Usage of the coreceptors CCR-5, CCR-3, and CXCR-4 by primary and cell line-adapted human immunodeficiency virus type 2.

The chemokine receptors CCR-5 and CXCR-4, and possibly CCR-3, are the principal human immunodeficiency virus type 1 (HIV-1) coreceptors, apparently interacting with HIV-1 envelope, in association with CD4. Cell lines coexpressing CD4 and these chemokine receptors were infected with a panel of seven primary HIV-2 isolates passaged in peripheral blood mononuclear cells (PBMC) and three laboratory HIV-2 strains passaged in T-cell lines. The CCR-5, CCR-3, and CXCR-4 coreceptors could all be used by HIV-2. The ability to use CXCR-4 represents a major difference between HIV-2 and the closely related simian immunodeficiency viruses. Most HIV-2 strains using CCR-5 could also use CCR-3, sometimes with similar efficiencies. As observed for HIV-1, the usage of CCR-5 or CCR-3 was observed principally for HIV-2 strains derived from asymptomatic individuals, while HIV-2 strains derived from AIDS patients used CXCR-4. However, there were several exceptions, and the patterns of coreceptor usage seemed more complex for HIV-2 than for HIV-1. The two T-tropic HIV-2 strains tested used CXCR-4 and not CCR-5, while T-tropic HIV-1 can generally use both. Moreover, among five primary HIV-2 strains all unable to use CXCR-4, three could replicate in CCR-5-negative PBMC, which has not been reported for HIV-1. These observations suggest that the CCR-5 coreceptor is less important for HIV-2 than for HIV-1 and indicate that HIV-2 can use other cell entry pathways and probably other coreceptors. One HIV-2 isolate replicating in normal or CCR-5-negative PBMC failed to infect CXCR-4+ cells or the U87MG-CD4 and sMAGI cell lines, which are permissive to infection by HIV-2 but not by HIV-1. This suggests the existence of several HIV-2-specific coreceptors, which are differentially expressed in cell lines and PBMC.     

Multiple extracellular domains of CCR-5 contribute to human immunodeficiency virus type 1 entry and fusion.

Human immunodeficiency virus type 1 (HIV-1) entry is governed by the interaction of the viral envelope glycoprotein (Env) with its receptor. The HIV-1 receptor is composed of two molecules, the CD4 binding receptor and a coreceptor. The seven-membrane-spanning chemokine receptor CCR-5 is one of the coreceptors used by primary isolates of HIV-1. We demonstrate that the mouse homolog of CCR-5 (mCCR-5) does not function as an HIV-1 coreceptor. A set of chimeras of human CCR-5 and mCCR-5 was studied for Env-induced cell fusion and HIV-1 infection. Using the HIV-1ADA envelope glycoprotein in a syncytium formation assay, we show that replacement of any fragment containing extracellular domains of mCCR-5 by its human counterparts is sufficient to allow Env-induced fusion. Conversely, replacement of any fragment containing human extracellular domains by its murine counterpart did not lead to coreceptor function loss. These results show that several domains of CCR-5 participate in coreceptor function. In addition, using a panel of primary nonsyncytium-inducing and syncytium-inducing isolates that use CCR-5 or both CXCR-4 and CCR-5 as coreceptors, we show that the latter dual-tropic isolates are less tolerant to changes in CCR-5 than strains with a more restricted coreceptor use. Thus, different strains are likely to have different ways of interacting with the CCR-5 coreceptor.     

Role of the first and third extracellular domains of CXCR-4 in human immunodeficiency virus coreceptor activity.

