Interactions of CCR5 and CXCR4 with CD4 and gp120 in human blood monocyte-derived dendritic cells

Dendritic cells (DC) and macrophages play an important role in the generation of immune responses and transmission of HIV infection. It has been recently found that, in the presence of gp120, CD4 can be efficiently coimmunoprecipitated by anti-CXCR4 antibodies from lymphocytes and monocytes but not from blood monocyte-derived macrophages. The gp120-CD4-CXCR4 complex formation paralleled the ability for these cell types to support X4 (LAV) HIV-1 envelope glycoprotein (Env)-mediated fusion. Here we report that, unlike macrophages but similar to lymphocytes and monocytes, human blood monocyte-derived DC allow efficient complex formation among the HIV-1 coreceptor CXCR4, the primary receptor CD4, and the Env gp120 (LAV) which parallels their fusion ability with cells expressing HIV-1 Env (LAV). In addition, DC behaved similarly to macrophages, lymphocytes, and monocytes in their ability to support formation of complexes between CD4 and the other major HIV-1 coreceptor CCR5 even in the absence of gp120 as demonstrated by CD4 coimmunoprecipitation with anti-CCR5 antibodies. Further, the amount of gp120-CD4-CXCR4 (or CCR5) complexes was proportional to the extent of cell fusion mediated by the HIV-1 Env (LAV or JRFL, respectively). These results demonstrate that of all the major types of host cells important for HIV-1 infection, the first central stage in the entry mechanism, the formation of gp120-CD4-coreceptor complexes, is not impaired except for the formation of the gp120-CD4-CXCR4 complex in macrophages. Therefore, for most CD4+ target cells restraint(s) on productive HIV-1 infection appears to occur at stages of the virus life cycle subsequent to the gp120-CD4-coreceptor complex formation.     

Immunopathogenesis of Dengue hemorrhagic fever

To test the hypothesis that inefficient interactions of CXCR4 with CD4 and gp120 could affect HIV-1 entry, we incubated macrophages, monocytes, and lymphocytes with gp120 and coimmunoprecipitated CD4 by using anti-CXCR4 antibodies. CD4 was efficiently coimmunoprecipitated in lymphocytes and monocytes but not in macrophages. Overexpression of CD4 in macrophages resulted in detection of CD4-CXCR4 and gp120-CD4-CXCR4 complexes in parallel with the restoration of macrophage fusion susceptibility. These results suggest a mechanism of resistance to entry of some X4 HIV-1 strains into macrophages and a method for dissection of the initial stages of HIV entry.     

Caspase-dependent apoptosis of cells expressing the chemokine receptor CXCR4 is induced by cell membrane-associated human immunodeficiency virus type 1 envelope glycoprotein (gp120)

Human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins interact with CD4 and chemokine receptors on T cells to deliver signals that trigger either activation, anergy, or apoptosis. However, the molecular mechanisms driving these responses remain poorly understood. In this study we demonstrate that apoptosis is induced upon HIV-1 envelope binding to the chemokine receptor CXCR4. Cells expressing a mutant form of CXCR4 with a C-terminal deletion were also sensitive to HIV-1 envelope-mediated apoptosis, indicating that the cytoplasmic tail of CXCR4 is not required to induce the apoptotic pathway. The specificity of this process was analyzed using several inhibitors of gp120-CD4-CXCR4 interaction. Monoclonal antibodies directed against the gp120-binding site on CD4 (ST4) and against CXCR4 (MAB173) prevented the apoptotic signal in a dose-dependent manner. The cell death program was also inhibited by SDF-1alpha, the natural ligand of CXCR4, and by suramin, a G protein inhibitor that binds with a high affinity to the V3 loop of HIV-1 gp120 envelope protein. These results highlight the role played by gp120-binding on CXCR4 to trigger programmed cell death. Next, we investigated the intracellular signal involved in gp120-induced apoptosis. This cell death program was insensitive to pertussis toxin and did not involve activation of the stress- and apoptosis-related MAP kinases p38(MAPK) and SAPK/JNK but was inhibited by a broad spectrum caspase inhibitor (z-VAD.fmk) and a relatively selective inhibitor of caspase 3 (z-DEVD.fmk). Altogether, our results demonstrate that HIV induces a caspase-dependent apoptotic signaling pathway through CXCR4.     

