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-1β and SDF-1α, 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-1_DS-br, HIV-1_RC-br, and HIV-1_YU-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.     

Cytokine Regulation of Human Immunodeficiency Virus Type 1 Entry and Replication in Human Monocytes/Macrophages through Modulation of CCR5 Expression

Human macrophages express chemokine receptors that act as coreceptors for human immunodeficiency virus type 1 (HIV-1) and are major targets for HIV-1 infection in vivo. The effects of cytokines on HIV-1 infection of macrophages and on the expression of CCR5, the principal coreceptor for macrophage-tropic viruses, have now been investigated. Expression of CCR5 on the surface of freshly isolated human monocytes was virtually undetectable by flow cytometry with the monoclonal antibody 5C7. However, after culture of monocytes for 48 h in serum-free medium, approximately 30% of the resulting macrophages expressed CCR5 and the cells were susceptible to infection by macrophage-tropic HIV-1. Addition of either macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) to the cultures markedly increased both the extent of HIV-1 entry and replication as well as surface expression of CCR5. In contrast, addition of the T-helper 2 (Th2) cell-derived cytokine interleukin-4 (IL-4) or IL-13 prevented the expression of CCR5 induced by culture in medium alone, and IL-4 inhibited virus entry, replication, and cytopathicity under these conditions. IL-4 or IL-13 also prevented the stimulatory effects of M-CSF or GM-CSF on CCR5 expression as well as HIV-1 entry and replication. In addition, IL-4 reversed the increase in CCR5 expression induced by pretreatment of cells with M-CSF. Although IL-10 also inhibits HIV-1 replication in macrophages, it did not suppress surface CCR5 expression induced by colony-stimulating factors. These results indicate that the cytokine environment determines the susceptibility of macrophages to HIV-1 infection by various mechanisms, one of which is the regulation of HIV-1 coreceptor expression.     

CCR5 Expression Correlates with Susceptibility of Maturing Monocytes to Human Immunodeficiency Virus Type 1 Infection

The chemokine receptor CCR5 and to a lesser extent CCR3 and CCR2b have been shown to serve as coreceptors for human immunodeficiency virus type 1 (HIV-1) entry into blood- or tissue-derived macrophages. Therefore, we examined the expression of the chemokine receptors CCR1, CCR2b, CCR3, CCR5, and CXCR4 as RNAs or as membrane-expressed antigens in monocytes maturing into macrophages and correlated these results with the susceptibility of macrophages to HIV-1 infection, as measured by their concentrations of extracellular p24 antigen and levels of intracellular HIV DNA by quantitative PCR. There was little change in levels of CCR1, CCR2b, and CCR5 RNAs. CCR3 RNA and surface antigen were undetectable throughout maturation of adherent monocytes over 10 days. CXCR4 RNA and membrane antigen were strongly expressed in newly adherent monocytes, but their levels declined at day 7. The amounts of CCR5 RNA remained stable, but the amounts of CCR5 antigen increased from undetectable to peak levels at day 7 and then declined slightly at day 10. Levels of susceptibility to laboratory (HIV-1_BaL) and clinical strains of HIV-1 showed parallel kinetics, peaking at day 7 and then decreasing at days 10 to 14. The concordance of levels of HIV DNA and p24 antigen suggested that the changes in susceptibility with monocyte maturation were at or immediately after entry and correlated well with CCR5 expression and inversely with CXCR4 expression.     