The CXCR-4 chemokine receptor and CD4 behave as coreceptors for cell line-adapted human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and for dual-tropic HIV strains, which also use the CCR-5 coreceptor. The cell line-adapted HIV-1 strains LAI and NDK and the dual-tropic HIV-2 strain ROD were able to infect CD4+ cells expressing human CXCR-4, while only LAI was able to infect cells expressing the rat homolog of CXCR-4. This strain selectivity was addressed by using human-rat CXCR-4 chimeras. All chimeras tested mediated LAI infection, but only those containing the third extracellular domain (e3) of human CXCR-4 mediated NDK and ROD infection. The e3 domain might be required for the functional interaction of NDK and ROD, but not LAI, with CXCR-4. Alternatively, LAI might also interact with e3 but in a different way. Monoclonal antibody 12G5, raised against human CXCR-4, did not stain cells expressing rat CXCR-4. Chimeric human-rat CXCR-4 allowed us to map the 12G5 epitope in the e3 domain. The ability of 12G5 to neutralize infection by certain HIV-1 and HIV-2 strains is also consistent with the role of e3 in the coreceptor activity of CXCR-4. The deletion of most of the amino-terminal extracellular domain (e1) abolished the coreceptor activity of human CXCR-4 for ROD and NDK but not for LAI. These results indicate that HIV strains have different requirements for their interaction with CXCR-4. They also suggest differences in the interaction of dual-tropic HIV with CCR-5 and CXCR-4.     

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.     

CD4, CXCR-4, and CCR-5 dependencies for infections by primary patient and laboratory-adapted isolates of human immunodeficiency virus type 1.

We have used a focal infectivity method to quantitatively analyze the CD4, CXCR-4, and CCR-5 dependencies for infections by diverse primary patient (PR) and laboratory-adapted (LA) isolates of human immunodeficiency virus type 1 (HIV-1). Infectivities of T-cell-tropic viruses were analyzed in a panel of HeLa-CD4 cell clones that have distinct quantities of CD4 and in human astroglioma U87MG-CD4 cells that express a large quantity of CD4 and become highly susceptible to infection after transfection with a CXCR-4 expression vector. The latter analysis indicated that PR as well as LA T-cell-tropic viruses efficiently employ CXCR-4 as a coreceptor in an optimal human cell line that contains abundant CD4. Previous uncertainties regarding coreceptor usage by PR T-cell-tropic HIV-1 isolates may therefore have derived from the assay conditions. As reported previously, unrelated LA and PR T-cell-tropic HIV-1 isolates differ in infectivities for the HeLa-CD4 clonal panel, with LA viruses infecting all clones equally and PR viruses infecting the clones in proportion to cellular CD4 quantities (D. Kabat, S. L. Kozak, K. Wherly, and B. Chesebro, J. Virol. 68:2570-2577, 1994). To analyze the basis for this difference, we used the HeLa-CD4 panel to compare a molecularly cloned T-cell-tropic PR virus (ELI1) with six of its variants that grow to different extents in CD4-positive leukemic cell lines and that differ only at specific positions in their gp120 and gp41 envelope glycoproteins. All mutations in gp120 or gp41 that contributed to laboratory adaptation preferentially enhanced infectivity for cells that had little CD4 and thereby decreased the CD4 dependencies of the infections. There was a close correlation between abilities of T-cell-tropic ELI viruses to grow in an expanded repertoire of leukemic cell lines, the reduced CD4 dependencies of their infections of the HeLa-CD4 panel, and their sensitivities to inactivation by soluble CD4 (sCD4). Since all of the ELI viruses can efficiently use CXCR-4 as a coreceptor, we conclude that an increase in viral affinity for CD4 rather than a switch in coreceptor specificity is principally responsible for laboratory adaption of T-cell-tropic HIV-1. Syncytium-inducing activities of the ELI viruses, especially when analyzed on cells with low amounts of CD4, were also highly correlated with their laboratory-adapted properties. Results with macrophage-tropic HIV-1 were strikingly different in both coreceptor and CD4 dependencies. When assayed in HeLa-CD4 cells transfected with an expression vector for CCR-5, macrophage-tropic HIV-1 isolates that had been molecularly cloned shortly after removal from patients were equally infectious for cells that had low or high CD4 quantities. Moreover, despite their substantial infectivities for cells that had only a trace of CD4, macrophage-tropic isolates were relatively resistant to inactivation by sCD4. We conclude that T-cell-tropic PR viruses bind weakly to CD4 and preferentially infect cells that coexpress CXCR-4 and large amounts of CD4. Their laboratory adaptation involves corresponding increases in affinities for CD4 and in abilities to infect cells that have relatively little CD4. In contrast, macrophage-tropic HIV-1 appears to interact weakly with CD4 although it can infect cells that coexpress CCR-5 and small quantities of CD4. We propose that cooperative binding of macrophage-tropic HIV-1 onto CCR-5 and CD4 may enhance virus adsorption and infectivity for cells that have only a trace of CD4.     

