Microglia express CCR5, CXCR4, and CCR3, but of these, CCR5 is the principal coreceptor for human immunodeficiency virus type 1 dementia isolates

Microglia are the main human immunodeficiency virus (HIV) reservoir in the central nervous system and most likely play a major role in the development of HIV dementia (HIVD). To characterize human adult microglial chemokine receptors, we analyzed the expression and calcium signaling of CCR5, CCR3, and CXCR4 and their roles in HIV entry. Microglia expressed higher levels of CCR5 than of either CCR3 or CXCR4. Of these three chemokine receptors, only CCR5 and CXCR4 were able to transduce a signal in microglia in response to their respective ligands, MIP-1beta and SDF-1alpha, as recorded by single-cell calcium flux experiments. We also found that CCR5 is the predominant coreceptor used for infection of human adult microglia by the HIV type 1 dementia isolates HIV-1DS-br, HIV-1RC-br, and HIV-1YU-2, since the anti-CCR5 antibody 2D7 was able to dramatically inhibit microglial infection by both wild-type and single-round luciferase pseudotype reporter viruses. Anti-CCR3 (7B11) and anti-CXCR4 (12G5) antibodies had little or no effect on infection. Last, we found that virus pseudotyped with the DS-br and RC-br envelopes can infect cells transfected with CD4 in conjunction with the G-protein-coupled receptors APJ, CCR8, and GPR15, which have been previously implicated in HIV entry.     

CXCR4 as a functional coreceptor for human immunodeficiency virus type 1 infection of primary macrophages

The coreceptors used by primary syncytium-inducing (SI) human immunodeficiency virus type 1 isolates for infection of primary macrophages were investigated. SI strains using only CXCR4 replicated equally well in macrophages with or without CCR5 and were inhibited by several different ligands for CXCR4 including SDF-1 and bicyclam derivative AMD3100. SI strains that used a broad range of coreceptors including CCR3, CCR5, CCR8, CXCR4, and BONZO infected CCR5-deficient macrophages about 10-fold less efficiently than CCR5(+) macrophages. Moreover, AMD3100 blocked infection of CCR5-negative macrophages by these strains. Our results therefore demonstrate that CXCR4, as well as CCR5, is used for infection of primary macrophages but provide no evidence for the use of alternative coreceptors.     

Patterns of CCR5, CXCR4, and CCR3 usage by envelope glycoproteins from human immunodeficiency virus type 1 primary isolates

Coreceptor usage by Envs from diverse primary human immunodeficiency virus type 1 isolates was analyzed by a vaccinia virus-based expression and assay system. Usage of recombinant CCR5 and CXCR4 correlated closely with fusogenicity toward macrophages and T-cell lines expressing endogenous coreceptors. Surprisingly, recombinant CCR3 was utilized by most primary and T-cell-line-adapted Envs. Endogenous CXCR4 in macrophages was functional as a coreceptor.     

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

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

CCR5- and CXCR4-utilizing strains of human immunodeficiency virus type 1 exhibit differential tropism and pathogenesis in vivo

CCR5-utilizing (R5) and CXCR4-utilizing (X4) strains of human immunodeficiency virus type 1 (HIV-1) have been studied intensively in vitro, but the pathologic correlates of such differential tropism in vivo remain incompletely defined. In this study, X4 and R5 strains of HIV-1 were compared for tropism and pathogenesis in SCID-hu Thy/Liv mice, an in vivo model of human thymopoiesis. The X4 strain NL4-3 replicates quickly and extensively in thymocytes in the cortex and medulla, causing significant depletion. In contrast, the R5 strain Ba-L initially infects stromal cells including macrophages in the thymic medulla, without any obvious pathologic consequence. After a period of 3 to 4 weeks, Ba-L infection slowly spreads through the thymocyte populations, occasionally culminating in thymocyte depletion after week 6 of infection. During the entire time of infection, Ba-L did not mutate into variants capable of utilizing CXCR4. Therefore, X4 strains are highly cytopathic after infection of the human thymus. In contrast, infection with R5 strains of HIV-1 can result in a two-phase process in vivo, involving apparently nonpathogenic replication in medullary stromal cells followed by cytopathic replication in thymocytes.     