Macrophage tropism of human immunodeficiency virus type 1 isolates from brain and lymphoid tissues predicts neurotropism independent of coreceptor specificity

The viral determinants that underlie human immunodeficiency virus type 1 (HIV-1) neurotropism are unknown, due in part to limited studies on viruses isolated from brain. Previous studies suggest that brain-derived viruses are macrophage tropic (M-tropic) and principally use CCR5 for virus entry. To better understand HIV-1 neurotropism, we isolated primary viruses from autopsy brain, cerebral spinal fluid, blood, spleen, and lymph node samples from AIDS patients with dementia and HIV-1 encephalitis. Isolates were characterized to determine coreceptor usage and replication capacity in peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages (MDM), and microglia. Env V1/V2 and V3 heteroduplex tracking assay and sequence analyses were performed to characterize distinct variants in viral quasispecies. Viruses isolated from brain, which consisted of variants that were distinct from those in lymphoid tissues, used CCR5 (R5), CXCR4 (X4), or both coreceptors (R5X4). Minor usage of CCR2b, CCR3, CCR8, and Apj was also observed. Primary brain and lymphoid isolates that replicated to high levels in MDM showed a similar capacity to replicate in microglia. Six of 11 R5 isolates that replicated efficiently in PBMC could not replicate in MDM or microglia due to a block in virus entry. CD4 overexpression in microglia transduced with retroviral vectors had no effect on the restricted replication of these virus strains. Furthermore, infection of transfected cells expressing different amounts of CD4 or CCR5 with M-tropic and non-M-tropic R5 isolates revealed a similar dependence on CD4 and CCR5 levels for entry, suggesting that the entry block was not due to low levels of either receptor. Studies using TAK-779 and AMD3100 showed that two highly M-tropic isolates entered microglia primarily via CXCR4. These results suggest that HIV-1 tropism for macrophages and microglia is restricted at the entry level by a mechanism independent of coreceptor specificity. These findings provide evidence that M-tropism rather than CCR5 usage predicts HIV-1 neurotropism.     

Potent, broad-spectrum inhibition of human immunodeficiency virus type 1 by the CCR5 monoclonal antibody PRO 140

CCR5 serves as a requisite fusion coreceptor for clinically relevant strains of human immunodeficiency virus type 1 (HIV-1) and provides a promising target for antiviral therapy. However, no study to date has examined whether monoclonal antibodies, small molecules, or other nonchemokine agents possess broad-spectrum activity against the major genetic subtypes of HIV-1. PRO 140 (PA14) is an anti-CCR5 monoclonal antibody that potently inhibits HIV-1 entry at concentrations that do not affect CCR5's chemokine receptor activity. In this study, PRO 140 was tested against a panel of primary HIV-1 isolates selected for their genotypic and geographic diversity. In quantitative assays of viral infectivity, PRO 140 was compared with RANTES, a natural CCR5 ligand that can inhibit HIV-1 entry by receptor downregulation as well as receptor blockade. Despite their divergent mechanisms of action and binding epitopes on CCR5, low nanomolar concentrations of both PRO 140 and RANTES inhibited infection of primary peripheral blood mononuclear cells (PBMC) by all CCR5-using (R5) viruses tested. This is consistent with there being a highly restricted pattern of CCR5 usage by R5 viruses. In addition, a panel of 25 subtype C South African R5 viruses were broadly inhibited by PRO 140, RANTES, and TAK-779, although approximately 30-fold-higher concentrations of the last compound were required. Interestingly, significant inhibition of a dualtropic subtype C virus was also observed. Whereas PRO 140 potently inhibited HIV-1 replication in both PBMC and primary macrophages, RANTES exhibited limited antiviral activity in macrophage cultures. Thus CCR5-targeting agents such as PRO 140 can demonstrate potent and genetic-subtype-independent anti-HIV-1 activity.     

TNF-alpha inhibits HIV-1 replication in peripheral blood monocytes and alveolar macrophages by inducing the production of RANTES and decreasing C-C chemokine receptor 5 (CCR5) expression.

