CD4-independent, CCR5-dependent simian immunodeficiency virus infection and chemotaxis of human cells

Most simian immunodeficiency virus (SIV), human immunodeficiency virus type 2 (HIV-2), and HIV-1 infection of host peripheral blood mononuclear cells (PBMCs) is CD4 dependent. In some cases, X4 HIV-1 chemotaxis is CD4 independent, and cross-species transmission might be facilitated by CD4-independent entry, which has been demonstrated for some SIV strains in CD4(-) non-T cells. As expected for CCR5-dependent virus, SIV required CD4 on rhesus and pigtail macaque PBMCs for infection and chemotaxis. However, SIV induced the chemotaxis of human PBMCs in a CD4-independent manner. Furthermore, in contrast to the results of studies using transfected human cell lines, SIV did not require CD4 binding to productively infect primary human PBMCs. CD4-independent lymphocyte and macrophage infection may facilitate cross-species transmission, while reacquisition of CD4 dependence may confer a selective advantage for the virus within new host species.     

Functional dissection of CCR5 coreceptor function through the use of CD4-independent simian immunodeficiency virus strains

With rare exceptions, all simian immunodeficiency virus (SIV) strains can use CCR5 as a coreceptor along with CD4 for viral infection. In addition, many SIV strains are capable of using CCR5 as a primary receptor to infect CD4-negative cells such as rhesus brain capillary endothelial cells. By using coupled fluorescence-activated cell sorter (FACS) and infection assays, we found that even very low levels of CCR5 expression could support CD4-independent virus infection. CD4-independent viruses represent valuable tools for finely dissecting interactions between Env and CCR5 which may otherwise be masked due to the stabilization of these contacts by Env-CD4 binding. Based on the ability of SIV Env to bind to and mediate infection of cells expressing CCR5 chimeras and mutants, we identified the N terminus of CCR5 as a critical domain for direct Env binding and for supporting CD4-independent virus infection. However, the activity of N-terminal domain CCR5 mutants could be rescued by the presence of CD4, indicating that other regions of CCR5 are important for post-binding events that lead to viral entry. Rhesus CCR5 supported CD4-independent infection and direct Env binding more efficiently than did human CCR5 due to a single amino acid difference in the N terminus. Interestingly, uncleaved, oligomeric SIV Env protein bound to both CD4 and CCR5 less efficiently than did monomeric gp120. Finally, several mutations present in chronically infected monkey populations are shown to decrease the ability of CCR5 to serve as a primary viral receptor for the SIV isolates examined.     

N-linked glycosylation of CXCR4 masks coreceptor function for CCR5-dependent human immunodeficiency virus type 1 isolates

The chemokine receptors CXCR4 and CCR5 are the principal coreceptors for infection of X4 and R5 human immunodeficiency virus type 1 (HIV-1) isolates, respectively. Here we report on the unexpected observation that the removal of the N-linked glycosylation sites in CXCR4 potentially allows the protein to serve as a universal coreceptor for both X4 and R5 laboratory-adapted and primary HIV-1 strains. We hypothesize that this alteration unmasks existing common extracellular structures reflecting a conserved three-dimensional similarity of important elements of CXCR4 and CCR5 that are involved in HIV envelope glycoprotein (Env) interaction. These results may have far-reaching implications for the differential recognition of cell type-dependent glycosylated CXCR4 by HIV-1 isolates and their evolution in vivo. They also suggest a possible explanation for the various observations of restricted virus entry in some cell types and further our understanding of the framework of elements that represent the Env-coreceptor contact sites.     

Mobility of the human immunodeficiency virus (HIV) receptor CD4 and coreceptor CCR5 in living cells: implications for HIV fusion and entry events

The sequence of events leading to human immunodeficiency virus fusion and entry likely involves the recruitment of multiple receptor and coreceptor proteins to a specific complex by the viral envelope. Using fluorescence recovery after photobleaching technology, we find that both CD4 and CCR5 are mobile in the cell membrane. Interestingly, our findings also suggest that the seven-span transmembrane coreceptor is significantly more mobile than CD4 and requires membrane cholesterol for mobility.     

Two distinct CCR5 domains can mediate coreceptor usage by human immunodeficiency virus type 1.

