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.     

Primary Human Immunodeficiency Virus Type 2 (HIV-2) Isolates Infect CD4-Negative Cells via CCR5 and CXCR4: Comparison with HIV-1 and Simian Immunodeficiency Virus and Relevance to Cell Tropism In Vivo

Cell surface receptors exploited by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) for infection are major determinants of tropism. HIV-1 usually requires two receptors to infect cells. Gp120 on HIV-1 virions binds CD4 on the cell surface, triggering conformational rearrangements that create or expose a binding site for a seven-transmembrane (7TM) coreceptor. Although HIV-2 and SIV strains also use CD4, several laboratory-adapted HIV-2 strains infect cells without CD4, via an interaction with the coreceptor CXCR4. Moreover, the envelope glycoproteins of SIV of macaques (SIV(MAC)) can bind to and initiate infection of CD4(-) cells via CCR5. Here, we show that most primary HIV-2 isolates can infect either CCR5(+) or CXCR4(+) cells without CD4. The efficiency of CD4-independent infection by HIV-2 was comparable to that of SIV, but markedly higher than that of HIV-1. CD4-independent HIV-2 strains that could use both CCR5 and CXCR4 to infect CD4(+) cells were only able to use one of these receptors in the absence of CD4. Our observations therefore indicate (i) that HIV-2 and SIV envelope glycoproteins form a distinct conformation that enables contact with a 7TM receptor without CD4, and (ii) the use of CD4 enables a wider range of 7TM receptors to be exploited for infection and may assist adaptation or switching to new coreceptors in vivo. Primary CD4(-) fetal astrocyte cultures expressed CXCR4 and supported replication by the T-cell-line-adapted ROD/B strain. Productive infection by primary X4 strains was only triggered upon treatment of virus with soluble CD4. Thus, many primary HIV-2 strains infect CCR5(+) or CXCR4(+) cell lines without CD4 in vitro. CD4(-) cells that express these coreceptors in vivo, however, may still resist HIV-2 entry due to insufficient coreceptor concentration on the cell surface to trigger fusion or their expression in a conformation nonfunctional as a coreceptor. Our study, however, emphasizes that primary HIV-2 strains carry the potential to infect CD4(-) cells expressing CCR5 or CXCR4 in vivo.     

Constitutive association of cell surface CCR5 and CXCR4 in the presence of CD4

Chemokine receptors CCR5 and CXCR4 are the major coreceptors of HIV-1 infection and also play fundamental roles in leukocyte trafficking, metastasis, angiogenesis, and embyogenesis. Here, we show that transfection of CCR5 into CXCR4 and CD4 expressing 3T3 cells enhances the cell surface level of CXCR4. In CCR5 high expressing cells, cell surface level of CXCR4 was incompletely modulated in the presence of the CXCR4 ligand CXCL12/SDF-1alpha. CCR5 was resistant to ligand-dependent modulation with the CCR5 ligand CCL5/RANTES. Confocal laser microscopy revealed that CCR5 was colocalized with CXCR4 on the cell surface. In CD4 expressing CCR5 and CXCR4 double positive NIH 3T3 cells, immunoprecipitation followed by Western blot analysis revealed that CCR5 was associated with CXCR4 and CD4. CXCR4 and CCR5 were not co-immunoprecipitated in cells expressing CCR5 and CXCR4 but without CD4 expression. Compared to NIH 3T3CD4 cells expressing CXCR4, the entry of an HIV-1 X4 isolate (HCF) into NIH 3T3CD4 expressing both CXCR4 and CCR5 was reduced. Our data indicate that chemokine receptors interact with each other, which may modulate chemokine-chemokine receptor interactions and HIV-1 coreceptor functions.     

