Inefficient formation of a complex among CXCR4, CD4 and gp120 in U937 clones resistant to X4 gp120-gp41-mediated fusion

Certain subclones (designated as minus clones) of the promonocytic U937 cell line do not support efficient infection and fusion mediated by T cell line adapted (TCLA) X4 HIV-1 gp120-gp41 (Env) although the CXCR4 and CD4 concentrations at their surfaces are similar to those at the surfaces of clones susceptible to HIV-1 entry (plus clones) (H. Moriuchi et al., J. Virol. 71, 9664-9671, 1997). To test the hypothesis that inefficient formation of gp120-CD4-CXCR4 complexes could contribute to the mechanism of resistance to Env-mediated fusion in the minus clones, we incubated plus and minus cells with HIV-1 LAI gp120 and coimmunoprecipitated CD4 by using anti-CXCR4 antibodies. The gp120 induced inefficient coimmunoprecipitation of CD4 in the minus clones but not in the plus ones. Overexpression of CD4 resulted in significant restoration of the minus clones' susceptibility to fusion in parallel with an increase in the amount of the gp120-CD4-CXCR4 complexes. These results not only suggest that the resistance to TCLA X4 HIV-1 entry in the U937 minus clones is due to the inability of these cells to efficiently form complexes among CD4, gp120, and CXCR4, but also provide a direct evidence for the correlation between fusion and the cell surface concentration of the complexes among CXCR4, CD4, and gp120. These data and similar recent observations in macrophages suggest that inefficient complex formation among CXCR4, CD4, and gp120 could be a general mechanism of cell resistance to gp120-gp41-mediated fusion and a major determinant of HIV-1 evolution in vivo.     

SDF-1alpha regulates HIV-1-gp120-induced changes in CD79b surface expression and Ig production in activated human B cells

Binding of HIV-1 glycoprotein (gp120) to activated B cells of HIV-infected and HIV-uninfected subjects induces increased cell proliferation, cAMP generation, immunoglobulin (Ig) production and downregulation of the invariant chain, CD79b, of the B-cell receptor. We present evidence that the stromal cell-derived factor-1alpha (SDF-1alpha), itself a B-cell stimulant, reversed gp120-driven downregulation of CD79b in CD40- and IL-4-activated purified HIV-1 seronegative human peripheral blood B cells. SDF-1alpha augmented gp120-induced Ig production, downregulated CXCR4 receptor expression, and alone, exerted no effect on CD79b surface expression, reversed the gp120-induced downregulation of CD79b. These SDF-1alpha-modulated B-cell responses were specifically abrogated by an anti-SDF-1alpha antibody. These data suggest that SDF-1alpha plays an important regulatory role in the altered B-cell responses seen in HIV-1 infection. Further, these findings may enhance the understanding of the pathophysiology of HIV-1 infection and suggest a strategy utilizing SDF-1alpha or related molecules as an anti-HIV therapy.     

N-linked glycosylation in the CXCR4 N-terminus inhibits binding to HIV-1 envelope glycoproteins

CXCR4 is a co-receptor along with CD4 for human immunodeficiency virus type 1 (HIV-1). We investigated the role of N-linked glycosylation in the N-terminus of CXCR4 in binding to HIV-1 gp120 envelope glycoproteins. Gp120s from CXCR4 (X4) and CCR5 (R5) using HIV-1 strains bound more efficiently to non-N-glycosylated than to N-glycosylated CXCR4 proteoliposomes in a CD4-dependent manner. Similar results were observed in binding studies using non-N-glycosylated or N-glycosylated CXCR4 expressed on cells. Mutation of the N-glycosylation site N11 in CXCR4 (N11Q-CXCR4) enhanced CD4-dependent binding of X4 and R5 gp120s and allowed more efficient entry of viruses pseudotyped with X4 or R5 HIV-1 envelope glycoproteins. However, the binding of R5 gp120 to N11Q-CXCR4 and entry of R5 HIV-1 viruses into cells expressing N11Q-CXCR4 were 20- and 100- to 1000-fold less efficient, respectively, than the levels achieved using X4 gp120 or X4 HIV-1 viruses. Binding of stromal cell-derived factor (SDF)-1alpha, the natural ligand of CXCR4, and SDF-1alpha-induced signaling were reduced by the N11Q mutation. These findings demonstrate that N-glycosylation at N11 inhibits the binding of CXCR4 to X4 and R5 HIV-1 gp120, and provide a better understanding of the structural elements of CXCR4 involved in HIV-1 Env-co-receptor interactions.     

Functional complementation of the envelope hypervariable V3 loop of human immunodeficiency virus type 1 subtype B by the subtype E V3 loop.

The hypervariable V3 loop within gp120 of human immunodeficiency virus type 1 (HIV-1) is the major determinant of cell tropism and the entry coreceptor usage of the virus. However, the information obtained thus far has been from only subtype B from North America and Europe, and little is known about other subtypes whose V3 amino acids differ by as much as 50% from subtype B V3. In this study, we examined the functional potential of the V3 element of the HIV-1 subtype E, the most crucial variant causing the AIDS epidemic throughout southeast Asia. A panel of HIV-1LAI recombinants was constructed by the overlap extension method, by which the LAI V3 loop was precisely replaced by that of the subtype E nonsyncytium-inducing (NSI) or syncytium-inducing (SI) variant. All of the recombinant viruses infected peripheral blood mononuclear cells, whereas only those with SI V3 infected MT2 cells, a CD4(+) T cell line. Consistently, the SI V3 recombinants used CXCR4, while the NSI V3 recombinants used CCR5 for infection of HOS-CD4(+) cells. Finally, only the NSI V3 sequence conferred CC-chemokine sensitivity on the parental virus. The data support the notion that the HIV-1 V3 loop consists of a relatively independent domain in gp120 and suggest that the subtype E V3 loop indeed contains the functional element to dictate the cell tropism, coreceptor preference, and chemokine sensitivity of the virus. These findings are of immediate importance in understanding V3 structure-function relationship and for examining phenotypic evolution of HIV-1 subtype E.     