The chemokine receptor CCR5 is the major fusion coreceptor for macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1). To define the structures of CCR5 that can support envelope (Env)-mediated membrane fusion, we analyzed the activity of homologs, chimeras, and mutants of human CCR5 in a sensitive gene reporter cell-cell fusion assay. Simian, but not murine, homologs of CCR5 were fully active as HIV-1 fusion coreceptors. Chimeras between CCR5 and divergent chemokine receptors demonstrated the existence of two distinct regions of CCR5 that could be utilized for Env-mediated fusion, the amino-terminal domain and the extracellular loops. Dual-tropic Env proteins were particularly sensitive to alterations in the CCR5 amino-terminal domain, suggesting that this domain may play a pivotal role in the evolution of coreceptor usage in vivo. We identified individual residues in both functional regions, Asp-11, Lys-197, and Asp-276, that contribute to coreceptor function. Deletion of a highly conserved cytoplasmic motif rendered CCR5 incapable of signaling but did not abrogate its ability to function as a coreceptor, implying the independence of fusion and G-protein-mediated chemokine receptor signaling. Finally, we developed a novel monoclonal antibody to CCR5 to assist in future studies of CCR5 expression.     

Simian immunodeficiency viruses of diverse origin can use CXCR4 as a coreceptor for entry into human cells

Primary simian immunodeficiency virus (SIV) isolated from sooty mangabey (SIVsm [n = 6]), stumptail (SIVstm [n = 1]), mandrill (SIVmnd [n = 1]), and African green (SIVagm [n = 1]) primates were examined for their ability to infect human cells and for their coreceptor requirements. All isolates infected human peripheral blood mononuclear cells (PBMCs) from a CCR5(+/+) donor, and seven of eight isolates tested also infected CCR5(-/-) PBMCs. Analysis of coreceptor utilization using GHOST and U87 cell lines revealed that all of the isolates tested used CCR5 and the orphan receptors STRL33 and GPR15. Coreceptors such as CCR2b, CCR3, CCR8, and CX3CR1 were also utilized by some primary SIV isolates. More importantly, we found that CXCR4 was used as a coreceptor by the SIVstm, the SIVagm, and four of the SIVsm isolates in GHOST and U87 cells. These data suggest that primary SIV isolates from diverse primate species can utilize CXCR4 for viral entry, similar to what has been described for human immunodeficiency viruses.     

Identification of residues of CXCR4 critical for human immunodeficiency virus coreceptor and chemokine receptor activities

CXCR4 is a G-coupled receptor for the stromal cell-derived factor (SDF-1) chemokine, and a CD4-associated human immunodeficiency virus type 1 (HIV-1) coreceptor. These functions were studied in a panel of CXCR4 mutants bearing deletions in the NH(2)-terminal extracellular domain (NT) or substitutions in the NT, the extracellular loops (ECL), or the transmembrane domains (TMs). The coreceptor activity of CXCR4 was markedly impaired by mutations of two Tyr residues in NT (Y7A/Y12A) or at a single Asp residue in ECL2 (D193A), ECL3 (D262A), or TMII (D97N). These acidic residues could engage electrostatical interactions with basic residues of the HIV-1 envelope protein gp120, known to contribute to the selectivity for CXCR4. The ability of CXCR4 mutants to bind SDF-1 and mediate cell signal was consistent with the two-site model of chemokine-receptor interaction. Site I involved in SDF-1 binding but not signaling was located in NT with particular importance of Glu(14) and/or Glu(15) and Tyr(21). Residues required for both SDF-1 binding and signaling, and thus probably part of site II, were identified in ECL2 (Asp(187)), TMII (Asp(97)), and TMVII (Glu(288)). The first residues () of NT also seem required for SDF-1 binding and signaling. A deletion in the third intracellular loop abolished signaling, probably by disrupting the coupling with G proteins. The identification of CXCR4 residues involved in the interaction with both SDF-1 and HIV-1 may account for the signaling activity of gp120 and has implications for the development of antiviral compounds.     

CCR5 signal transduction in macrophages by human immunodeficiency virus and simian immunodeficiency virus envelopes

The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1beta. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1alpha, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages.     

Primary, syncytium-inducing human immunodeficiency virus type 1 isolates are dual-tropic and most can use either Lestr or CCR5 as coreceptors for virus entry.

