensitive to neutralization by the panel of V3-directed MAbs used in these assays. However, the primary isolate 168P remained refractory to neutralization regardless of coreceptor pathway taken. Our findings suggest that envelope protein structure, and not coreceptor utilization, is the primary determinant of differential neutralization sensitivity in PI and TCLA viruses.

Coreceptor utilization by pedigreed PI and TCLA viruses.

Cross-sectional surveys of coreceptor use have shown that primary SI isolates generally utilize CXCR4 and CCR5 coreceptors, whereas unrelated laboratory-adapted isolates utilize only CXCR4 (1, 6, 7, 10–12, 14, 33, 38). We wished to confirm this trend in a longitudinal study of adaptation. We previously described the adaptation of the SI primary isolate 168P to persistent growth in the FDA/H9 T-cell line and the concomitant development of neutralization sensitivity in the resulting TCLA virus 168C (37). In the present study, the ability of these pedigreed viruses to utilize specific coreceptors was tested by infection of U87 human glioma cell lines expressing CD4 (U87-CD4) and the specific coreceptor (19).For this assay, virus stocks were prepared from cell culture supernatants of phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBLs) (168P) or FDA/H9 cells (168C) and standardized to yield a submaximal number of foci of infection on U87-CD4-CXCR4 cells (approximately 100 to 200 foci/96-well microplate culture). To confirm coreceptor specificity, in some assays CCR5 chemokines (each at 500 ng/ml) were added to cells 1 h prior to infection. After 2 days of incubation, cell monolayers were fixed with methanol-acetone and immunochemically stained with HIV immunoglobulin (HIVIG) (29), anti-human ABC kit (Biomeda Corp.), and diaminobenzidine substrate.Figure ​Figure11 confirms the ability of the SI 168P virus to utilize both CXCR4 and CCR5 and the subsequent loss of this latter specificity in the 168C TCLA virus. Infection was dependent on coreceptor expression, and both PI and TCLA viruses could also utilize CCR3 (data not presented). Open in a separate windowFIG. 1Coreceptor utilization by pedigreed PI and TCLA 168 viruses. U87-CD4 cell lines expressing CXCR4 (▪) or CCR5 () were used to define the ability of 168P and 168C viruses to utilize the respective coreceptor. CCR5 utilization was further tested by the addition to U87-CD4-CCR5 cells of CCR5-specific chemokines (RANTES, MIP-1α, and MIP-1β; R&D Systems) (□). For details, see text. ∗, no foci were observed.In keeping with the determined coreceptor specificity, infection could be blocked by addition of coreceptor-specific ligands. Thus, 168P virus infection of CCR5-expressing cells was blocked by the CCR5-specific ligands RANTES, MIP-1α, and MIP-1β (1, 6, 10–12) (Fig. ​(Fig.1).1). Similarly, infection of CXCR4-expressing U87-CD4 cells by either virus could be blocked by the CXCR4-specific chemokine ligand SDF-1 (3, 27) (data not presented).

Coreceptor pathway and neutralization sensitivity.

In previous work, we demonstrated that the PI 168P virus is refractory to neutralization by HIV MN gp120 vaccine sera and by several well-characterized V3-directed murine MAbs which strongly neutralize infectivity of the TCLA 168C virus (37). In the present study, we extended the panel of MAbs to include two V3-directed human MAbs, 257-D and 268-D (17). These well-characterized human MAbs recognize core epitopes at the crown of the V3 loop of gp120 (KRIHI and HIGPGR, respectively), linear sequences known to be present in both 168P and 168C envelope proteins (37). These epitope predictions were confirmed by gp120 capture enzyme-linked immunosorbent assay (ELISA) (26) which demonstrated equal binding to envelope protein in detergent-solubilized 168P and 168C virions (data not presented). Sensitivity to neutralization by these human MAbs was determined in a standard assay using PHA-activated PBLs (37). MAbs 257-D and 268-D were found to potently neutralize 168C but fail to neutralize 168P (Fig. ​(Fig.2).2). This pattern of neutralization sensitivity is similar to that previously described for the V3-directed murine MAb 50.1 (30, 36, 37). Open in a separate windowFIG. 2Neutralization sensitivity of 168 viruses in PBL culture. Virus neutralization assays in PHA-stimulated PBL culture were performed as previously described (37). 168P (○, •) and 168C (□, ▪) virus stocks were standardized to yield submaximal extents of virus spread during the 5-day infection. CCR5-specific chemokines (•, ▪) were added as described for Fig. ​Fig.1.1. The V3-directed MAbs are indicated. p24 antigen was determined by p24 antigen capture ELISA (SAIC Frederick) and was normalized to infected cell control values (168P, 190 ng/ml [170 ng/ml with chemokines]; 168C, 36 ng/ml [33 ng/ml with chemokines]).To examine whether sensitivity to neutralization was affected by the coreceptor pathway utilized in infection of PBLs, we used inhibitory concentrations of CCR5-specific chemokine ligands RANTES, MIP-1α, and MIP-1β in order to restrict infection to the CXCR4 pathway. Addition of these chemokines to the PBL cultures did not affect virus growth, nor did it affect sensitivity to neutralization by the V3-directed human MAbs (Fig. ​(Fig.2).2). To the extent that CCR5 blockade was complete, these results suggest that the simple availability of the CCR5 pathway is not a factor in the resistance of PI viruses to neutralization.To strengthen this conclusion, we examined neutralization sensitivity in human U87-CD4 cell lines expressing only CXCR4 or CCR5. Using this method, we confirmed that the SI 168P virus remained refractory to neutralization by human MAbs 257-D and 268-D as well as by the murine MAb 50.1, regardless of whether infection occurred via CXCR4 or CCR5 (Fig. ​(Fig.3).3). These results suggest that availability of the CCR5 pathway is not a primary determinant for the resistance of PI viruses to neutralization. The TCLA 168C virus utilized CXCR4 only and was sensitive to neutralization. Open in a separate windowFIG. 3Neutralization sensitivity of 168 viruses in U87-CD4 cell lines expressing CCR5 or CXCR4 coreceptor. 168P (○, •) and 168C (▪) viruses were used to infect U87-CD4 cell lines expressing CXCR4 (•, ▪) or CCR5 (○) as described for Fig. ​Fig.1.1. The V3-directed MAbs were incubated with virus for 1 h prior to infection.