Dendritic cells preferentially transfer CXCR4-using human immunodeficiency virus type 1 variants to CD4+ T lymphocytes in trans

Human immunodeficiency virus type 1 (HIV-1) preferentially utilizes the CCR5 coreceptor for target cell entry in the acute phase of infection, while later in disease progression the virus switches to the CXCR4 coreceptor in approximately 50% of patients. In response to HIV-1 the adaptive immune response is triggered, and antibody (Ab) production is elicited to block HIV-1 entry. We recently determined that dendritic cells (DCs) can efficiently capture Ab-neutralized HIV-1, restore infectivity, and transmit infectious virus to target cells. Here, we tested the effect of Abs on trans transmission of CCR5 or CXCR4 HIV-1 variants. We observed that transmission of HIV-1 by immature as well as mature DCs was significantly higher for CXCR4- than CCR5-tropic viral strains. Additionally, neutralizing Abs directed against either the gp41 or gp120 region of the envelope such as 2F5, 4E10, and V3-directed Abs inhibited transmission of CCR5-tropic HIV-1, whereas Ab-treated CXCR4-tropic virus demonstrated unaltered or increased transmission. To further study the effects of coreceptor usage we tested molecularly cloned HIV-1 variants with modifications in the envelope that were based on longitudinal gp120 V1 and V3 variable loop sequences from a patient progressing to AIDS. We observed that DCs preferentially facilitated infection of CD4⁺ T lymphocytes of viral strains with an envelope phenotype found late in disease. Taken together, our results illustrate that DCs transmit CXCR4-tropic HIV-1 much more efficiently than CCR5 strains; we hypothesize that this discrimination could contribute to the in vivo coreceptor switch after seroconversion and could be responsible for the increase in viral load.     

Enhanced infection of an X4 strain of HIV-1 due to capping and colocalization of CD4 and CXCR4 induced by capsianoside G, a diterpene glycoside

We investigated whether capsianosides, diterpene glycosides, extracted from Capsicum plants could affect human immunodeficiency virus type 1 (HIV-1) infection. Significant effect on virus infection in MAGI/CCR5 cells was neither observed for the X4 virus by capsianosides II, XI, and A, nor for an R5 virus by capsianoside G. Apparent enhancement of X4 HIV-1 infection by capsianoside G was observed and exclusively related to the usage of the CXCR4 coreceptor. The capsianoside G-treated cells had no change in the expression level of CD4, CXCR4, and CCR5, however, colocalization and capping of CD4 and CXCR4, but not of CD4 and CCR5 was observed. Our results suggested that capsianoside G enhanced X4 virus infection at the level of viral penetration through the capping and colocalization of receptors needed for infection.     

Expression and Use of Human Immunodeficiency Virus Type 1 Coreceptors by Human Alveolar Macrophages

Human immunodeficiency virus type 1 (HIV-1) requires, in addition to CD4, coreceptors of the CC or CXC chemokine families for productive infection of T cells and cells of the monocyte-macrophage lineage. Based on the hypothesis that coreceptor expression on alveolar macrophages (AM) may influence HIV-1 infection of AM in the lung, this study analyzes the expression and utilization of HIV-1 coreceptors on AM of healthy individuals. AM were productively infected with five different primary isolates of HIV-1. Levels of surface expression of CCR5, CXCR4, and CD4 were low compared to those of blood monocytes, but CCR3 was not detectable. mRNA for CCR5, CXCR4, CCR2, and CCR3 were all detectable, but to varying degrees and with variability among donors. Expression of CCR5, CXCR4, and CCR2 mRNA was downregulated following stimulation with lipopolysaccharide (LPS). In contrast, secretion of the chemokines RANTES, MIP-1alpha, and MIP-1beta was upregulated with LPS stimulation. Interestingly, HIV-1 replication was diminished following LPS stimulation. Infection of AM with HIV-1 in the presence of the CC chemokines demonstrated blocking of infection. Together, these studies demonstrate that AM can be infected by a variety of primary HIV-1 isolates, AM express a variety of chemokine receptors, the dominant coreceptor used for HIV entry into AM is CCR5, the expression of these receptors is dependent on the state of activation of AM, and the ability of HIV-1 to infect AM may be modulated by expression of the chemokine receptors and by chemokines per se.     