A panel of primary syncytium-inducing (SI) human immunodeficiency virus type 1 isolates that infected several CD4+ T-cell lines, including MT-2 and C8166, were tested for infection of blood-derived macrophages. Infectivity titers for C8166 cells and macrophages demonstrated that primary SI strains infected macrophages much more efficiently than T-cell line-adapted HIV-1 strains such as LAI and RF. These primary SI strains were therefore dual-tropic. Nine biological clones of two SI strains, prepared by limiting dilution, had macrophage/C8166 infectivity ratios similar to those of their parental viruses, indicating that the dual-tropic phenotype was not due to a mixture of non-SI/macrophage-tropic and SI/T-cell tropic viruses. We tested whether the primary SI strains used either Lestr (fusin) or CCR5 as coreceptors. Infection of cat CCC/CD4 cells transiently expressing Lestr supported infection by T-cell line-adapted strains including LAI, whereas CCC/CD4 cells expressing CCR5 were sensitive to primary non-SI strains as well as to the molecularly cloned strains SF-162 and JR-CSF. Several primary SI strains, as well as the molecularly cloned dual-tropic viruses 89.6 and GUN-1, infected both Lestr+ and CCR5+ CCC/CD4 cells. Thus, these viruses can choose between Lestr and CCR5 for entry into cells. Interestingly, some dual-tropic primary SI strains that infected Lestr+ cells failed to infect CCR5+ cells, suggesting that these viruses may use an alternative coreceptor for infection of macrophages. Alternatively, CCR5 may be processed or presented differently on cat cells so that entry of some primary SI strains but not others is affected.     

CD4+ CCR5+ T-cell dynamics during simian immunodeficiency virus infection of Chinese rhesus macaques

Simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) provides a reliable model to study the relationship between lentivirus replication, cellular immune responses, and CD4⁺ T-cell dynamics. Here we investigated, using SIVmac251-infected RMs of a Chinese genetic background (which experience a slower disease progression than Indian RMs), the dynamics of CD4⁺ CCR5⁺ T cells, as this subset of memory/activated CD4⁺ T cells is both a preferential target of virus replication and a marker of immune activation. As expected, we observed that the number of circulating CD4⁺ CCR5⁺ T cells decreases transiently at the time of peak viremia. However, at 60 days postinfection, i.e., when set-point viremia is established, the level of CD4⁺ CCR5⁺ T cells was increased compared to the baseline level. Interestingly, this increase correlated with faster disease progression, higher plasma viremia, and early loss of CD4⁺ T-cell function, as measured by CD4⁺ T-cell count, the fraction of memory CD4⁺ T cells, and the recall response to purified protein derivative. Taken together, these data show a key difference between the dynamics of the CD4⁺ CCR5⁺ T-cell pool (and its relationship with disease progression) in Chinese RMs and those described in previous reports for Indian SIVmac251-infected RMs. As the SIV-associated changes in the CD4⁺ CCR5⁺ T-cell pool reflect the opposing forces of SIV replication (which reduces this cellular pool) and immune activation (which increases it), our data suggest that in SIV-infected Chinese RMs the impact of immune activation is more prominent than that of virus replication in determining the size of the pool of CD4⁺ CCR5⁺ T cells in the periphery. As progression of HIV infection in humans also is associated with a relative expansion of the level of CD4⁺ CCR5⁺ T cells, we propose that SIV infection of Chinese RMs is a very valuable and important animal model for understanding the pathogenesis of human immunodeficiency virus infection.     

Utilization of C-C chemokine receptor 5 by the envelope glycoproteins of a pathogenic simian immunodeficiency virus, SIVmac239.

We examined chemokine receptors for the ability to facilitate the infection of CD4-expressing cells by viruses containing the envelope glycoproteins of a pathogenic simian immunodeficiency virus, SIVmac239. Expression of either human or simian C-C chemokine receptor CCR5 allowed the SIVmac239 envelope glycoproteins to mediate virus entry and cell-to-cell fusion. Thus, distantly related immunodeficiency viruses such as SIV and the primary human immunodeficiency virus type 1 isolates can utilize CCR5 as an entry cofactor.     