Molecularly cloned PI and TCLA envelope genes.

To understand better the changes that accompany adaptation and those that determine coreceptor utilization and neutralization sensitivity, we molecularly cloned the envelope genes of the 168P and 168C viruses. High-fidelity XL PCR (rTth and Vent DNA polymerases; PE Applied Biosystems) and primers envA and envN (15) were used to amplify a 3.1-kb region of proviral DNA encoding the rev and envelope genes. PCR products were isolated by unidirectional T/A cloning in the eucaryotic expression vector pCR3.1-Uni (Invitrogen). Expression in pCR3.1-Uni is driven by the cytomegalovirus immediate-early promoter. Multiple clones were isolated from each virus, and transient transfection studies in COS-7 cells confirmed the surface expression and fusion competence of all clones tested (data not presented).DNA sequence analysis demonstrated that all 168C molecular clones analyzed encoded the three adaptation-associated amino acid changes previously identified by PCR sequencing of the 168C virus population (V2, I166R; C2, I282N; and V3, G318R) (37). Two molecular clones of each 168P and 168C envelope were subjected to complete DNA sequence analysis (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF035532","term_id":"2827997","term_text":"AF035532"}}AF035532 to {"type":"entrez-nucleotide","attrs":{"text":"AF035534","term_id":"2828001","term_text":"AF035534"}}AF035534). Molecular clones 168C23 and 168C60 were identical throughout the envelope gene. Molecular clones 168P5 and 168P23 differed from each other and from the previously determined sequence at four to five positions distinct from those associated with adaptation. These scattered changes within the primary virus quasispecies are considered inconsequential at the present level of analysis; the significance of the three adaptation-associated changes is under separate investigation.Functional analysis of these molecularly cloned envelope genes was performed by incorporation of the molecularly cloned envelope protein into pseudotyped HIV virions. We used an envelope-defective provirus derived from the molecularly cloned NL4-3 provirus (kindly provided by I. S. Y. Chen, University of California, Los Angeles). The pNLthyΔBgl provirus (28) contains a BglII-BglII deletion within the envelope gene and a substitution of the viral nef gene with a cDNA encoding the murine Thy1.2 cell surface protein. The simian virus 40 ori was subsequently introduced into the plasmid to generate pSVNLthyΔBgl (27a). Cotransfection of COS-7 cells (16, 20) with pSVNLthyΔBgl provirus and the envelope expression plasmid resulted in the production of pseudotyped HIV virions. Culture supernatants were harvested 3 days posttransfection, filtered, and used to infect U87-CD4 cell lines expressing coreceptor. Cells infected by virions bearing the complementing envelope protein were identified by immunostaining for murine Thy1.2 or HIV proteins.As anticipated, the molecularly cloned envelope proteins recapitulated the coreceptor specificity of the parental virus population (see the legend to Fig. ​Fig.4).4). Pseudotyped virions containing 168C60 were able to infect only U87-CD4 cells expressing CXCR4, while virions containing 168P23 envelope were able to infect U87-CD4 cells expressing either CCR5 or CXCR4. Thus, the viral envelope protein appears to be the major, if not sole, determinant of viral coreceptor use. These findings also indicate that dual coreceptor use is a direct property of the envelope protein complex and not a result of a mixture of distinct envelope proteins in the SI virus population. This conclusion is corroborated by the failure of CCR5-specific chemokine ligands to diminish 168P virus infection in PBL culture (Fig. ​(Fig.22).Open in a separate windowFIG. 