The pathogenesis of HIV-1 infection is influenced by the immunoregulatory responses of the host. Macrophages present in the lymphoid tissue are susceptible to infection with HIV-1, but are relatively resistant to its cytopathic effects and serve as a reservoir for the virus during the course of disease. Previous investigators have demonstrated that increased serum levels of TNF-alpha contribute to the clinical symptoms of AIDS and that TNF-alpha stimulates the production of HIV-1 in chronically infected lymphocytic and monocytic cell lines by increasing HIV-1 gene expression. Although previous studies have suggested that TNF-alpha may increase HIV-1 infection of primary human mononuclear cells, some recent studies have indicated that TNF-alpha suppresses HIV-1 infection of macrophages. We now demonstrate that TNF-alpha suppresses HIV-1 replication in freshly infected peripheral blood monocytes (PBM) and alveolar macrophages (AM) in a dose-dependent manner. As TNF-alpha has been shown to increase the production of C-C chemokine receptor (CCR5)-binding chemokines under certain circumstances, we hypothesized that TNF-alpha inhibits HIV-1 replication by increasing the expression of these HIV-suppressive factors. We now show that TNF-alpha treatment of PBM and AM increases the production of the C-C chemokine, RANTES. Immunodepletion of RANTES alone or in combination with macrophage inflammatory protein-1alpha and -1beta block the ability of TNF-alpha to suppress viral replication in PBM and AM. In addition, we found that TNF-alpha treatment reduces CCR5 expression on PBM and AM. These findings suggest that TNF-alpha plays a significant role in inhibiting monocytotropic strains of HIV-1 by two distinct, but complementary, mechanisms.     

Envelope glycoproteins from human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus can use human CCR5 as a coreceptor for viral entry and make direct CD4-dependent interactions with this chemokine receptor.

Several members of the chemokine receptor family have recently been identified as coreceptors, with CD4, for entry of human immunodeficiency virus type 1 (HIV-1) into target cells. In this report, we show that the envelope glycoproteins of several strains of HIV-2 and simian immunodeficiency virus (SIV) employ the same chemokine receptors for infection. Envelope glycoproteins from HIV-2 use CCR5 or CXCR4, while those from several strains of SIV use CCR5. Our data indicate also that some viral envelopes can use more than one coreceptor for entry and suggest that some of these coreceptors remain to be identified. To further understand how different envelope molecules use CCR5 as an entry cofactor, we show that soluble purified envelope glycoproteins (SU component) from CCR5-tropic HIV-1, HIV-2, and SIV can compete for binding of iodinated chemokine to CCR5. The competition is dependent on binding of the SU glycoprotein to cell surface CD4 and implies a direct interaction between envelope glycoproteins and CCR5. This interaction is specific since it is not observed with SU glycoprotein from a CXCR4-tropic virus or with a chemokine receptor that is not competent for viral entry (CCR1). For HIV-1, the interaction can be inhibited by antibodies specific for the V3 loop of SU. Soluble CD4 was found to potentiate binding of the HIV-2 ST and SIVmac239 envelope glycoproteins to CCR5, suggesting that a CD4-induced conformational change in SU is required for subsequent binding to CCR5. These data suggest a common fundamental mechanism by which structurally diverse HIV-1, HIV-2, and SIV envelope glycoproteins interact with CD4 and CCR5 to mediate viral entry.     

Blocking HIV-1 infection via CCR5 and CXCR4 receptors by acting in trans on the CCR2 chemokine receptor

The identification of chemokine receptors as HIV-1 coreceptors has focused research on developing strategies to prevent HIV-1 infection. We generated CCR2-01, a CCR2 receptor-specific monoclonal antibody that neither competes with the chemokine CCL2 for binding nor triggers signaling, but nonetheless blocks replication of monotropic (R5) and T-tropic (X4) HIV-1 strains. This effect is explained by the ability of CCR2-01 to induce oligomerization of CCR2 with the CCR5 or CXCR4 viral coreceptors. HIV-1 infection through CCR5 and CXCR4 receptors can thus be prevented in the absence of steric hindrance or receptor downregulation by acting in trans on a receptor that is rarely used by the virus to infect cells.     

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.     

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.     

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 Δ32 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 Δ32 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.     