CD4-binding site alterations in CCR5-using HIV-1 envelopes influencing gp120-CD4 interactions and fusogenicity

CD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, we characterized functionally diverse R5 envelope (Env) clones (n = 16) to elucidate potential structural alterations within the gp120 CD4bs that influence Env function. Initially, we showed that the magnitude of gp120-CD4-binding correlates with increased fusogenicity and reduced CD4 dependence. Analysis of three-dimensional gp120 structural models revealed two CD4bs variants, D279 and N362, that were associated with reduced CD4 dependence. Further structural analysis showed that a wider aperture of the predicted CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was associated with amino acid alterations within V5 and correlated with increased gp120-CD4 binding and increased fusogenicity. Our results provide evidence that the gp120 V5 loop may alter CD4bs conformation and contribute to increased gp120-CD4 interactions and Env fusogenicity.     

Identification of determinants of interaction between CXCR4 and gp120 of a dual-tropic HIV-1DH12 isolate.

Using a panel of chimeric viruses and their chimeric envelope glycoproteins, we have previously reported that the V1/V2 or the V3 regions of a dual-tropic primary human immunodeficiency virus type 1 (HIV-1) isolate (HIV-1DH12) could individually confer CXCR4 usage when introduced into the backbone of a macrophage-tropic (M-tropic) virus isolate (HIV-1AD8). In this study, chimeric CXCR4-CXCR2 chemokine receptors were employed to identify the determinants involved in the interaction between CXCR4 and the dual-tropic HIV-1DH12 gp120. Our results indicate that (i) HIV-1DH12 gp120 interacts primarily with the extracellular domains 1 (E1) and 2 (E2) of CXCR4, (ii) the V1/V2 and the V3 regions interact with different domains of CXCR4, and (iii) the V1/V2 region plays a more critical role in the interaction between CXCR4 and HIV-1DH12 gp120. Combining our data and those of others suggests that the pattern of CXCR4 usage is highly dependent on HIV-1 isolates. In addition, an M-tropic virus may evolve to become dual-tropic by first acquiring the ability to interact with CXCR4 through the V1/V2 region of gp120.     

V3 loop region of the HIV-1 gp120 envelope protein is essential for virus infectivity.

The mechanism by which HIV-1 mediates cell fusion and penetrates target cells, subsequent to receptor (CD4) binding, is not well understood. However, neutralizing antibodies, which recognize the principal neutralizing determinants of the gp120 envelope protein (the V3 loop region, residues 296 to 331), have been shown to effectively block cell fusion and virus infectivity independent of the initial gp120-CD4 binding. To investigate the role of the V3 loop in an HIV infection, a series of site-specific mutations were introduced into the HIV-1 envelope gene. Specifically, each residue (312 to 315) in the strongly conserved tetrapeptide sequence, GPGR, which is positioned in the center of the V3 loop domain was individually altered. The processing, transport, and CD4 binding properties of the mutant envelope proteins were comparable to those of the wild-type protein, however, none of the mutants were able to form syncytia in the HeLa-T4 assay. Molecular HIV-1 clones containing mutations altering the G312, G314, or R315 residues produced noninfectious virions, whereas a clone with a P313A mutation was found to be infectious. These results demonstrate that certain V3 loop mutations can be lethal and clearly indicate that this region of the HIV-1 gp120 protein is essential for virus infectivity.     

A human immunodeficiency virus Env inducible transcription system to examine consequences of gp120 expression

According to several studies, the HIV-1 envelope gp120 protein and the co-receptor CXCR4 play an essential role in HIV-1 induced cell toxicity. Characterisation of the CD4-independent m7NDK isolate provided the opportunity of studying the effects of direct interactions between m7NDK gp120 and CXCR4. Therefore, an inducible expression system was designed enabling synthesis of HIV-1 Env proteins upon doxycycline induction. Analysis of the expression of the env gene of the m7NDK HIV-1 isolate revealed, unexpectedly, that even long-term expression of m7NDK gp120 did not result in cytotoxycity in CXCR4-positive or -negative cell lines. This is the first report of a CD4-independent HIV-1-protein inducible expression regulated through the Tet-On system and by an alternative splicing. Env inducible expression cell lines could constitute a useful cellular tool to undertake analysis of HIV Env protein expression.     

Role of the gp120 inner domain beta-sandwich in the interaction between the human immunodeficiency virus envelope glycoprotein subunits

The inner domain of the human immunodeficiency virus (HIV-1) gp120 glycoprotein has been proposed to mediate the noncovalent interaction with the gp41 transmembrane envelope glycoprotein. We used mutagenesis to investigate the functional importance of a conserved beta-sandwich located within the gp120 inner domain. Changes in aliphatic residues lining a hydrophobic groove on the surface of the beta-sandwich decreased the association of the gp120 and gp41 glycoproteins. Other changes in the base of the hydrophobic groove resulted in envelope glycoproteins that were structurally intact and able to bind receptors, but were inefficient in mediating either syncytium formation or virus entry. These results support a model in which the beta-sandwich in the gp120 inner domain contributes to gp120-gp41 contacts, thereby maintaining the integrity of the envelope glycoprotein complex and allowing adjustments in the gp120-gp41 interaction required for membrane fusion.     