4Neutralization sensitivity of pseudotyped virions in U87-CD4 cell lines expressing CCR5 or CXCR4 coreceptor. Pseudotyped virions were derived by cotransfection of COS-7 cells with pSVNLthyΔBgl provirus and plasmid expressing 168P23 (○, •) or 168C60 (▪) envelope protein. Virion preparations were incubated with U87-CD4 cell lines expressing CXCR4 (•, ▪) or CCR5 (○) as described for Fig. ​Fig.1;1; V3-directed MAbs were added as indicated. The number of foci was normalized to control values (60 to 100 foci/well for U87-CD4-CXCR4 cells; 10 foci/well for U87-CD4-CCR5 cells). ∗, no foci were observed.Finally, we wished to determine the neutralization sensitivity of pseudotyped virions containing the molecularly cloned 168P23 and 168C60 envelope proteins and to confirm that coreceptor pathway is not a primary determinant of neutralization sensitivity. We found that infection of U87-CD4-CXCR4 cells by pseudotyped virions containing 168C60 envelope protein was sensitive to neutralization by MAbs 257-D, 268-D, and 50.1 at concentrations comparable to those determined in assays using 168C virus (Fig. ​(Fig.4).4). Pseudotyped virions containing 168P23 envelope protein remained refractory to neutralization by all three V3-directed MAbs, regardless of the coreceptor expressed by the U87-CD4 cell line. In summary, we examined the relationship between coreceptor utilization and sensitivity to neutralization by V3-directed MAbs. The observed dichotomy in the sensitivity to neutralization of PI and TCLA viruses had suggested a discrete difference between these viruses, and we tested one hypothesis: that PI viruses are refractory to neutralization as a result of their unique ability to utilize the CCR5 coreceptor. We examined neutralization sensitivity of a well-characterized SI primary isolate under experimental conditions wherein the virus was forced to utilize either CCR5 or CXCR4 for infection. We showed that coreceptor pathway is not a direct determinant of neutralization sensitivity. The primary virus envelope protein remained refractory to neutralization by V3-directed MAbs regardless of the coreceptor pathway utilized. Similarly, coreceptor utilization did not affect neutralization sensitivity by soluble CD4 (34) or HIVIG (data not presented).In discarding the otherwise attractive hypothesis that PI viruses escape neutralization through their unique ability to utilize CCR5, we are left to consider the as yet undefined structural differences between the envelope protein complex of PI and TCLA viruses. Several studies have suggested that critical determinants in the envelope protein of PI viruses are less accessible than those of TCLA viruses and that it is this differential access that determines neutralization sensitivity (reviewed in reference 25). By contrast, our studies have indicated similar binding of V3-directed MAbs to PBLs infected with neutralization-resistant isolate 168P or neutralization-sensitive isolate 168C (37). Thus, the basis for the differential neutralization sensitivity of PI and TCLA viruses remains unresolved.Our present studies also do not address whether changes in coreceptor utilization and/or neutralization sensitivity are necessarily linked as a consequence of adaptation. The analysis of independently derived PI and TCLA viruses may allow further separation of these viral phenotypes. Subsequent dissection of the amino acid changes that distinguish pedigreed PI and TCLA envelope proteins will help to define the structural bases underlying the changes that accompany adaptation.     

Antibody binding and neutralization of primary and T-cell line-adapted isolates of human immunodeficiency virus type 1

The relative resistance of human immunodeficiency virus type 1 (HIV-1) primary isolates (PIs) to neutralization by a wide range of antibodies remains a theoretical and practical barrier to the development of an effective HIV vaccine. One model to account for the differential neutralization sensitivity between Pls and laboratory (or T-cell line-adapted [TCLA]) strains of HIV suggests that the envelope protein (Env) complex is made more accessible to antibody binding as a consequence of adaptation to growth in established cell lines. Here, we revisit this question using genetically related PI and TCLA viruses and molecularly cloned env genes. By using complementary techniques of flow cytometry and virion binding assays, we show that monoclonal antibodies targeting the V3 loop, CD4-binding site, CD4-induced determinant of gp120, or the ectodomain of gp41 bind equally well to PI and TCLA Env complexes, despite large differences in neutralization outcome. The data suggest that the differential neutralization sensitivity of PI and TCLA viruses may derive not from differences in the initial antibody binding event but rather from differences in the subsequent functioning of the PI and TCLA Envs during virus entry. An understanding of these as yet undefined differences may enhance our ability to generate broadly neutralizing HIV vaccine immunogens.     