Stromal down-regulation of macrophage CD4/CCR5 expression and NF-κB activation mediates HIV-1 non-permissiveness in intestinal macrophages

Tissue macrophages are derived exclusively from blood monocytes, which as monocyte-derived macrophages support HIV-1 replication. However, among human tissue macrophages only intestinal macrophages are non-permissive to HIV-1, suggesting that the unique microenvironment in human intestinal mucosa renders lamina propria macrophages non-permissive to HIV-1. We investigated this hypothesis using blood monocytes and intestinal extracellular matrix (stroma)-conditioned media (S-CM) to model the exposure of newly recruited monocytes and resident macrophages to lamina propria stroma, where the cells take up residence in the intestinal mucosa. Exposure of monocytes to S-CM blocked up-regulation of CD4 and CCR5 expression during monocyte differentiation into macrophages and inhibited productive HIV-1 infection in differentiated macrophages. Importantly, exposure of monocyte-derived macrophages simultaneously to S-CM and HIV-1 also inhibited viral replication, and sorted CD4+ intestinal macrophages, a proportion of which expressed CCR5+, did not support HIV-1 replication, indicating that the non-permissiveness to HIV-1 was not due to reduced receptor expression alone. Consistent with this conclusion, S-CM also potently inhibited replication of HIV-1 pseudotyped with vesicular stomatitis virus glycoprotein, which provides CD4/CCR5-independent entry. Neutralization of TGF-β in S-CM and recombinant TGF-β studies showed that stromal TGF-β inhibited macrophage nuclear translocation of NF-κB and HIV-1 replication. Thus, the profound inability of intestinal macrophages to support productive HIV-1 infection is likely the consequence of microenvironmental down-regulation of macrophage HIV-1 receptor/coreceptor expression and NF-κB activation.     

Grape seed extract proanthocyanidins downregulate HIV-1 entry coreceptors,CCR2b,CCR3 and CCR5 gene expression by normal peripheral blood mononuclear cells

Flavonoids and related polyphenols, in addition to their cardioprotective, anti-tumor, anti-inflammatory, anti-carcinogenic and anti-allergic activities, also possess promising anti-HIV effects. Recent studies documented that the beta-chemokine receptors, CCR2b, CCR3 and CCR5, and the alpha-chemokine receptors, CXCR1, CXCR2 and CXCR4 serve as entry coreceptors for HIV-1. Although flavonoids and polyphenolic compounds elicit anti-HIV effects such as inhibition of HIV-1 expression and virus replication, the molecular mechanisms underlying these effects remain to be clearly elucidated. We hypothesize that flavonoids exert their anti-HIV effects, possibly by interfering at the HIV co-receptor level. We investigated the effect of flavonoid constituents of a proprietary grape seed extract (GSE) on the expression of HIV-1 coentry receptors by immunocompetent mononuclear leukocytes. Our results showed that GSE significantly downregulated the expression of the HIV-1 entry co-receptors, CCR2b, CCR3 and CCR5 in normal PBMC in a dose dependent manner. Further, GSE treated cultures showed significantly lower number of CCR3 positive cells as quantitated by flow cytometry analysis which supports RT-PCR gene expression data. Investigations of the mechanisms underlying the anti-HIV-1 effects of grape seed extracts may help to identify promising natural products useful in the prevention and/or amelioration of HIV-1 infection.     

Lipopolysaccharide inhibits HIV-1 infection of monocyte- derived macrophages through direct and sustained down-regulation of CC chemokine receptor 5