The role of dendritic cells in the infection of CD4+ T cells with the human immunodeficiency virus: use of dendritic cells from individuals homozygous for the delta32CCR5 allele as a model

Despite exposure to multiple strains of both macrophage (M)-tropic and T cell (T)-tropic HIV, primary infection is largely restricted to relatively homogeneous M-tropic virus. Since dendritic cells (DCs) play a pivotal role in the early events of HIV infection, several studies have focused on the role of DCs in this restriction. It has been proposed that DCs are more efficiently infected with M-tropic versus T-tropic viruses; however, the infectability of DCs and the relevance of their infectability for inducing productive infection is controversial. It has also been suggested that variability in DC expression of coreceptors for M-tropic versus T-tropic virus could explain the restriction in the transmitting virus. Using HIV-pulsed DCs from individuals with a homozygous deletion in the CCR5 gene as a human "knockout" model, we demonstrate that infection of DCs per se is not necessary to promulgate infection in CD4+ T cells. The data also suggest that transmission of HIV to CD4+ T cells is not dependent on DC coreceptor expression.     

Determinants for sensitivity of human immunodeficiency virus coreceptor CXCR4 to the bicyclam AMD3100

The bicyclam AMD3100 is a potent and selective inhibitor of the replication of human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2). It was recently demonstrated that the compound inhibited HIV entry through CXCR4 but not through CCR5. Selectivity of AMD3100 for CXCR4 was further indicated by its lack of effect on HIV-1 and HIV-2 infection mediated by the CCR5, CCR3, Bonzo, BOB, and US28, coreceptors. AMD3100 completely blocked HIV-1 infection mediated by a mutant CXCR4 bearing a deletion of most of the amino-terminal extracellular domain. In contrast, relative resistance to AMD3100 was conferred by different single amino acid substitutions in the second extracellular loop (ECL2) or in the adjacent membrane-spanning domain, TM4. Only substitutions of a neutral residue for aspartic acid and of a nonaromatic residue for phenylalanine (Phe) were associated with drug resistance. This suggests a direct interaction of AMD3100 with these amino acids rather than indirect effects of their mutation on the CXCR4 structure. The interaction of aspartic acids of ECL2 and TM4 with AMD3100 is consistent with the positive charge of bicyclams, which might block HIV-1 entry by preventing electrostatic interactions between CXCR4 and the HIV-1 envelope protein gp120. Other features of AMD3100 must account for its high antiviral activity, in particular the presence of an aromatic linker between the cyclam units. This aromatic group might engage in hydrophobic interactions with the Phe-X-Phe motifs of ECL2 or TM4. These results confirm the importance of ECL2 for the HIV coreceptor activity of CXCR4.     

Rabbit cells expressing human CD4 and human CCR5 are highly permissive for human immunodeficiency virus type 1 infection

To evaluate the feasibility of using transgenic rabbits expressing CCR5 and CD4 as a small-animal model of human immunodeficiency virus type 1 (HIV) disease, we examined whether the expression of the human chemokine receptor (CCR5) and human CD4 would render a rabbit cell line (SIRC) permissive to HIV replication. Histologically, SIRC cells expressing CD4 and CCR5 formed multinucleated cells (syncytia) upon exposure to BaL, a macrophagetropic strain of HIV that uses CCR5 for cell entry. Intracellular viral capsid p24 staining showed abundant viral gene expression in BaL-infected SIRC cells expressing CD4 and CCR5. In contrast, neither SIRC cells expressing CD4 alone nor murine 3T3 cells expressing CCR5 and CD4 exhibited significant expression of p24. These stably transfected rabbit cells were also highly permissive for the production of virions upon infection by two other CCR5-dependent strains (JR-CSF and YU-2) but not by a CXCR4-dependent strain (NL4-3). The functional integrity of these virions was demonstrated by the successful infection of human peripheral blood mononuclear cells (PBMC) with viral stocks prepared from these transfected rabbit cells. Furthermore, primary rabbit PBMC were found to be permissive for production of infectious virions after circumventing the cellular entry step. These results suggest that a transgenic rabbit model for the study of HIV disease may be feasible.     