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.     

Direct visualization of HIV-1 entry: mechanisms and role of cell surface receptors.

Highly fluorescent virions of T- and M-tropic HIV-1 strains were obtained by incorporation of the viral accessory protein Vpr, fused to the green fluorescent protein, in trans. The fluorescent virions displayed normal morphology, were infectious, and could be used for direct visualization of HIV-1 attachment and trafficking in various cell lines. More than 90% of the viral particles were found to enter the cells by direct membrane fusion in T-cells, CD4+ HeLa cells, and macrophages. Visualizing HIV-1 attachment and entry in the absence or presence of CD4 and/or the appropriate coreceptors indicated that CD4 is the major receptor for virus attachment in the case of JR-CSF and NL-4-3 HIV-1 isolates; however, the coreceptors are required for membrane fusion. Internalization of the coreceptor CXCR4 inhibited entry, but did not prevent virus binding suggesting that transient downregulation of the coreceptor(s) may not be the most efficient way of blocking HIV infection in vivo.     

Preferential infection of dendritic cells during human immunodeficiency virus type 1 infection of blood leukocytes

Human immunodeficiency virus type 1 (HIV-1) transmission by the parenteral route is similar to mucosal transmission in the predominance of virus using the CCR5 coreceptor (R5 virus), but it is unclear whether blood dendritic cells (DCs), monocytes, or T cells are the cells initially infected. We used ex vivo HIV-1 infection of sorted blood mononuclear cells to model the in vivo infection of blood leukocytes. Using quantitative real-time PCR to detect full-length HIV-1 DNA, both sorted CD11c⁺ myeloid and CD11c⁻ plasmacytoid DCs were more frequently infected than other blood mononuclear cells, including CD16⁺ or CD14⁺ monocytes or resting CD4⁺ T cells. There was a strong correlation between CCR5 coreceptor use and preferential DC infection across a range of HIV-1 isolates. After infection of unsorted blood mononuclear cells, HIV-1 was initially detected in the CD11c⁺ DCs and later in other leukocytes, including clustering DCs and activated T cells. DC infection with R5 virus was productive, as shown by efficient transmission to CD4⁺ T cells in coculture. Blood DCs infected with HIV-1 in vitro and cultured alone expressed only low levels of multiply spliced HIV-1 RNA unless cocultured with CD4⁺ T cells. Early selective infection of immature blood DCs by R5 virus and upregulation of viral expression during DC-T-cell interaction and transmission provide a potential pathway for R5 selection following parenteral transmission.     

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.     

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.     

Roles of CD4 and coreceptors in binding, endocytosis, and proteolysis of gp120 envelope glycoproteins derived from human immunodeficiency virus type 1.