It is now well established that HIV-1 requires interactions with both CD4 and a chemokine receptor on the host cell surface for efficient infection. The expression of the CCR5 chemokine receptor in human macrophages facilitates HIV-1 entry into these cells, which are considered important in HIV pathogenesis not only as viral reservoirs but also as modulators of altered inflammatory function in HIV disease and AIDS. LPS, a principal constituent of Gram-negative bacterial cell walls, is a potent stimulator of macrophages and has been shown to inhibit HIV infection in this population. We now present evidence that one mechanism by which LPS mediates its inhibitory effect on HIV-1 infection is through a direct and unusually sustained down-regulation of cell-surface CCR5 expression. This LPS-mediated down-regulation of CCR5 expression was independent of de novo protein synthesis and differed from the rapid turnover of these chemokine receptors observed in response to two natural ligands, macrophage-inflammatory protein-1alpha and -1beta. LPS did not act by down-regulating CCR5 mRNA (mRNA levels actually increased slightly after LPS treatment) or by enhancing the degradation of internalized receptor. Rather, the observed failure of LPS-treated macrophages to rapidly restore CCR5 expression at the cell-surface appeared to result from altered recycling of chemokine receptors. Taken together, our results suggest a novel pathway of CCR5 recycling in LPS-stimulated human macrophages that might be targeted to control HIV-1 infection.     

CD4-independent infection of astrocytes by human immunodeficiency virus type 1: requirement for the human mannose receptor

Human immunodeficiency virus type 1 (HIV-1) infection occurs in the central nervous system and causes a variety of neurobehavioral and neuropathological disorders. Both microglia, the residential macrophages in the brain, and astrocytes are susceptible to HIV-1 infection. Unlike microglia that express and utilize CD4 and chemokine coreceptors CCR5 and CCR3 for HIV-1 infection, astrocytes fail to express CD4. Astrocytes express several chemokine coreceptors; however, the involvement of these receptors in astrocyte HIV-1 infection appears to be insignificant. In the present study using an expression cloning strategy, the cDNA for the human mannose receptor (hMR) was found to be essential for CD4-independent HIV-1 infectivity. Ectopic expression of functional hMR rendered U87.MG astrocytic cells susceptible to HIV-1 infection, whereas anti-hMR serum and hMR-specific siRNA blocked HIV-1 infection in human primary astrocytes. In agreement with these findings, hMR bound to HIV-1 virions via the abundant and highly mannosylated sugar moieties of HIV-1 envelope glycoprotein gp120 in a Ca(2+)-dependent fashion. Moreover, hMR-mediated HIV-1 infection was dependent upon endocytic trafficking as assessed by transmission electron microscopy, as well as inhibition of viral entry by endosomo- and lysosomotropic drugs. Taken together, these results demonstrate the direct involvement of hMR in HIV-1 infection of astrocytes and suggest that HIV-1 interaction with hMR plays an important role in HIV-1 neuropathogenesis.     

A Tyrosine-Rich Region in the N Terminus of CCR5 Is Important for Human Immunodeficiency Virus Type 1 Entry and Mediates an Association between gp120 and CCR5

Human immunodeficiency virus type 1 (HIV-1) requires the presence of specific chemokine receptors in addition to CD4 to enter target cells. The chemokine receptor CCR5 is used by the macrophage-tropic strains of HIV-1 that predominate during the asymptomatic stages of infection. Here we identify a small tyrosine-rich region of CCR5 proximal to the N-terminal cysteine that is critical for entry of macrophage-tropic and dual-tropic variants of HIV-1. HIV-1 infection of cells expressing CCR5 mutants with changes in this region was substantially reduced compared with the infection of cells bearing wild-type CCR5. Simian immunodeficiency virus (SIV_mac239) entry was also ablated on a subset of these mutants but enhanced on others. These differences in virus entry were correlated with the relative ability of soluble, monomeric HIV-1 and SIV_mac239 gp120 glycoproteins to bind the CCR5 mutants. These results identify a region of CCR5 that is necessary for the physical association of the gp120 envelope glycoprotein with CCR5 and for HIV-1 infection.     