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

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

Evolution of HIV-1 coreceptor usage through interactions with distinct CCR5 and CXCR4 domains

The chemokine receptor CXCR4 functions as a fusion coreceptor for T cell tropic and dual-tropic HIV-1 strains. To identify regions of CXCR4 that are important for coreceptor function, CXCR4-CXCR2 receptor chimeras were tested for the ability to support HIV-1 envelope (env) protein-mediated membrane fusion. Receptor chimeras containing the first and second extracellular loops of CXCR4 supported fusion by T tropic and dual-tropic HIV-1 and HIV-2 strains and binding of a monoclonal antibody to CXCR4, 12G5, that blocks CXCR4-dependent infection by some virus strains. The second extracellular loop of CXCR4 was sufficient to confer coreceptor function to CXCR2 for most virus strains tested but did not support binding of 12G5. Truncation of the CXCR4 cytoplasmic tail or mutation of a conserved DRY motif in the second intracellular loop did not affect coreceptor function, indicating that phosphorylation of the cytoplasmic tail and the DRY motif are not required for coreceptor function. The results implicate the involvement of multiple CXCR4 domains in HIV-1 coreceptor function, especially the second extracellular loop, though the structural requirements for coreceptor function were somewhat variable for different env proteins. Finally, a hybrid receptor in which the amino terminus of CXCR4 was replaced by that of CCR5 was active as a coreceptor for M tropic, T tropic, and dual-tropic env proteins. We propose that dual tropism may evolve in CCR5-restricted HIV-1 strains through acquisition of the ability to utilize the first and second extracellular loops of CXCR4 while retaining the ability to interact with the CCR5 amino-terminal domain.     

Natural infection of a homozygous delta24 CCR5 red-capped mangabey with an R2b-tropic simian immunodeficiency virus

A homozygous 24-bp deletion (Delta24) was found in the CC chemokine receptor 5 (CCR5) of 11 out of 15 red-capped mangabeys (RCMs), Cercocebus torquatus torquatus, both in Africa and in an American zoo. The CCR5 Delta24 defect encompassed eight amino acids in frame in the fourth transmembrane region. Unexpectedly, RCM-009, one of 11 homozygotes (Delta24CCR5/ Delta24CCR5), was found to be naturally infected with a divergent simian immunodeficiency virus (SIV) strain, which was not R5-tropic, but used CCR2b (R2b) as its major coreceptor. SIVrcmGab1 was the only R2b-tropic SIV among other divergent SIVs tested. Cells transfected with the Delta24 CCR5 did not support entry of R5-tropic SIVmac, SIVcpz, SIVmne, HIV-2, or HIV-1, and were also inactive in signal transduction mediated by beta-chemokines. At 86.6%, the Delta24 allelic frequency was significantly higher than that of the 32-bp deletion found in humans. The Delta24 frequency was 4.1% in 34 sooty mangabeys (SMs), a geographically isolated subspecies that was naturally infected with R5-tropic SIV. Finding identical deletions in two mangabey subspecies separated for 10,000 years or more dates the Delta24 CCR5 deletion as ancient. However, the source of the selective pressure for the high rate of CCR5 deletion in RCMs remains to be determined. The high allelic frequency of the Delta24 CCR5 in RCMs, in comparison to that of SMs, suggests that R2b-tropism may have been acquired by SIVrcm, as an adaptation to CCR5 genetic defects appeared in its host.     

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.     

Inhibition of human immunodeficiency virus type 1 virion entry by dominant-negative Hck

To study the role of Src family tyrosine kinases in infection with human immunodeficiency virus type 1 (HIV-1), we constructed an Hck mutant, HckN, that hinders signaling from wild-type Hck. HIV-1 produced in HckN-expressing cells was significantly less infectious to HeLa-CD4-LTR-beta-gal (MAGI) cells than HIV-1 produced in mock-transfected cells. The inhibitory effect of HckN was compensated for by the expression of vesicular stomatitis virus G protein. Finally, we found that the HIV-1 produced in the HckN-expressing cells entered into the cells less efficiently than did the control HIV-1. These results suggest that the Src family tyrosine kinases regulate entry of HIV-1 into target cells.     

gp120 envelope glycoproteins of human immunodeficiency viruses competitively antagonize signaling by coreceptors CXCR4 and CCR5