Infections by human immunodeficiency virus type 1 (HIV-1) involve interactions of the viral envelope glycoprotein gp120 with CD4 and then with a coreceptor. R5 isolates of HIV-1 use CCR5 as a coreceptor, whereas X4 isolates use CXCR4. It is not known whether coreceptors merely trigger fusion of the viral and cellular membranes or whether they also influence the energetics of virus adsorption, the placement of the membrane fusion reaction, and the metabolism of adsorbed gp120. Surprisingly, the pathway for metabolism of adsorbed gp120 has not been investigated thoroughly in any cells. To address these issues, we used purified (125)I-gp120s derived from the R5 isolate BaL and from the X4 isolate IIIB as ligands for binding onto human cells that expressed CD4 alone or CD4 with a coreceptor. The gp120 preparations were active in forming ternary complexes with CD4 and the appropriate coreceptor. Moreover, the cellular quantities of CD4 and coreceptors were sufficient for efficient infections by the corresponding HIV-1 isolates. In these conditions, the kinetics and affinities of (125)I-gp120 adsorptions and their subsequent metabolisms were strongly dependent on CD4 but were not significantly influenced by CCR5 or CXCR4. After binding to CD4, the (125)I-gp120s slowly became resistant to extraction from the cell monolayers by pH 3.0 buffer, suggesting that they were endocytosed with half-times of 1-2 h. Within 20-30 min of endocytosis, the (125)I-gp120s were proteolytically degraded to small products that were shed into the media. The weak base chloroquine strongly inhibited (125)I-gp120 proteolysis and caused its intracellular accumulation, suggesting involvement of a low pH organelle. Results supporting these methods and conclusions were obtained by confocal immunofluorescence microscopy. We conclude that the energetics, kinetics, and pathways of (125)I-gp120 binding, endocytosis, and proteolysis are determined principally by CD4 rather than by coreceptors in cells that contain sufficient coreceptors for efficient infections. Therefore, the role of coreceptors in HIV-1 infections probably does not include steerage or subcellular localization of adsorbed virus.     

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.     

Endogenous Production of β-Chemokines by CD4+, but Not CD8+, T-Cell Clones Correlates with the Clinical State of Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals and May Be Responsible for Blocking Infection with NonSyncytium-Inducing HIV-1 In Vitro

Recent studies have demonstrated that the β-chemokines RANTES, MIP-1α, and MIP-1β suppress human immunodeficiency virus type 1 (HIV-1) replication in vitro and may play an important role in protecting exposed but uninfected individuals from HIV-1 infection. However, levels of β-chemokines in AIDS patients are comparable to and can exceed levels in nonprogressing individuals, indicating that global β-chemokine production may have little effect on HIV-1 disease progression. We sought to clarify the role of β-chemokines in nonprogressors and AIDS patients by examination of β-chemokine production and HIV-1 infection in patient T-lymphocyte clones established by herpesvirus saimiri immortalization. Both CD4⁺ and CD8⁺ clones were established, and they resembled primary T cells in their phenotypes and expression of activated T-cell markers. CD4⁺ T-cell clones from all patients had normal levels of mRNA-encoding CCR5, a coreceptor for non-syncytium-inducing (NSI) HIV-1. CD4⁺ clones from nonprogressors and CD8⁺ clones from AIDS patients secreted high levels of RANTES, MIP1α, and MIP-1β. In contrast, CD4⁺ clones from AIDS patients produced no RANTES and little or no MIP-1α or MIP-1β. The infection of CD4⁺ clones with the NSI HIV-1 strain ADA revealed an inverse correlation to β-chemokine production; clones from nonprogressors were poorly susceptible to ADA replication, but clones from AIDS patients were highly infectable. The resistance to ADA infection in CD4⁺ clones from nonprogressors could be partially reversed by treatment with anti-β-chemokine antibodies. These results indicate that CD4⁺ cells can be protected against NSI-HIV-1 infection in culture through endogenously produced factors, including β-chemokines, and that β-chemokine production by CD4⁺, but not CD8⁺, T cells may constitute one mechanism of disease-free survival for HIV-1-infected individuals.     

Intrinsic Human Immunodeficiency Virus Type 1 Resistance of Hematopoietic Stem Cells Despite Coreceptor Expression