Cytolysis by CCR5-using human immunodeficiency virus type 1 envelope glycoproteins is dependent on membrane fusion and can be inhibited by high levels of CD4 expression

T-tropic (X4) and dualtropic (R5X4) human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins kill primary and immortalized CD4(+) CXCR4(+) T cells by mechanisms involving membrane fusion. However, because much of HIV-1 infection in vivo is mediated by M-tropic (R5) viruses whose envelope glycoproteins use CCR5 as a coreceptor, we tested a panel of R5 and R5X4 envelope glycoproteins for their ability to lyse CCR5(+) target cells. As is the case for CXCR4(+) target cells, HIV-1 envelope glycoproteins expressed by single-round HIV-1 vectors killed transduced CD4(+) CCR5(+) cells in a membrane fusion-dependent manner. Furthermore, a CD4-independent R5 HIV-1 envelope glycoprotein was able to kill CD4-negative target cells expressing CCR5, demonstrating that CD4 is not intrinsically required for the induction of death. Interestingly, high levels of CD4 expression protected cells from lysis and syncytium formation mediated by the HIV-1 envelope glycoproteins. Immunoprecipitation experiments showed that high levels of CD4 coexpression inhibited proteolytic processing of the HIV-1 envelope glycoprotein precursor gp160. This inhibition could be overcome by decreasing the CD4 binding ability of gp120. Studies were also undertaken to investigate the ability of virion-bound HIV-1 envelope glycoproteins to kill primary CD4(+) T cells. However, neither X4 nor R5X4 envelope glycoproteins on noninfectious virions caused death in primary CD4(+) T cells. These results demonstrate that the interaction of CCR5 with R5 HIV-1 envelope glycoproteins capable of inducing membrane fusion leads to cell lysis; overexpression of CD4 can inhibit cell killing by limiting envelope glycoprotein processing.     

Transmembrane envelope glycoproteins of human immunodeficiency virus type 2 and simian immunodeficiency virus SIV-mac exist as homodimers.

An 80-kilodalton glycoprotein (gp80) was produced in human immunodeficiency virus type 2 (HIV-2)-infected cells along with three envelope glycoproteins that we have recently reported: the extracellular glycoprotein (gp125), the envelope glycoprotein precursor (gp140), and the transient dimeric form of the precursor (gp300). gp125 and gp80 were detectable after the synthesis of gp140 and the formation of gp300. Using a specific monoclonal antibody, we showed here that gp80 is a dimeric form of the transmembrane glycoprotein gp36 of HIV-2. Dimerization of the envelope glycoprotein precursor and dimeric forms of the transmembrane glycoproteins were also observed in cells infected with simian immunodeficiency virus (SIV-mac), a virus closely related to HIV-2. Under routine conditions of our experiments (i.e., extraction by 1% Triton X-100 before polyacrylamide gel electrophoresis in sodium dodecyl sulfate [SDS]), monomeric forms of the transmembrane glycoprotein of HIV-2 and SIV-mac were only seldomly observed. Dimeric forms of the envelope precursors and the transmembrane glycoproteins are probably stabilized by extraction in the nonionic detergent Triton X-100 since such dimeric forms resist dissociation during subsequent electrophoresis in the presence of the ionic detergent SDS. However, the dissociation of these dimeric forms might occur when samples are prepared by extraction directly in 1% SDS or by incubation of the purified dimers at acidic pH. Dimerization of the envelope precursor might be required for its processing to give the mature envelope proteins, whereas the transmembrane dimer might be essential for optimal structure of the virion and thus its infectivity.     

Dynamics of CCR5 expression by CD4(+) T cells in lymphoid tissues during simian immunodeficiency virus infection

Early viral replication and profound CD4(+) T-cell depletion occur preferentially in intestinal tissues of macaques infected with simian immunodeficiency virus (SIV). Here we show that a much higher percentage of CD4(+) T cells in the intestine express CCR5 compared with those found in the peripheral blood, spleen, or lymph nodes. In addition, the selectivity and extent of the CD4(+) T-cell loss in SIV infection may depend upon these cells coexpressing CCR5 and having a "memory" phenotype (CD45RA(-)). Following intravenous infection with SIVmac251, memory CD4(+) CCR5(+) T cells were selectively eliminated within 14 days in all major lymphoid tissues (intestine, spleen, and lymph nodes). However, the effect on CD4(+) T-cell numbers was most profound in the intestine, where cells of this phenotype predominate. The CD4(+) T cells that remain after 14 days of infection lacked CCR5 and/or were naive (CD45RA(+)). Furthermore, when animals in the terminal stages of SIV infection (with AIDS) were examined, virtually no CCR5-expressing CD4(+) T cells were found in lymphoid tissues, and all of the remaining CD4(+) T cells were naive and coexpressed CXCR4. These findings suggest that chemokine receptor usage determines which cells are targeted for SIV infection and elimination in vivo.     