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.     

Role of V3 independent domains on a dualtropic human immunodeficiency virus type 1 (HIV-1) envelope gp120 in CCR5 coreceptor utilization and viral infectivity

The molecular mechanism of human immunodeficiency virus type 1 (HIV-1) entry into cells involves specific interactions between the viral envelope glycoprotein gp120 and two target cell proteins, CD4 and either CCR5 or CXCR4 chemokine receptors. In order to delineate the functional role of HIV-1 gp120 subdomains of dualtropic strains in CCR5 coreceptor usage, we used a panel of chimeric viruses in which the V1/V2 and V3 domains of gp120 from the dualtropic HIV-1(KMT) isolate were introduced either alone or in combination into the T-tropic HIV-1(NL4-3) background. These chimeric constructs were employed in cell-cell fusion and cell-free virus infectivity assays using cell lines expressing CD4 and the CCR5 chemokine receptor. In both assays, the V3 domain of HIV-1(KMT) but not the V1/V2 domain proved to be the principal determinant of CCR5 coreceptor usage. However, in the cell-free viral infectivity assay although a chimeric virus with a combined V1/V2 and V3 domains of HIV-1(KMT) efficiently fused with coreceptor expressing cells, yet its infectivity was markedly diminished in CCR5 as well as CXCR4 expressing cells. Restoring a comparable level of infection of such chimeric virus required the C3-V5 domain from HIV-1(KMT) to be introduced. Our present findings confirmed that the V3 domain is the major determinant of fusion activity and cellular tropism, and demonstrated a dispensable role for the V1/V2 domain. In addition the C3-V5 domain appeared to play an important role in viral infectivity when the corresponding V1/V2 and V3 domains are present.     

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.     

The HIV-1 Tat protein selectively enhances CXCR4 and inhibits CCR5 expression in megakaryocytic K562 cells

The hematopoietic compartments act as long-term reservoirs for human immunodeficiency virus type-1 (HIV-1). Although hematopoietic progenitor cells (HPCs) are rarely infectable, HPCs committed to the megakaryocytic lineage can be infected and support a productive infection by both the X4 and R5 strains of HIV-1. Indeed, in contrast to the CD34+ progenitors, the lineage-committed HPCs express high levels of the HIV-1 co-receptors, CXCR4 and CCR5. The HIV-1 transactivator (Tat) protein has been shown to alter co-receptor expression in T lymphocytes and macrophages. We hypothesized that Tat may regulate co-receptor expression in lineage-specific HPCs as well. We have monitored the effects of Tat protein on co-receptor expression and on lineage-specific differentiation, using the HPC cell line, K562. Butyric acid (BA)-induced erythroid differentiation in K562 cells was suppressed by 1-100 ng/ml of Tat, as evident from a 70-80% decrease in hemoglobin (Hb) production and a 10-30-fold decrease in glycophorin-A expression. However, Tat treatment enhanced phorbol myristate acetate (PMA)-induced megakaryocytic differentiation, as evident from a 180-210% increase in 3H-serotonin uptake and a 5-12-fold increase in CD61 expression. Tat did not significantly alter co-receptor expression in erythroid cells. However, Tat co-treatment profoundly effected both CXCR4 and CCR5 gene expression and protein levels in megakaryocytic cells. In PMA-stimulated cells, Tat increased CXCR4 and decreased in CCR5 expression, this was potentiated in cells chronically exposed to Tat. In conclusion, Tat protein suppresses erythroid and facilitates megakaryocytic differentiation of K562 cells. In megakaryocytic cells, Tat differentially effected CXCR4 and CCR5 expression. Because megakaryocytes may play a crucial role in HIV-1 infectivity in viral reservoirs, our findings implicate a role for Tat protein in dictating co-receptor usage in lineage-committed HPCs.     