Interactions of human immunodeficiency virus type 1 (HIV-1) with hematopoietic stem cells may define restrictions on immune reconstitution following effective antiretroviral therapy and affect stem cell gene therapy strategies for AIDS. In the present study, we demonstrated mRNA and cell surface expression of HIV-1 receptors CD4 and the chemokine receptors CCR-5 and CXCR-4 in fractionated cells representing multiple stages of hematopoietic development. Chemokine receptor function was documented in subsets of cells by calcium flux in response to a cognate ligand. Productive infection by HIV-1 via these receptors was observed with the notable exception of stem cells, in which case the presence of CD4, CXCR-4, and CCR-5, as documented by single-cell analysis for expression and function, was insufficient for infection. Neither productive infection, transgene expression, nor virus entry was detectable following exposure of stem cells to either wild-type HIV-1 or lentivirus constructs pseudotyped in HIV-1 envelopes of macrophage-tropic, T-cell-tropic, or dualtropic specificity. Successful entry into stem cells of a vesicular stomatitis virus G protein-pseudotyped HIV-1 construct demonstrated that the resistance to HIV-1 was mediated at the level of virus-cell membrane fusion and entry. These data define the hematopoietic stem cell as a sanctuary cell which is resistant to HIV-1 infection by a mechanism independent of receptor and coreceptor expression that suggests a novel means of cellular protection from HIV-1.     

Naïve and Memory CD4 T Cells Differ in Their Susceptibilities to Human Immunodeficiency Virus Type 1 Infection following CD28 Costimulation: Implications for Transmission and Pathogenesis

In vitro evidence suggests that memory CD4⁺ cells are preferentially infected by human immunodeficiency virus type 1 (HIV-1), yet studies of HIV-1-infected individuals have failed to detect preferential memory cell depletion. To explore this paradox, we stimulated CD45RA⁺ CD4⁺ (naïve) and CD45RO⁺ CD4⁺ (memory) cells with antibodies to CD3 and CD28 and infected them with either CCR5-dependent (R5) or CXCR4-dependent (X4) HIV-1 isolates. Naïve CD4⁺ cells supported less X4 HIV replication than their memory counterparts. However, naïve cells were susceptible to R5 viral infection, while memory cells remained resistant to infection and viral replication. As with the unseparated cells, mixing the naïve and memory cells prior to infection resulted in cells resistant to R5 infection and highly susceptible to X4 infection. While both naïve and memory CD4⁺ subsets downregulated CCR5 expression in response to CD28 costimulation, only the memory cells produced high levels of the β-chemokines RANTES, MIP-1α, and MIP-1β upon stimulation. Neutralization of these β-chemokines rendered memory CD4⁺ cells highly sensitive to infection with R5 HIV-1 isolates, indicating that downregulation of CCR5 is not sufficient to mediate complete protection from CCR5 strains of HIV-1. These results indicate that susceptibility to R5 HIV-1 isolates is determined not only by the level of CCR5 expression but also by the balance of CCR5 expression and β-chemokine production. Furthermore, our results suggest a model of HIV-1 transmission and pathogenesis in which naïve rather than memory CD4⁺ T cells serve as the targets for early rounds of HIV-1 replication.     

CD4 binding affinity determines human immunodeficiency virus type 1-induced alpha interferon production in plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDC) are major producers of type I interferons (IFN) in response to human immunodeficiency virus type 1 (HIV-1) infection. To better define the underlying mechanisms, we studied the magnitude of alpha IFN (IFN-α) induction by recombinant viruses containing changes in the Env protein that impair or disrupt CD4 binding or expressing primary env alleles with differential coreceptor tropism. We found that the CD4 binding affinity but not the viral coreceptor usage is critical for the attachment of autofluorescing HIV-1 to PDC and for subsequent IFN-α induction. Our results illustrate the importance of the gp120-CD4 interaction in determining HIV-1-induced immune stimulation via IFN-α production.     

Genetic Subtype-Independent Inhibition of Human Immunodeficiency Virus Type 1 Replication by CC and CXC Chemokines