The interaction of CD4 with HIV-1 gp120

CD4 is an integral cell surface glycoprotein that is able to enhance T cell specific antigen responses when it interacts with its physiological ligand, class II major histocompatibility (MHC) molecules. In addition, CD4 is a specific cell-surface receptor for the human immunodeficiency virus-1 (HIV-1). Infection by HIV-1 is initiated by the binding of the envelope glycoprotein, gp120, to the first domain of CD4. The binding of CD4 to class II MHC is inhibited by gp120, one possible mechanism for immunosuppression in AIDS patients. In addition, the CD4/gp120 interaction may directly inhibit T cell function. Recently we have synthesized small molecules (CPFs) that specifically inhibit this interaction. CPFs bind to gp120 and prevent the binding of gp120 to CD4, and also inhibit the infectivity of HIV-1.     

Increased CXCR4-dependent HIV-1 fusion in activated T cells: role of CD4/CXCR4 association

Activation of peripheral CD4+ T cells resulted in augmented fusion with X4 human immunodeficiency virus type 1 (HIV-1) envelope-expressing cells without parallel increases in the surface expression of CD4 or CXC chemokine receptor 4 (CXCR4). Our study used biochemical methods and biological assays to correlate the increased fusion potential of activated T cells with changes in CXCR4 isoforms and CD4-CXCR4 association. Western blot analyses of CXCR4, precipitated from resting T cells, identified several CXCR4 species with molecular weights of 47, 50, 62, and 98 kDa. After 24 h stimulation with phytohemagglutinin/interleukin-2, a marked reduction was seen in the 47-kDa, with a concomitant increase in the amounts of 50 and 62-64 kDa CXCR4. T cell activation also induced an increase in the coprecipitation of CXCR4 with CD4. The 62-kDa CXCR4 predominantly coprecipitated with CD4 and was shown to be ubiquitinated. Stripping of CD4 from the cell surface with pronase treatment prior to cell lysis only partially reduced coprecipitation of CD4 with the 62-kDa CXCR4, revealing a pool of intracellular CD4-CXCR4 complexes. Coprecipitation of CXCR4 with CD4 was reduced in activated cells treated with Brefeldin A and Monensin, suggesting that late endosomes play a role in intracellular association of CXCR4 with CD4. Confocal microscopy confirmed the colocalization of CD4 and CXCR4 within CD63+ endocytic compartments. These findings demonstrated a correlation between the enhanced susceptibility of activated T cells to HIV-1 fusion and accumulation of ubiquitinated 62-64 kDa CXCR4 species, which preferentially associated with CD4. The CD4-CXCR4 complexes may shuttle between late endosomes and the cell surface.     

Glycans and proteoglycans are involved in the interactions of human immunodeficiency virus type 1 envelope glycoprotein and of SDF-1alpha with membrane ligands of CD4(+) CXCR4(+) cells

We demonstrate that human immunodeficiency virus HIV-1(LAI) envelope glycoprotein 120 (gp120(LAi)) specifically interacts with several membrane ligands on lymphoid CEM or monocytic U937 cells in addition to its previously identified receptor, CD4, and CXCR4, its coreceptor. In its native state, gp120(LAI) is able to elicit specific multimolecular complexes with these membrane ligands at the surface of the cells; most of the interactions are abolished by mannan or heparin but not by dextran. Similarly, stromal cell-derived factor (SDF)-1alpha interacts not only with CXCR4 expressed by CXCR4(+) CD4(+) U937, CEM, and HOS-CD4(+) CXCR4(+) cells but also with CD4 expressed by intact U937, CEM, and HOS-CD4(+) CXCR4(+/-) cells or electroblotted onto Immobilon. SDF-1alpha binding to CD4(+) CXCR4(+/-) cells, or soluble CD4 electroblotted onto Immobilon, is significantly inhibited by sCD4, whereas truncated sCD4 lacking D3 and D4 domains had no significant effect, which indicates that SDF-1 binds to CD4 but at regions different from the HIV-gp120-binding site. Heparin and mannan also inhibit SDF-1alpha binding to intact CD4(+) CXCR4(+/-) cells, and electroblotted soluble CD4. Heparitinase treatment of such cells reduced SDF-1alpha binding. These data demonstrate that glycans and glycosaminoglycans are directly or indirectly involved in the interactions of HIV-1 gp120(LAI) and of SDF-1alpha with membrane ligands of CD4(+) CXCR4(+) cells and thus could play a role both in HIV-1 infection and in the physiology of SDF-1alpha.     

DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d3) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d3. In addition, both sCD4-gp120 and sCD4-gp120-mC3d3 bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d3 or sCD4-gp120-mC3d3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d3-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d3 had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.     

Macrophage activation through CCR5- and CXCR4-mediated gp120-elicited signaling pathways

Macrophages are major targets for infection by human immunodeficiency virus type 1 (HIV-1). In addition to their role as productive viral reservoirs, inappropriate activation of infected and uninfected macrophages appears to contribute to pathogenesis. HIV-1 infection requires initial interactions between the viral envelope surface glycoprotein gp120, the cell-surface protein CD4, and a chemokine receptor CCR5 or CXCR4. Besides their role in HIV-1 entry, CCR5 and CXCR4 are G protein-coupled receptors that can activate multiple intracellular signaling pathways. HIV-1 gp120 has been shown to activate signaling pathways through the chemokine receptors in several cell types including lymphocytes, neurons, and astrocytes. In some cell types, these consequences may cause cellular injury. In this review, we highlight our data demonstrating diverse signaling events that occur in primary human macrophages in response to gp120/chemokine receptor interactions. These responses include K+, Cl-, and nonselective cation currents, intracellular Ca2+ increases, and activation of several kinases including the focal adhesion-related tyrosine kinase Pyk2, mitogen-activated protein kinases (MAPK), and phosphoinositol-3 kinase. Activation of the MAPK leads to gp120-induced expression of chemokines such as monocyte chemoattractant protein-1 and macrophage-inflammatory protein-1beta and the proinflammatory cytokine tumor necrosis factor alpha. These responses establish a complex cytokine network, which may enhance or suppress HIV-1 replication. In addition, dysregulation of macrophage function by gp120/chemokine receptor signaling may contribute to local inflammation and injury and further recruit additional inflammatory and/or target cells. Targeting these cellular signaling pathways may have benefit in controlling inflammatory sequelae of HIV infection such as in neurological disease.     

In vivo alteration of humoral responses to HIV-1 envelope glycoprotein gp120 by antibodies to the CD4-binding site of gp120

The binding of antibodies to the CD4-binding site (CD4bs) of the HIV-1 envelope glycoprotein gp120 has been shown to induce gp120 to undergo conformational changes that can expose and/or shield specific epitopes on gp120. Here, we study alterations in the antigenicity and immunogenicity of gp120 when complexed with human monoclonal antibodies (mAbs) specific for the CD4bs of gp120. The data showed that gp120 bound by anti-CD4bs mAbs had enhanced reactivity with mAbs to the V3 and N-terminal regions, but not with mAb to the C terminus. Moreover, mice immunized with the gp120/anti-CD4bs mAb complexes produced higher titers of gp120-specific serum IgG and IgA than mice immunized with uncomplexed gp120 or other gp120/mAb complexes. Notably, the enhanced antibody production was directed against V3 and correlated with better exposure of V3 on the gp120/anti-CD4bs mAb complexes. The higher antibody reactivity was evident against the homologous V3(LAI) peptide, but not against heterologous V3 peptides. Potent neutralization activity against HIV-1(LAI) was also observed in the sera from mice immunized with gp120/anti-CD4bs mAb complexes, although the sera exhibited poor neutralizing activities against other viruses tested. These results indicate that the anti-CD4bs antibodies alter the antigenicity and immunogenicity of gp120, leading to enhanced production of anti-gp120 antibodies directed particularly against the V3 region.     

Epitope mapping and characterization of a novel CD4-induced human monoclonal antibody capable of neutralizing primary HIV-1 strains

Human immunodeficiency virus (HIV-1) enters target cells by binding its gp120 exterior envelope glycoprotein to CD4 and one of the chemokine receptors, CCR5 or CXCR4. CD4-induced (CD4i) antibodies bind gp120 more efficiently after CD4 binding and block the interaction with the chemokine receptor. Examples of CD4i antibodies are limited, and the prototypes of the CD4i antibodies exhibit only weak neutralizing activity against primary, clinical HIV-1 isolates. Here we report the identification of a novel antibody, E51, that exhibits CD4-induced binding to gp120 and neutralizes primary HIV-1 more efficiently than the prototypic CD4i antibodies. The E51 antibody blocks the interaction of gp120-CD4 complexes with CCR5 and binds to a highly conserved, basic gp120 element composed of the beta 19-strand and surrounding structures. Thus, on primary HIV-1 isolates, this gp120 region, which has been previously implicated in chemokine receptor binding, is accessible to a subset of CD4i antibodies.     