Human immunodeficiency virus type 1 coreceptor switching: V1/V2 gain-of-fitness mutations compensate for V3 loss-of-fitness mutations

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is mediated by the virus envelope binding to CD4 and the conformationally altered envelope subsequently binding to one of two chemokine receptors. HIV-1 envelope glycoprotein (gp120) has five variable loops, of which three (V1/V2 and V3) influence the binding of either CCR5 or CXCR4, the two primary coreceptors for virus entry. Minimal sequence changes in V3 are sufficient for changing coreceptor use from CCR5 to CXCR4 in some HIV-1 isolates, but more commonly additional mutations in V1/V2 are observed during coreceptor switching. We have modeled coreceptor switching by introducing most possible combinations of mutations in the variable loops that distinguish a previously identified group of CCR5- and CXCR4-using viruses. We found that V3 mutations entail high risk, ranging from major loss of entry fitness to lethality. Mutations in or near V1/V2 were able to compensate for the deleterious V3 mutations and may need to precede V3 mutations to permit virus survival. V1/V2 mutations in the absence of V3 mutations often increased the capacity of virus to utilize CCR5 but were unable to confer CXCR4 use. V3 mutations were thus necessary but not sufficient for coreceptor switching, and V1/V2 mutations were necessary for virus survival. HIV-1 envelope sequence evolution from CCR5 to CXCR4 use is constrained by relatively frequent lethal mutations, deep fitness valleys, and requirements to make the right amino acid substitution in the right place at the right time.     

Evidence for Common Structural Determinants of Human Immunodeficiency Virus Type 1 Coreceptor Activity Provided through Functional Analysis of CCR5/CXCR4 Chimeric Coreceptors

Human immunodeficiency virus type 1 (HIV-1) infection in vivo is dependent upon the interaction of the viral envelope glycoprotein gp120 with CC chemokine receptor 5 (CCR5) or CXC chemokine receptor 4 (CXCR4). To study the determinants of the gp120-coreceptor association, we generated a set of chimeric HIV-1 coreceptors which express all possible combinations of the four extracellular domains of CCR5 and CXCR4. Stable U87 astroglioma cell lines expressing CD4 and individual chimeric coreceptor proteins were tested against a variety of R5, X4, and R5X4 envelope glycoproteins and virus strains for their ability to support HIV-1-mediated cell fusion and infection, respectively. Each of the cell lines promoted fusion with cells expressing an HIV envelope glycoprotein, except for U87.CD4.5455, which presents the first extracellular loop (ECL1) and flanking sequences of CXCR4 in the context of CCR5. However, all of the chimeric coreceptors allowed productive infection by one or more of the viral strains tested. Viral phenotype was a predictive factor for the observed activity of the chimeric molecules; X4 and R5X4 HIV strains utilized a majority of the chimeras, while R5 strains were limited in their ability to infect cells expressing these chimeric molecules. The expression of CCR5 ECL2 within the CXCR4 backbone supported infection by an R5 primary isolate, but no chimeras bearing the N terminus of CCR5 exhibited activity with R5 strains. Remarkably, the introduction of any CXCR4 domain into the CCR5 backbone was sufficient to allow utilization by multiple X4 strains. However, critical determinants within ECL2 and/or ECL3 of CXCR4 were apparent for all X4 viruses upon replacement of these domains in CXCR4 with CCR5 sequences. Unexpectedly, chimeric coreceptor-facilitated entry was blocked in all cases by the presence of the CXCR4-specific inhibitor AMD3100. Our data provide proof that CCR5 contains elements that support usage by X4 viral strains and demonstrate that the gp120 interaction sites of CCR5 and CXCR4 are structurally related.     

An alteration of human immunodeficiency virus gp41 leads to reduced CCR5 dependence and CD4 independence

Human immunodeficiency virus (HIV) type 1 infection requires functional interactions of the viral surface (gp120) glycoprotein with cell surface CD4 and a chemokine coreceptor (usually CCR5 or CXCR4) and of the viral transmembrane (gp41) glycoprotein with the target cell membrane. Extensive genetic variability, generally in gp120 and the gp41 ectodomain, can result in altered coreceptor use, fusion kinetics, and neutralization sensitivity. Here we describe an R5 HIV variant that, in contrast to its parental virus, infects T-cell lines expressing low levels of cell surface CCR5. This correlated with an ability to infect cells in the absence of CD4, increased sensitivity to a neutralizing antibody recognizing the coreceptor binding site of gp120, and increased resistance to the fusion inhibitor T-20. Surprisingly, these properties were determined by alterations in gp41, including the cytoplasmic tail, a region not previously shown to influence coreceptor use. These data indicate that HIV infection of cells with limiting levels of cell surface CCR5 can be facilitated by gp41 sequences that are not exposed on the envelope ectodomain yet induce allosteric changes in gp120 that facilitate exposure of the CCR5 binding site.     