Increased CCR5 affinity and reduced CCR5/CD4 dependence of a neurovirulent primary human immunodeficiency virus type 1 isolate

Most human immunodeficiency virus type 1 (HIV-1) viruses in the brain use CCR5 as the principal coreceptor for entry into a cell. However, additional phenotypic characteristics are necessary for HIV-1 neurotropism. Furthermore, neurotropic strains are not necessarily neurovirulent. To better understand the determinants of HIV-1 neurovirulence, we isolated viruses from brain tissue samples from three AIDS patients with dementia and HIV-1 encephalitis and analyzed their ability to induce syncytia in monocyte-derived macrophages (MDM) and neuronal apoptosis in primary brain cultures. Two R5X4 viruses (MACS1-br and MACS1-spln) were highly fusogenic in MDM and induced neuronal apoptosis. The R5 viruses UK1-br and MACS2-br are both neurotropic. However, only UK1-br induced high levels of fusion in MDM and neuronal apoptosis. Full-length Env clones from UK1-br required lower CCR5 and CD4 levels than Env clones from MACS2-br to function efficiently in cell-to-cell fusion and single-round infection assays. UK1-br Envs also had a greater affinity for CCR5 than MACS2-br Envs in binding assays. Relatively high levels of UK1-br and MACS2-br Envs bound to CCR5 in the absence of soluble CD4. However, these Envs could not mediate CD4-independent infection, and MACS2-br Envs were unable to mediate fusion or infection in cells expressing low levels of CD4. The UK1-br virus was more resistant than MACS2-br to inhibition by the CCR5-targeted inhibitors TAK-779 and Sch-C. UK1-br was more sensitive than MACS2-br to neutralization by monoclonal antibodies (2F5 and immunoglobulin G1b12 [IgG1b12]) and CD4-IgG2. These results predict the presence of HIV-1 variants with increased CCR5 affinity and reduced dependence on CCR5 and CD4 in the brains of some AIDS patients with central nervous system disease and suggest that R5 variants with increased CCR5 affinity may represent a pathogenic viral phenotype contributing to the neurodegenerative manifestations of AIDS.     

Envelope conformational changes induced by human immunodeficiency virus type 1 attachment inhibitors prevent CD4 binding and downstream entry events

BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes.     

Amino acid 324 in the simian immunodeficiency virus SIVmac V3 loop can confer CD4 independence and modulate the interaction with CCR5 and alternative coreceptors

The V3 loop of the simian immunodeficiency virus (SIV) envelope protein (Env) largely determines interactions with viral coreceptors. To define amino acids in V3 that are critical for coreceptor engagement, we functionally characterized Env variants with amino acid substitutions at position 324 in V3, which has previously been shown to impact SIV cell tropism. These changes modulated CCR5 engagement and, in some cases, allowed the efficient usage of CCR5 in the absence of CD4. The tested amino acid substitutions had highly differential effects on viral infectivity. Eleven of sixteen substitutions disrupted entry via CCR5 or the alternative coreceptor GPR15. Nevertheless, most of these variants replicated in the macaque T-cell line 221-89 and some also replicated in rhesus macaque peripheral blood monocytes, suggesting that efficient usage of CCR5 and GPR15 on cell lines is not a prerequisite for SIV replication in primary cells. Four variants showed enhanced entry into the macaque sMagi reporter cell line. However, sMagi cells did not express appreciable amounts of CCR5 and GPR15 mRNA, and entry into these cells was not efficiently blocked by a small-molecule CCR5 antagonist, suggesting that sMagi cells express as-yet-unidentified entry cofactors. In summary, we found that a single amino acid at position 324 in the SIV Env V3 loop can modulate both the efficiency and the types of coreceptors engaged by Env and allow for CD4-independent fusion in some cases.