We have studied the breadth and potency of the inhibitory actions of the CC chemokines macrophage inhibitory protein 1α (MIP-1α), MIP-1β, and RANTES against macrophage-tropic (M-tropic) primary isolates of human immunodeficiency virus type 1 (HIV-1) and of the CXC chemokine stromal cell-derived factor 1α against T-cell-tropic (T-tropic) isolates, using mitogen-stimulated primary CD4⁺ T cells as targets. There was considerable interisolate variation in the sensitivity of HIV-1 to chemokine inhibition, which was especially pronounced for the CC chemokines and M-tropic strains. However, this variation was not obviously dependent on the genetic subtype (A through F) of the virus isolates. Peripheral blood mononuclear cell donor-dependent variation in chemokine inhibition potency was also observed. Among the CC chemokines, the rank order for potency (from most to least potent) was RANTES, MIP-1β, MIP-1α. Some M-tropic isolates, unexpectedly, were much more sensitive to RANTES than to MIP-1β, whereas other isolates showed sensitivities comparable to those of these two chemokines. Down-regulation of the CCR5 and CXCR4 receptors occurred in cells treated with the cognate chemokines and probably contributes to anti-HIV-1 activity. Thus, for CCR5, the rank order for down-regulation was also RANTES, MIP-1β, MIP-1α.     

Multiple Residues Contribute to the Inability of Murine CCR-5 To Function as a Coreceptor for Macrophage-Tropic Human Immunodeficiency Virus Type 1 Isolates

Infection of CD4-positive cells by human immunodeficiency virus type 1 (HIV-1) requires functional interaction of the viral envelope protein with a coreceptor belonging to the chemokine receptor family of seven-membrane-spanning receptors. For the majority of macrophage-tropic HIV-1 isolates, the physiologically relevant coreceptor is the human CCR-5 (hCCR-5) receptor. Although the murine homolog of CCR-5 (mCCR-5) is unable to mediate HIV-1 infection, chimeric hCCR-5/mCCR-5 molecules containing single extracellular domains derived from hCCR-5 are effective coreceptors for certain macrophage-tropic HIV-1 isolates. Here, we have sought to identify residues in hCCR-5 critical for HIV-1 infection by substitution of mCCR-5-derived residues into the context of functional chimeric hCCR-5/mCCR-5 receptor molecules. Using this strategy, we demonstrate that residues 7, 13, and 15 in the first extracellular domain and residue 180 in the third extracellular domain of CCR-5 are important for HIV-1 envelope-mediated membrane fusion. Of interest, certain substitutions, for example, at residues 184 and 185 in the third extracellular domain, have no phenotype when introduced individually but strongly inhibit hCCR-5 coreceptor function when present together. We hypothesize that these changes, which do not preclude chemokine receptor function, may inhibit a conformational transition in hCCR-5 that contributes to HIV-1 infection. Finally, we report that substitution of glycine for valine at residue 5 in CCR-5 can significantly enhance the level of envelope-dependent cell fusion by expressing cells. The diversity of the mutant phenotypes observed in this mutational analysis, combined with their wide distribution across the extracellular regions of CCR-5, emphasizes the complexity of the interaction between HIV-1 envelope and coreceptor.Infection of cells by human immunodeficiency virus type 1 (HIV-1) requires interaction of the viral envelope protein with not only CD4 but also a second cell surface molecule, termed a coreceptor (reviewed in reference 19). Coreceptor usage varies significantly among different HIV-1 isolates, although all known coreceptors are members of the G-protein-coupled chemokine receptor family of seven-membrane-spanning receptors. The primary coreceptor used by non-syncytium-inducing, macrophage-tropic (M-tropic) HIV-1 isolates, which constitute the majority of primary isolates, is CCR-5 (1, 6, 8, 12, 27). In contrast, syncytium-inducing, T-cell-line-adapted (T-tropic) HIV-1 isolates predominantly use CXCR-4 as a coreceptor (13). Other chemokine receptors utilized by a small percentage of generally dualtropic HIV-1 isolates include CCR-2b and CCR-3 (6, 11). The importance of two orphan chemokine receptors, termed Bonzo/STRL33 and BOB/GPR15, in infection by HIV-1 remains to be established, although these proteins were recently shown to serve as coreceptors for several simian immunodeficiency virus and HIV-2 isolates (2, 9). The critical importance of CCR-5 for infection by primary, M-tropic HIV-1 isolates, however, has been highlighted by the finding that a small percentage of humans lack a functional CCR-5 gene and as a result appear highly, although not completely, resistant to infection by HIV-1 (17, 22). Importantly, primary T cells derived from such individuals are refractory to infection by M-tropic HIV-1 isolates in vitro (17, 22, 27), thus demonstrating that CCR-5 is the physiologically relevant coreceptor for the majority of primary isolates.At present, relatively little is known about how the viral envelope and coreceptor interact, although it appears clear that interaction is dependent upon a prior conformational shift induced by binding of the envelope gp120 subunit to CD4 (24, 26). This in turn is believed to lead to the formation of a ternary complex, consisting of gp120, coreceptor, and CD4, on the surface of the target cell (15, 24, 26). It is unknown how this protein complex then induces the fusion of the viral and host cell membranes, although the envelope gp41 subunit is believed to play a critical role at this stage.An important unresolved question is the identity of the amino acid residues in gp120 and the coreceptor that interact during infection. However, it is well established that HIV-1 tropism, and hence coreceptor usage, is largely controlled by a small number of residues located in the envelope V3 loop (6, 14, 23, 25). Efforts to identify residues in the CCR-5 coreceptor involved in mediating infection have thus far largely focused on the functional analysis of chimeric receptors generated with human CCR-5 (hCCR-5) and a chemokine receptor lacking coreceptor function, such as the murine CCR-5 homolog (mCCR-5) (3, 5, 20, 21). These studies have led to three major conclusions. Firstly, the residues in hCCR-5 involved in mediating HIV-1 infection are diffuse, being located on at least three of the four extracellular domains of CCR-5. Secondly, these residues are functionally redundant, so that several distinct regions of hCCR-5 can suffice independently to confer coreceptor function when substituted into mCCR-5. Lastly, different HIV-1 envelope proteins interact differently with CCR-5, such that CCR-5 residues important for mediating fusion by one envelope protein may be largely irrelevant to the interaction of CCR-5 with a second envelope protein. Overall, these data demonstrate that the envelope–CCR-5 interaction is likely to be highly complex and to involve the interaction of multiple residues in both proteins.As noted above, the mCCR-5 chemokine receptor, despite extensive sequence similarity to hCCR-5, fails to function as an HIV-1 coreceptor (3, 5, 20). Therefore, it is apparent that one or more of the 20 extracellular residues that differ between mCCR-5 and hCCR-5 must contribute to the interaction with the HIV-1 envelope protein. Using mutational analysis in the context of chimeric mCCR-5/hCCR-5 receptors, we have now identified several residues, located in three of the four extracellular domains of hCCR-5, that play roles in mediating infection by HIV-1.     