Coevolution of RANTES sensitivity and mode of CCR5 receptor use by human immunodeficiency virus type 1 of the R5 phenotype

The evolution of human immunodeficiency virus type 1 (HIV-1) coreceptor use has been described as the acquisition of CXCR4 use linked to accelerated disease progression. However, CXCR4-using virus can be isolated only from approximately one-half of individuals with progressive HIV-1 disease. The other half continue to yield only CCR5-using viruses (R5 phenotype) throughout the course of disease. In the present work, the use of receptor chimeras between CCR5 and CXCR4 allowed us to study the evolution of HIV-1 with the R5 phenotype, which was not revealed by studies of wild-type coreceptor use. All together, 246 isolates (173 with the R5 phenotype) from 31 individuals were tested for their ability to infect cells through receptor chimeras. R5(narrow) virus was able to use only wild-type CCR5, whereas R5(broad(1)) to R5(broad(3)) viruses were able to use one to three chimeric receptors, respectively. Broad use of chimeric receptors was interpreted as an increased flexibility in the mode of receptor use. R5(broad) isolates showed higher infectivity in cells expressing wild-type CCR5 than R5(narrow) isolates. Also, the increased flexibility of R5(broad) isolates was concomitant with a lower sensitivity to inhibition by the CC chemokine RANTES. Our results indicate a close relationship between HIV-1 phenotypic changes and the pathogenic process, since the mode and efficiency of CCR5 use as well as the decrease in the RANTES sensitivities of isolated viruses are significantly correlated with CD4(+)-T-cell decline in a patient. One possible explanation is that ligand competition at the CCR5 receptor or changed CCR5 availability may shape the outcome of HIV-1 infection.     

Replication-competent variants of human immunodeficiency virus type 2 lacking the V3 loop exhibit resistance to chemokine receptor antagonists

Entry of human immunodeficiency virus type 1 (HIV-1) and HIV-2 requires interactions between the envelope glycoprotein (Env) on the virus and CD4 and a chemokine receptor, either CCR5 or CXCR4, on the cell surface. The V3 loop of the HIV gp120 glycoprotein plays a critical role in this process, determining tropism for CCR5- or CXCR4-expressing cells, but details of how V3 interacts with these receptors have not been defined. Using an iterative process of deletion mutagenesis and in vitro adaptation of infectious viruses, variants of HIV-2 were derived that could replicate without V3, either with or without a deletion of the V1/V2 variable loops. The generation of these functional but markedly minimized Envs required adaptive changes on the gp120 core and gp41 transmembrane glycoprotein. V3-deleted Envs exhibited tropism for both CCR5- and CXCR4-expressing cells, suggesting that domains on the gp120 core were mediating interactions with determinants shared by both coreceptors. Remarkably, HIV-2 Envs with V3 deletions became resistant to small-molecule inhibitors of CCR5 and CXCR4, suggesting that these drugs inhibit wild-type viruses by disrupting a specific V3 interaction with the coreceptor. This study represents a proof of concept that HIV Envs lacking V3 alone or in combination with V1/V2 that retain functional domains required for viral entry can be derived. Such minimized Envs may be useful in understanding Env function, screening for new inhibitors of gp120 core interactions with chemokine receptors, and designing novel immunogens for vaccines.     

V3 Recombinants Indicate a Central Role for CCR5 as a Coreceptor in Tissue Infection by Human Immunodeficiency Virus Type 1

Binding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 to both CD4 and one of several chemokine receptors (coreceptors) permits entry of virus into target cells. Infection of tissues may establish latent viral reservoirs as well as cause direct pathologic effects that manifest as clinical disease such as HIV-associated dementia. We sought to identify the critical coreceptors recognized by HIV-1 tissue-derived strains as well as to correlate these coreceptor preferences with site of infection and dementia diagnosis. To reconstitute coreceptor use, we cloned HIV-1 envelope V3 sequences encoding the primary determinants of coreceptor specificity from 13 brain-derived and 6 colon-derived viruses into an isogenic (NL4-3) viral background. All V3 recombinants utilized the chemokine receptor CCR5 uniformly and efficiently as a coreceptor but not CXCR4, BOB/GPR15, or Bonzo/STRL33. Other receptors such as CCR3, CCR8, and US28 were inefficiently and variably used as coreceptors by various envelopes. CCR5 without CD4 present did not allow for detectable infection by any of the tested recombinants. In contrast to the pathogenic switch in coreceptor specificity frequently observed in comparisons of blood-derived viruses early after HIV-1 seroconversion and after onset of AIDS, the characteristics of these V3 recombinants suggest that CCR5 is a primary coreceptor for brain- and colon-derived viruses regardless of tissue source or diagnosis of dementia. Therefore, tissue infection may not depend significantly on viral envelope quasispeciation to broaden coreceptor range but rather selects for CCR5 use throughout disease progression.     