Expression of CCR5 Increases during Monocyte Differentiation and Directly Mediates Macrophage Susceptibility to Infection by Human Immunodeficiency Virus Type 1

The stage of differentiation and the lineage of CD4⁺ cells profoundly affect their susceptibility to infection by human immunodeficiency virus type 1 (HIV-1). While CD4⁺ T lymphocytes in patients are readily susceptible to HIV-1 infection, peripheral blood monocytes are relatively resistant during acute or early infection, even though monocytes also express CD4 and viral strains with macrophage (M)-tropic phenotypes predominate. CCR5, the main coreceptor for M-tropic viruses, clearly contributes to the ability of CD4⁺ T cells to be infected. To determine whether low levels of CCR5 expression account for the block in infection of monocytes, we examined primary monocyte lineage cells during differentiation. Culturing of blood monocytes for 5 days led to an increase in the mean number of CCR5-positive cells from <20% of monocytes to >80% of monocyte-derived macrophages (MDM). Levels of CCR5 expression per monocyte were generally lower than those on MDM, perhaps below a minimum threshold level necessary for efficient infection. Productive infection may be restricted to the small subset of monocytes that express relatively high levels of CCR5. Steady-state CCR5 mRNA levels also increased four- to fivefold during MDM differentiation. Infection of MDM by M-tropic HIV-1_JRFL resulted in >10-fold-higher levels of p24, and MDM harbored >30-fold more HIV-1 DNA copies than monocytes. In the presence of the CCR5-specific monoclonal antibody (MAb) 2D7, virus production and cellular levels of HIV-1 DNA were decreased by >80% in MDM, indicating a block in viral entry. There was a direct association between levels of CCR5 and differentiation of monocytes to macrophages. Levels of CCR5 were related to monocyte resistance and macrophage susceptibility to infection because infection by the M-tropic strain HIV-1_JRFL could be blocked by MAb 2D7. These results provide direct evidence that CCR5 functions as a coreceptor for HIV-1 infection of primary macrophages.     

Inhibition of human immunodeficiency virus replication by a dual CCR5/CXCR4 antagonist

Here we report that the N-pyridinylmethyl cyclam analog AMD3451 has antiviral activity against a wide variety of R5, R5/X4, and X4 strains of human immunodeficiency virus type 1 (HIV-1) and HIV-2 (50% inhibitory concentration [IC(50)] ranging from 1.2 to 26.5 microM) in various T-cell lines, CCR5- or CXCR4-transfected cells, peripheral blood mononuclear cells (PBMCs), and monocytes/macrophages. AMD3451 also inhibited R5, R5/X4, and X4 HIV-1 primary clinical isolates in PBMCs (IC(50), 1.8 to 7.3 microM). A PCR-based viral entry assay revealed that AMD3451 blocks R5 and X4 HIV-1 infection at the virus entry stage. AMD3451 dose-dependently inhibited the intracellular Ca(2+) signaling induced by the CXCR4 ligand CXCL12 in T-lymphocytic cells and in CXCR4-transfected cells, as well as the Ca(2+) flux induced by the CCR5 ligands CCL5, CCL3, and CCL4 in CCR5-transfected cells. The compound did not interfere with chemokine-induced Ca(2+) signaling through CCR1, CCR2, CCR3, CCR4, CCR6, CCR9, or CXCR3 and did not induce intracellular Ca(2+) signaling by itself at concentrations up to 400 microM. In freshly isolated monocytes, AMD3451 inhibited the Ca(2+) flux induced by CXCL12 and CCL4 but not that induced by CCL2, CCL3, CCL5, and CCL7. The CXCL12- and CCL3-induced chemotaxis was also dose-dependently inhibited by AMD3451. Furthermore, AMD3451 inhibited CXCL12- and CCL3L1-induced endocytosis in CXCR4- and CCR5-transfected cells. AMD3451, in contrast to the specific CXCR4 antagonist AMD3100, did not inhibit but enhanced the binding of several anti-CXCR4 monoclonal antibodies (such as clone 12G5) at the cell surface, pointing to a different interaction with CXCR4. AMD3451 is the first low-molecular-weight anti-HIV agent with selective HIV coreceptor, CCR5 and CXCR4, interaction.