CXCR4 and CCR5 regulation and expression patterns on T- and monocyte-macrophage cell lineages: implications for susceptibility to infection by HIV-1

Chemokine receptor expression may vary dramatically among cell subsets. Therefore, the stage of differentiation and the lineage of CD4 cells may profoundly affect their susceptibility to infection by human immunodeficiency virus type 1 (HIV-1). However, the mechanisms of coreceptor competition for association with HIV-1 glycoproteins remain unknown. Here, we propose mathematical models that address the interdependence of the concentrations of CD4 and CCR5 for efficient infection by M-tropic HIV-1 as well as additional complications originated by coreceptor competition caused by posttranslational modifications that positively or negatively affect the coreceptor ability to form complexes with CD4 and/or HIV-1 envelope. Furthermore, since CCR5 and CXCR4 expression on human leukocytes designate these cells as HIV-1 potential targets, the expression of the major HIV-1 coreceptors are also dynamically modeled/quantified as function of the stage of cell differentiation. Results show that although coreceptor competition degree has limited influence on R5 strain infectivity, the infectivity of CXCR4-using isolates strongly depends on the CD4 expression, according to the coreceptor competition model proposed in Lee et al. [J. Virol. 74(11) (2000) 5016]. Understanding the role of in vivo alterations in CD4, CCR5 and CXCR4 densities on HIV-1 cell entry may help the development of optimal control strategies for AIDS pathogenesis.