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.     

Genetically Divergent Strains of Human Immunodeficiency Virus Type 2 Use Multiple Coreceptors for Viral Entry

Several members of the seven-transmembrane chemokine receptor family have been shown to serve, with CD4, as coreceptors for entry by human immunodeficiency virus type 1 (HIV-1). While coreceptor usage by HIV-1 primary isolates has been studied by several groups, there is only limited information available concerning coreceptor usage by primary HIV-2 isolates. In this study, we have analyzed coreceptor usage of 15 primary HIV-2 isolates, using lymphocytes from a donor with nonfunctional CCR5 (CCR5 −/−; homozygous 32-bp deletion). Based on the infections of PBMCs, seven of these primary isolates had an absolute requirement for CCR5 expression, whereas the remaining eight exhibited a broader coreceptor usage. All CCR5-requiring isolates were non-syncytium inducing, whereas isolates utilizing multiple coreceptors were syncytium inducing. Blocking experiments using known ligands for chemokine receptors provided indirect evidence for additional coreceptor utilization by primary HIV-2 isolates. Analysis of GHOST4 cell lines expressing various chemokine receptors (CCR1, CCR2b, CCR3, CCR4, CCR5, CXCR4, BONZO, and BOB) further defined specific coreceptor usage of primary HIV-2 isolates. The receptors used included CXCR4, CCR1-5, and the recently described receptors BONZO and BOB. However, the efficiency at which the coreceptors were utilized varied greatly among the various isolates. Analysis of V3 envelope sequences revealed no specific motif that correlated with coreceptor usage. Our data demonstrate that primary HIV-2 isolates are capable of using a broad range of coreceptors for productive infection in vitro. Additionally, our data suggest that expanded coreceptor usage by HIV-2 may correlate with disease progression.     

Follicular dendritic cell-mediated up-regulation of CXCR4 expression on CD4 T cells and HIV pathogenesis

Follicular dendritic cells (FDCs) represent a major reservoir of HIV, and active infection occurs surrounding these cells, suggesting that this microenvironment is highly conducive to virus transmission. Because CD4 T cells around FDCs in germinal centers express the HIV coreceptor, CXCR4, whereas CD4 lymphocytes in many other sites do not, it prompted the hypothesis that FDCs may increase CXCR4 expression on CD4 T cells, thereby facilitating infection. To test this, HIV receptor/coreceptor expression was determined on CD4 T cells cultured with or without FDCs, and its consequence to infection was assessed by measuring virus binding and entry. FDCs had little effect on CCR5 or CD4 expression but increased CXCR4 expression on CD4 T cells. FDC-mediated up-regulation of CXCR4 on CD4 T cells occurred by 24 h and was sustained for at least 96 h in vitro, and FDC-CD4 T cell contact was necessary. Importantly, increased CXCR4 expression directly correlated with increased binding and entry of HIV-1 X4 isolates. Furthermore, CD4(+)CD57(+) germinal center T cells expressed high levels of CXCR4 and supported enhanced entry of X4 HIV compared with other CD4 T cells from the same tissue. Thus, in addition to serving as a reservoir of infectious virus, FDCs render surrounding germinal center T cells highly susceptible to infection with X4 isolates of HIV-1.     