Identification of a subset of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus strains able to exploit an alternative coreceptor on untransformed human brain and lymphoid cells

The chemokine receptors CCR5 and CXCR4 are the major coreceptors for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). At least 12 other chemokine receptors or close relatives support infection by particular HIV and SIV strains on CD4(+) transformed indicator cell lines in vitro. However, the role of these alternative coreceptors in vivo is presently thought to be insignificant. Infection of cell lines expressing high levels of recombinant CD4 and coreceptors thus does not provide a true indication of coreceptor use in vivo. We therefore tested primary untransformed cell cultures that lack CCR5 and CXCR4, including astrocytes and brain microvascular endothelial cells (BMVECs), for naturally expressed alternative coreceptors functional for HIV and SIV infection. An adenovirus vector (Ad-CD4) was used to express CD4 in CD4(-) astrocytes and thus confer efficient infection if a functional coreceptor is present. Using a large panel of viruses with well-defined coreceptor usage, we identified a subset of HIV and SIV strains able to infect two astrocyte cultures derived from adult brain tissue. Astrocyte infection was partially inhibited by several chemokines, indicating a role for the chemokine receptor family in the observed infection. BMVECs were weakly positive for CD4 but negative for CCR5 and CXCR4 and were susceptible to infection by the same subset of isolates that infected astrocytes. BMVEC infection was efficiently inhibited by the chemokine vMIP-I, implicating one of its receptors as an alternative coreceptor for HIV and SIV infection. Furthermore, we tested whether the HIV type 1 and type 2 strains identified were able to infect peripheral blood mononuclear cells (PBMCs) via an alternative coreceptor. Several strains replicated in Delta32/Delta32 CCR5 PBMCs with CXCR4 blocked by AMD3100. This AMD3100-resistant replication was also sensitive to vMIP-I inhibition. The nature and potential role of this alternative coreceptor(s) in HIV infection in vivo is discussed.     

Improved coreceptor usage prediction and genotypic monitoring of R5-to-X4 transition by motif analysis of human immunodeficiency virus type 1 env V3 loop sequences

Early in infection, human immunodeficiency virus type 1 (HIV-1) generally uses the CCR5 chemokine receptor (along with CD4) for cellular entry. In many HIV-1-infected individuals, viral genotypic changes arise that allow the virus to use CXCR4 (either in addition to CCR5 or alone) as an entry coreceptor. This switch has been associated with an acceleration of both CD3(+) T-cell decline and progression to AIDS. While it is well known that the V3 loop of gp120 largely determines coreceptor usage and that positively charged residues in V3 play an important role, the process of genetic change in V3 leading to altered coreceptor usage is not well understood. Further, the methods for biological phenotyping of virus for research or clinical purposes are laborious, depend on sample availability, and present biosafety concerns, so reliable methods for sequence-based "virtual phenotyping" are desirable. We introduce a simple bioinformatic method of scoring V3 amino acid sequences that reliably predicts CXCR4 usage (sensitivity, 84%; specificity, 96%). This score (as determined on the basis of position-specific scoring matrices [PSSM]) can be interpreted as revealing a propensity to use CXCR4 as follows: known R5 viruses had low scores, R5X4 viruses had intermediate scores, and X4 viruses had high scores. Application of the PSSM scoring method to reconstructed virus phylogenies of 11 longitudinally sampled individuals revealed that the development of X4 viruses was generally gradual and involved the accumulation of multiple amino acid changes in V3. We found that X4 viruses were lost in two ways: by the dying off of an established X4 lineage or by mutation back to low-scoring V3 loops.     

Role of Naturally Occurring Basic Amino Acid Substitutions in the Human Immunodeficiency Virus Type 1 Subtype E Envelope V3 Loop on Viral Coreceptor Usage and Cell Tropism