Amino acid residues of the human immunodeficiency virus type I gp120 critical for the binding of rat and human neutralizing antibodies that block the gp120-sCD4 interaction.

We have characterized the discontinuous epitopes recognized by two rat and three human neutralizing monoclonal antibodies (mAb) by examining the effect of single amino acid changes in conserved residues of gp120 on mAb recognition. A human mAb derived from an infected individual, 448D, and two rat mAbs, 39.13g and 39.3b, respectively, derived by immunization with native recombinant gp120, recognize similar epitopes. Recognition of the envelope glycoproteins by these mAbs was affected by changes in gp120 amino acid residues 88, 113, 117, 257, 368, or 370. The gp120 amino acids 257, 368, and 370 have previously been reported to be important for CD4 binding, which is consistent with the ability of these mAbs to block the gp120-CD4 interaction. Residues 88, 113, and 117 are not thought to be important for CD4 binding, suggesting that the antibody epitopes overlap, but are distinct from, the CD4 binding region. We also found that some alterations in gp120 residues 88, 117, 368, or 421 reduced the ability of polyclonal sera from HIV-1-infected individuals to inhibit the interaction of the mutant gp120 glycoproteins with soluble CD4. Thus, changes in the HIV-1 gp120 glycoprotein that minimally affect the receptor binding may allow escape from neutralizing antibodies directed against the CD4 binding region.     

Human immunodeficiency virus type-1 envelope glycoprotein gp120 induces expression of fusion regulatory protein (FRP)-1/CD98 on CD4+ T cells: a possible regulatory mechanism of HIV-induced syncytium formation

Syncytium formation is one of major cytopathic effects of human immunodeficiency virus (HIV) infection, and requires the interaction of CD4 molecules on uninfected cells with HIV envelope glyoprotein gp120 expressed on HIV-infected cells. Recent evidence suggests chemokine receptors function as fusion cofactors. We have recently found that fusion regulatory protein (FRP)-1/ CD98 is involved in syncytium formation of HIV gp160-expressing U2ME-7 cells and TALL-1 cells persistently infected with HIV. However, resting lymphocytes were found to express no FRP-1 molecule. In this study, we demonstrated that recombinant gp120 (rpg120) has the ability to induce expression of FRP-1 on peripheral blood mononuclear cells (PBMC). Three-color flow cytometric analysis showed that rgp120-induced FRP-1 was expressed selectively on CD4⁺ T cells in a dose-dependent manner. FRP-1 expression level was maximum 3 days after addition of rgp120. Anti-CD4 and anti-gp120 antibodies blocked rgp120-induced FRP-1 expression. Co-cultivation of PBMC with HIV-1 gp160-expressing HeLa cells also resulted in the increased expression of FRP-1 on T cells. These results suggest that FRP-1 molecules are induced on CD4⁺ T cells via CD4-gp120 interaction and may play an important role in regulation of HIV-induced syncytium formation. Received: 27 September 1996     

Non-covalent complexes of HIV gp120 with CD4 and/or mAbs enhance activation of gp120-specific T clones and provide intermolecular help for anti-CD4 antibody production

The ‘dangerous liaison’ between CD4 and gp120 thatoffers the first entry opportunity to HIV may also provoke perturbationsof the immune control of the host with far-reaching immunopathologicalconsequences. We wondered whether a mechanism of intermolecularhelp (T help across the gap of a non-covalent bond, in contrastto the interamolecular help of carrier to hapten) could breakself-tolerance and be the cause of the frequent anti-CD4 autoantibodiesfound in AIDS patients. To determine whether this hypothesisdeserves further testing, we designed a series of in vitro andin vivo experiments of increasing complexity, focused on thepresentation of gp120 to specific T cells by antigen presentingcells (APC) exposed to the envelope protein in the form of non-covalentcomplexes. Bi-molecular complexes were constructed by allowinggp120 or gp160 to bind specific human mAbs. Tri-molecular complexeswere constructed by introducing CD4 as an intermediate ligandbetween gp120 and mouse mAbs specific for CD4. In all casesthe use of complexes did enhance the immunogenic capacity ofsubstimulatory doses of gp120 or gp160 by facilitating uptakeby APC via Fc receptor and consequent presentation to specifichuman T cell clones. Finally, help for the production in vivoof anti-CD4 antibodies was obtained from T lymphocytes specificfor gp120 when CD4-primed memory B cells were pulsed with CD4complexed with gp120, thus demonstrating in the mouse the entirecycle of intermolecular help via non-covalent interaction, andsetting the stage for future experiments on self-tolerance breakagein a human molecular context.