Dendritic Cell–T-Cell Interactions Support Coreceptor-Independent Human Immunodeficiency Virus Type 1 Transmission in the Human Genital Tract

Worldwide, human immunodeficiency virus (HIV) is transmitted predominantly by heterosexual contact. Here, we investigate for the first time, by examining mononuclear cells obtained from cervicovaginal tissue, the mechanisms whereby HIV type 1 (HIV-1) directly targets cells from the human genital tract. In contrast to earlier findings in mucosal models such as human skin, we demonstrate that the majority of T cells and macrophages but none or few dendritic cells (DC) express the HIV-1 coreceptor CCR5 in normal human cervicovaginal mucosa, whereas all three cell types express the coreceptor CXCR4. To understand the role of coreceptor expression on infectivity, mucosal mononuclear cells were infected with various HIV-1 isolates, using either CCR5 or CXCR4. Unstimulated T cells become rapidly, albeit nonproductively, infected with R5- and X4-tropic variants. However, DC and T cells form stable conjugates which permit productive infection by viruses of both coreceptor specificities. These results indicate that HIV-1 can exploit T-cell-DC synergism in the human genital tract to overcome potential coreceptor restrictions on DC and postentry blocks of viral replication in unactivated T cells. Thus, mononuclear cells infiltrating the genital mucosa are permissive for transmission of both R5- and X4-tropic HIV-1 variants, and selection of virus variants does not occur by differential expression of HIV-1 coreceptors on genital mononuclear cells.     

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.     

Expression and function of chemokine receptors on human thymocytes: implications for infection by human immunodeficiency virus type 1

The presence or absence of the receptor CD4 and the coreceptors CCR5 and CXCR4 restrict the cell tropism of human immunodeficiency virus type 1 (HIV-1). Despite the importance of thymic infection by HIV-1, conflicting reports regarding the expression of HIV-1 coreceptors on human thymocytes have not been resolved. We assayed the expression and function of the major HIV-1 coreceptors, CCR5 and CXCR4, as well as CCR4 and CCR7 as controls, on human thymocytes. We detected CCR5 on 2.5% of thymocytes, CXCR4 on 53% of the cells, and CCR4 on 16% and CCR7 on 11% of human thymocytes. Moreover, infection by R5 HIV-1 did not significantly induce expression of CCR5. We found that two widely used anti-CCR5 monoclonal antibodies cross-reacted with CCR8, which may account for discrepancies among published reports of CCR5 expression on primary cells. This cross-reactivity could be eliminated by deletion of amino acids 2 through 4 of CCR8. Chemotaxis assays showed that SDF-1, which binds CXCR4; MDC, which binds CCR4; and ELC, which binds CCR7, mediated significant chemotaxis of thymocytes. In contrast, MIP-1beta, whose receptor is CCR5, did not induce significant chemotaxis. Our results indicate that CXCR4, CCR4, CCR7, and their chemokine ligands may be involved in thymocyte migration during development in the thymus. CCR5 and its ligands, however, are likely not involved in these processes. Furthermore, the pattern of CCR5 and CXCR4 expression that we found may explain the greater susceptibility of human thymocytes to infection by HIV-1 isolates capable of using CXCR4 in cell entry compared to those that use only CCR5.     

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.     

Antigenically distinct conformations of CXCR4

The major human immunodeficiency virus type 1 (HIV-1) coreceptors are the chemokine receptors CCR5 and CXCR4. The patterns of expression of the major coreceptors and their use by HIV-1 strains largely explain viral tropism at the level of entry. However, while virus infection is dependent upon the presence of CD4 and an appropriate coreceptor, it can be influenced by a number of factors, including receptor concentration, affinity between envelope gp120 and receptors, and potentially receptor conformation. Indeed, seven-transmembrane domain receptors, such as CCR5, can exhibit conformational heterogeneity, although the significance for virus infection is uncertain. Using a panel of monoclonal antibodies (MAbs) to CXCR4, we found that CXCR4 on both primary and transformed T cells as well as on primary B cells exhibited considerable conformational heterogeneity. The conformational heterogeneity of CXCR4 explains the cell-type-dependent ability of CXCR4 antibodies to block chemotaxis to stromal cell-derived factor 1 alpha and to inhibit HIV-1 infection. In addition, the MAb most commonly used to study CXCR4 expression, 12G5, recognizes only a subpopulation of CXCR4 molecules on all primary cell types analyzed. As a result, CXCR4 concentrations on these important cell types have been underestimated to date. Finally, while the factors responsible for altering CXCR4 conformation are not known, we found that they do not involve CXCR4 glycosylation, sulfation of the N-terminal domain of CXCR4, or pertussis toxin-sensitive G-protein coupling. The fact that this important HIV-1 coreceptor exists in multiple conformations could have implications for viral entry and for the development of receptor antagonists.     