To assess the role of naturally occurring basic amino acid substitutions in the V3 loop of human immunodeficiency virus type 1 (HIV-1) subtype E on viral coreceptor usage and cell tropism, we have constructed a panel of chimeric viruses with mutant V3 loops of HIV-1 subtype E in the genetic background of HIV-1LAI. The arginine substitutions naturally occurring at positions 8, 11, and 18 of the V3 loop in an HIV-1 subtype E X4 strain were systematically introduced into that of an R5 strain to generate a series of V3 loop mutant chimera. These chimeric viruses were employed in virus infectivity assays using HOS-CD4 cells expressing either CCR5 or CXCR4, peripheral blood mononuclear cells, T-cell lines, or macrophages. The arginine substitution at position 11 of the V3 loop uniformly caused the loss of infectivity in HOS-CD4-CCR5 cells, indicating that position 11 is critical for utilization of CCR5. CXCR4 usage was conferred by a minimum of two arginine substitutions, regardless of combination, whereas arginine substitutions at position 8 and 11 were required for T-cell line tropism. Nonetheless, macrophage tropism was not conferred by the V3 loop of subtype E R5 strain per se. We found that the specific combinations of amino acid changes in HIV-1 subtype E env V3 loop are critical for determining viral coreceptor usage and cell tropism. However, the ability to infect HOS-CD4 cells through either CXCR4 or CCR5 is not necessarily correlated with T-cell or macrophage tropism, suggesting that cellular tropism is not dictated solely by viral coreceptor utilization.     

Patterns of Chemokine Receptor Fusion Cofactor Utilization by Human Immunodeficiency Virus Type 1 Variants from the Lungs and Blood

Human immunodeficiency virus type 1 (HIV-1) infection is highly compartmentalized, with distinct viral genotypes being found in the lungs, brain, and other organs compared with blood. CCR5 and CXCR4 are the principal HIV-1 coreceptors, and a number of other molecules support entry in vitro but their roles in vivo are uncertain. To address the relationship between tissue compartmentalization and the selective use of entry coreceptors, we generated functional env clones from primary isolates derived from the lungs and blood of three infected individuals and analyzed their use of the principal, secondary, orphan, and virus-encoded coreceptors for fusion. All Env proteins from lung viruses used CCR5 but not CXCR4, while those from blood viruses used CCR5 or CXCR4 or both. The orphan receptor APJ was widely used for fusion by Env proteins from both blood and lung viruses, but none used the cytomegalovirus-encoded receptor US28. Fusion mediated by the secondary coreceptors CCR2b, CCR3, CCR8, and CX3CR1 and orphan receptors GPR1, GPR15, and STRL33 was variable and heterogeneous, with relatively broad utilization by env clones from isolates of one subject but limited use by env clones from the other two subjects. However, there was no clear distinction between blood and lung viruses in secondary or orphan coreceptor fusion patterns. In contrast to fusion, none of the secondary or orphan receptors enabled efficient productive infection. These results confirm, at the level of cofactor utilization, previous observations that HIV-1 populations in the lungs and blood are biologically distinct and demonstrate diversity within lung-derived as well as blood-derived quasispecies. However, the heterogeneity in coreceptor utilization among clones from each isolate and the lack of clear distinction between lung- and blood-derived Env proteins argue against selective coreceptor utilization as a major determinant of compartmentalization.     

A Broad Range of Chemokine Receptors Are Used by Primary Isolates of Human Immunodeficiency Virus Type 2 as Coreceptors with CD4

Like human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), HIV-2 requires a coreceptor in addition to CD4 for entry into cells. HIV and SIV coreceptor molecules belong to a family of seven-transmembrane-domain G-protein-coupled receptors. Here we show that primary HIV-2 isolates can use a broad range of coreceptor molecules, including CCR1, CCR2b, CCR3, CCR4, CCR5, and CXCR4. Despite broad coreceptor use, the chemokine ligand SDF-1 substantially blocked HIV-2 infectivity of peripheral blood mononuclear cells, indicating that its receptor, CXCR4, was the predominant coreceptor for infection of these cells. However, expression of CXCR4 together with CD4 on some cell types did not confer susceptibility to infection by all CXCR4-using virus isolates. These data therefore indicate that another factor(s) influences the ability of HIV-2 to replicate in human cell types that express the appropriate receptors for virus entry.     

Role of CXCR4 in Cell-Cell Fusion and Infection of Monocyte-Derived Macrophages by Primary Human Immunodeficiency Virus Type 1 (HIV-1) Strains: Two Distinct Mechanisms of HIV-1 Dual Tropism