Signal transductions by the dual-function CXCR4 and CCR5 chemokine receptors/HIV type 1 (HIV-1) coreceptors were electrophysiologically monitored in Xenopus laevis oocytes that also coexpressed the viral receptor CD4 and a G protein-coupled inward-rectifying K+ channel (Kir 3.1). Large Kir 3.1-dependent currents generated in response to the corresponding chemokines (SDF-1alpha for CXCR4 and MIP-1alpha; MIP-1beta and RANTES for CCR5) were blocked by pertussis toxin, suggesting involvement of inhibitory guanine nucleotide-binding proteins. Prolonged exposures to chemokines caused substantial but incomplete desensitization of responses with time constants of 5-7 min and recovery time constants of 12-19 min. CXCR4 and CCR5 exhibited heterologous desensitization in this oocyte system, suggesting possible inhibition of a common downstream step in their signaling pathways. In contrast to chemokines, perfusion with monomeric or oligomeric preparations of the glycoprotein of Mr 120, 000 (gp120) derived from several isolates of HIV-1 did not activate signaling by CXCR4 or CCR5 regardless of CD4 coexpression. However, adsorption of the gp120 from a T-cell-tropic virus resulted in CD4-dependent antagonism of CXCR4 response to SDF-1alpha, whereas gp120 from macrophage-tropic viruses caused CD4-dependent antagonism of CCR5 response to MIP-1alpha. These antagonisms could be partially overcome by high concentrations of chemokines and were specific for coreceptors of the corresponding HIV-1 isolates, suggesting that they resulted from direct interactions of gp120-CD4 complexes with coreceptors and that they did not involve the desensitization pathway. These results indicate that monomeric or oligomeric gp120s specifically antagonize CXCR4 and CCR5 signaling in response to chemokines, but they do not exclude the possibility that gp120s might also function as weak agonists in some cells. The gp120-mediated disruption of CXCR4 and CCR5 signaling may contribute to AIDS pathogenesis.     

Protection against human immunodeficiency virus type 1 infection in persons with repeated exposure: evidence for T cell immunity in the absence of inherited CCR5 coreceptor defects

It has been hypothesized that protection against human immunodeficiency virus (HIV)-1 infection may result from either acquired host immunity, inheritance of a dysfunctional CCR5 HIV-1 coreceptor, or a low or attenuated virus inoculum. Thirty-seven HIV-1-uninfected persons engaging in repeated high-risk sexual activity with an HIV-1-infected partner were prospectively studied to determine the contribution of these factors in protecting against HIV-1 transmission. More than one-third (13/36) demonstrated HIV-1-specific cytotoxicity, and this activity significantly correlated with the wild type CCR5 genotype (P=.03). Only 1 subject (3%) demonstrated the homozygous CCR5 32-bp deletion (Delta32/Delta32). Median plasma HIV-1 RNA levels from 18 HIV-1-infected sex partners were not statistically different from those of matched infected control patients. These results indicate that inheritance of the Delta32 CCR5 mutation does not account for the majority of persistently HIV-1-resistant cases, and the presence of cellular immunity in these persons suggests either undetected infection or protective immunity.     

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.     

Interaction of human immunodeficiency virus type 1 envelope glycoprotein V3 loop with CCR5 and CD4 at the membrane of human primary macrophages

We show that infection of primary monocyte-derived macrophages (MDMs) and blood lymphocytes (PBLs) by human immunodeficiency virus type 1 (HIV-1) R5 strains, but not that of PBLs by X4 strain HIV-1LAI, is inhibited by beta-chemokines RANTES and MIP-1alpha. A biotinylated disulfide-bridged peptide mimicking the complete loop of clade B consensus V3 domain of gp120 (V3Cs), but not a biotinylated V3LAI peptide or a control beta-endorphin peptide of approximately the same molecular weight (MW), was found to bind specifically to MDM membrane proteins, in particular two proteins of 42 and 62 kDa migrating as sharp bands after electroblotting onto Immobilon, and this was specifically inhibited by anti-V3 antibodies. When biotinylated V3Cs was incubated with intact MDMs, which were then washed and lysed, and the resulting material was incubated with streptavidin-agarose beads and electroblotted onto Immobilon, fresh V3Cs also bound to proteins of the same molecular weight recovered in the V3Cs-interacting material. This binding was inhibited by anti-V3 antibodies, and no binding occurred with the control peptides. V3Cs also bound to soluble recombinant CD4, and CD4 monoclonal antibody Q4120 specifically recognized the V3Cs-interacting 62-kDa protein, which should thus correspond to CD4. Recombinant radiolabeled RANTES, MIP-1alpha, and MIP-1beta, but not IL-8, also bound to a 42-kDa protein on the membrane of MDMs as well as to the V3Cs-interacting 42-kDa protein, and excess unlabeled V3Cs inhibited such binding. This protein was also recognized by antibodies to CCR5, the RANTES/MIP-1alpha/MIP-1beta receptor. These data show that V3Cs binds to MDM membrane proteins that comprise CD4 and CCR5, and that multimolecular complexes involving at least gp120 V3, CD4, and CCR5 are formed on the surface of MDMs as part of V3-mediated postbinding events occurring during HIV-1 infection.