Influence of the CCR2-V64I Polymorphism on Human Immunodeficiency Virus Type 1 Coreceptor Activity and on Chemokine Receptor Function of CCR2b, CCR3, CCR5, and CXCR4

The chemokine receptors CCR5 and CXCR4 are used by human immunodeficiency virus type 1 (HIV-1) in conjunction with CD4 to infect cells. In addition, some virus strains can use alternative chemokine receptors, including CCR2b and CCR3, for infection. A polymorphism in CCR2 (CCR2-V64I) is associated with a 2- to 4-year delay in the progression to AIDS. To investigate the mechanism of this protective effect, we studied the expression of CCR2b and CCR2b-V64I, their chemokine and HIV-1 coreceptor activities, and their effects on the expression and receptor activities of the major HIV-1 coreceptors. CCR2b and CCR2b-V64I were expressed at similar levels, and neither molecule affected the expression or coreceptor activity of CCR3, CCR5, or CXCR4 in cotransfected cell lines. Peripheral blood mononuclear cells (PBMCs) from CCR2-V64I heterozygotes had normal levels of CCR2b and CCR5 but slightly reduced levels of CXCR4. CCR2b and CCR2b-V64I functioned equally well as HIV-1 coreceptors, and CCR2-V64I PBMCs were permissive for HIV-1 infection regardless of viral tropism. The MCP-1-induced calcium mobilization mediated by CCR2b signaling was unaffected by the polymorphism, but MCP-1 signaling mediated by either CCR2b- or CCR2-V64I-encoded receptors resulted in heterologous desensitization (i.e., limiting the signal response of other receptors) of both CCR5 and CXCR4. The heterologous desensitization of CCR5 and CXCR4 signaling by both CCR2 allele receptor types provides a mechanistic link that might help explain the in vivo effects of CCR2 gene variants on progression to AIDS as well as the reported antiviral activity of natural CCR2 ligands.     

Apoptosis of bystander T cells induced by human immunodeficiency virus type 1 with increased envelope/receptor affinity and coreceptor binding site exposure

Apoptosis of uninfected bystander CD4(+) T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) pathogenesis. The viral and host mechanisms that lead to bystander apoptosis are not well understood. To investigate properties of the viral envelope glycoproteins (Env proteins) that influence the ability of HIV-1 to induce bystander apoptosis, we used molecularly cloned viruses that differ only in specific amino acids in Env. The ability of these strains to induce bystander apoptosis was tested in herpesvirus saimiri-immortalized primary CD4(+) T cells (CD4/HVS), which resemble activated primary T cells. Changes in Env that increase affinity for CD4 or CCR5 or increase coreceptor binding site exposure enhanced the capacity of HIV-1 to induce bystander apoptosis following viral infection or exposure to nonreplicating virions. Apoptosis induced by HIV-1 virions was inhibited by CD4, CXCR4, and CCR5 antibodies or by the CXCR4 inhibitor AMD3100, but not the fusion inhibitor T20. HIV-1 virions with mutant Envs that bind CXCR4 but are defective for CD4 binding or membrane fusion induced apoptosis, whereas CXCR4 binding-defective mutants did not. These results demonstrate that HIV-1 virions induce apoptosis through a CXCR4- or CCR5-dependent pathway that does not require Env/CD4 signaling or membrane fusion and suggest that HIV-1 variants with increased envelope/receptor affinity or coreceptor binding site exposure may promote T-cell depletion in vivo by accelerating bystander cell death.