Dual-tropic human immunodeficiency virus type 1 (HIV-1) strains infect both primary macrophages and transformed T-cell lines. Prototype T-cell line-tropic (T-tropic) strains use CXCR4 as their principal entry coreceptor (X4 strains), while macrophagetropic (M-tropic) strains use CCR5 (R5 strains). Prototype dual tropic strains use both coreceptors (R5X4 strains). Recently, CXCR4 expressed on macrophages was found to support infection by certain HIV-1 isolates, including the dual-tropic R5X4 strain 89.6, but not by T-tropic X4 prototypes like 3B. To better understand the cellular basis for dual tropism, we analyzed the macrophage coreceptors used for Env-mediated cell-cell fusion as well as infection by several dual-tropic HIV-1 isolates. Like 89.6, the R5X4 strain DH12 fused with and infected both wild-type and CCR5-negative macrophages. The CXCR4-specific inhibitor AMD3100 blocked DH12 fusion and infection in macrophages that lacked CCR5 but not in wild-type macrophages. This finding indicates two independent entry pathways in macrophages for DH12, CCR5 and CXCR4. Three primary isolates that use CXCR4 but not CCR5 (tybe, UG021, and UG024) replicated efficiently in macrophages regardless of whether CCR5 was present, and AMD3100 blocking of CXCR4 prevented infection in both CCR5 negative and wild-type macrophages. Fusion mediated by UG021 and UG024 Envs in both wild-type and CCR5-deficient macrophages was also blocked by AMD3100. Therefore, these isolates use CXCR4 exclusively for entry into macrophages. These results confirm that macrophage CXCR4 can be used for fusion and infection by primary HIV-1 isolates and indicate that CXCR4 may be the sole macrophage coreceptor for some strains. Thus, dual tropism can result from two distinct mechanisms: utilization of both CCR5 and CXCR4 on macrophages and T-cell lines, respectively (dual-tropic R5X4), or the ability to efficiently utilize CXCR4 on both macrophages and T-cell lines (dual-tropic X4).     

R5X4 viruses are evolutionary, functional, and antigenic intermediates in the pathway of a simian-human immunodeficiency virus coreceptor switch

To examine the pathway of the coreceptor switching of CCR5-using (R5) virus to CXCR4-using (X4) virus in simian-human immunodeficiency virus SHIV(SF162P3N)-infected rhesus macaque BR24, analysis was performed on variants present at 20 weeks postinfection, the time when the signature gp120 V3 loop sequence of the X4 switch variant was first detected by PCR. Unexpectedly, circulating and tissue variants with His/Ile instead of the signature X4 V3 His/Arg insertions predominated at this time point. Phylogenetic analysis of the sequences of the C2 conserved region to the V5 variable loop of the envelope (Env) protein showed that viruses bearing HI insertions represented evolutionary intermediates between the parental SHIV(SF162P3N) and the final X4 HR switch variant. Functional analyses demonstrated that the HI variants were phenotypic intermediates as well, capable of using both CCR5 and CXCR4 for entry. However, the R5X4 intermediate virus entered CCR5-expressing target cells less efficiently than the parental R5 strain and was more sensitive to both CCR5 and CXCR4 inhibitors than either the parental R5 or the final X4 virus. It was also more sensitive than the parental R5 virus to antibody neutralization, especially to agents directed against the CD4 binding site, but not as sensitive as the late X4 virus. Significantly, the V3 loop sequence that determined CXCR4 use also conferred soluble CD4 neutralization sensitivity. Collectively, the data illustrate that, similar to human immunodeficiency virus type 1 (HIV-1) infection in individuals, the evolution from CCR5 to CXCR4 usage in BR24 transitions through an intermediate phase with reduced virus entry and coreceptor usage efficiencies. The data further support a model linking an open envelope gp120 conformation, better CD4 binding, and expansion to CXCR4 usage.     

Tat Protein Induces Human Immunodeficiency Virus Type 1 (HIV-1) Coreceptors and Promotes Infection with both Macrophage-Tropic and T-Lymphotropic HIV-1 Strains

Chemokine receptors CCR5 and CXCR4 are the primary fusion coreceptors utilized for CD4-mediated entry by macrophage (M)- and T-cell line (T)-tropic human immunodeficiency virus type 1 (HIV-1) strains, respectively. Here we demonstrate that HIV-1 Tat protein, a potent viral transactivator shown to be released as a soluble protein by infected cells, differentially induced CXCR4 and CCR5 expression in peripheral blood mononuclear cells. CCR3, a less frequently used coreceptor for certain M-tropic strains, was also induced. CXCR4 was induced on both lymphocytes and monocytes/macrophages, whereas CCR5 and CCR3 were induced on monocytes/macrophages but not on lymphocytes. The pattern of chemokine receptor induction by Tat was distinct from that by phytohemagglutinin. Moreover, Tat-induced CXCR4 and CCR5 expression was dose dependent. Monocytes/macrophages were more susceptible to Tat-mediated induction of CXCR4 and CCR5 than lymphocytes, and CCR5 was more readily induced than CXCR4. The concentrations of Tat effective in inducing CXCR4 and CCR5 expression were within the picomolar range and close to the range of extracellular Tat observed in sera from HIV-1-infected individuals. The induction of CCR5 and CXCR4 expression correlated with Tat-enhanced infectivity of M- and T-tropic viruses, respectively. Taken together, our results define a novel role for Tat in HIV-1 pathogenesis that promotes the infectivity of both M- and T-tropic HIV-1 strains in primary human leukocytes, notably in monocytes/macrophages.