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.     

The proline-rich region of HIV-1 Nef affects CXCR4-mediated chemotaxis in Jurkat T cells

T-cell chemotaxis constitutes an essential function of the immune response, since active secretion of chemokines controls homing and recruitment of leukocytes into tissues. Modification of chemotactic responses by HIV-1 may provide a mechanism to increase viral spread, and may be an important factor in HIV-1 disease progression and pathogenesis. One potent T-cell chemoattractant is SDF-1 alpha, the natural ligand for the HIV-1 co-receptor CXCR4. In addition, the HIV-1 gp120 molecule shares the chemotactic properties of several chemokines, including SDF-1 alpha. HIV-1 Nef is a pathogenic determinant and a virulence factor that has pleiotropic effects on immune cell processes and receptor signaling. In this study, the effects of Nef on T-cell migration to SDF-1 alpha, and on CXCR4 receptor signaling were examined. We report that disruption of the proline-rich region of Nef inhibits T-cell migration to SDF-1 alpha. This dominant negative effect indicates that Nef occupies a position in the CXCR4-mediated signaling pathway that is upstream of an SH3-dependent pathway. The results suggest that Nef may play an important role in homing of T cells during viral invasion in HIV-1 disease.     

Asymmetric HIV-1 co-receptor use and replication in CD4(+) T lymphocytes

Susceptibility to infection by the human immunodeficiency virus type-1 (HIV-1), both in vitro and in vivo, requires the interaction between its envelope (Env) glycoprotein gp120 Env and the primary receptor (R), CD4, and Co-R, either CCR5 or CXCR4, members of the chemokine receptor family. CCR5-dependent (R5) viruses are responsible for both inter-individual transmission and for sustaining the viral pandemics, while CXCR4-using viruses, usually dualtropic R5X4, emerge in ca. 50% of individuals only in the late, immunologically suppressed stage of disease. The hypothesis that such a major biological asymmetry is explained exclusively by the availability of cells expressing CCR5 or CXCR4 is challenged by several evidences. In this regard, binding of the HIV-1 gp120 Env to the entry R complex, i.e. CD4 and a chemokine R, leads to two major events: virion-cell membrane fusion and a cascade of cell signaling. While the fusion/entry process has been well defined, the role of R/Co-R signaling in the HIV-1 life cycle has been less characterized. Indeed, depending on the cellular model studied, the capacity of HIV-1 to trigger a flow of events favoring either its own latency or replication remains a debated issue. In this article, we will review the major findings related to the role of HIV R/Co-R signaling in the steps following viral entry and leading to viral spreading in CD4(+) T lymphocytes.     

Enhanced replication of R5 HIV-1 over X4 HIV-1 in CD4(+)CCR5(+)CXCR4(+) T cells

To enter human cells, HIV-1 usually uses CD4 and 1 of 2 coreceptors: CCR5 and CXCR4. Interestingly, even though CCR5 is expressed on far fewer T cells than is CXCR4, many patients in early- and late-stage HIV disease maintain high levels of CCR5-tropic (R5) viruses. We hypothesized that such high R5 viral loads may be sustained because, relative to CXCR4-tropic (X4) HIV-1 infection, R5 HIV-1 infection of permissive CD4(+)CCR5(+)CXCR4(+) T cells results in the production of significantly more infectious virus particles per target cell. To investigate this possibility, we compared the levels of virus production per target cell after isogenic R5 and X4 HIV-1 infection of 2 in vitro primary human lymphocyte culture systems: T-cell receptor-stimulated blood-derived CD4(+) T cells and tonsil histoculture (which requires no exogenous stimulation for ex vivo infection). We provide evidence that R5 HIV-1 does indeed compensate for a small target cell population by producing, on average, 5 to 10 times more infectious virus per CCR5(+) target cell than X4 HIV-1. This replicative advantage may contribute to the predominance of R5 HIV-1 in vivo.     

Effects of SDF-1alpha and gp120IIIB on apoptotic pathways in SK-N-SH neuroblastoma cells

CXCR4, a chemokine receptor constitutively expressed in the brain, binds both ligands, the chemokine SDF-1alpha and the HIV envelope glycoprotein gp120(IIIB). There seem to be intracellular differences between the neuronal apoptosis induced by SDF-1alpha and that induced by gp120(IIIB), but the apoptotic pathways involved have not been compared in human neuronal cells. In this study, we characterized the apoptotic intracellular pathways activated by neurotoxic concentrations of SDF-1alpha and gp120(IIIB) in human neuroblastoma cells SK-N-SH. SDF-1alpha (10 nM) and gp120(IIIB) (2 nM) induced similar levels of apoptosis after 24 h of incubation (49 +/- 4% and 48 +/- 3%, respectively, of the neurons were apoptotic). SDF1alpha-induced apoptosis was completely abolished by the inhibition of Src phosphorylation by PP2. Exposure to SDF-1alpha (10 nM) triggered an increase in Src phosphorylation, with a maximum after 20 min of incubation (1.80 +/- 0.24 times higher than control, P = 0.01). NMDA calcium flux was enhanced only if cells were incubated with SDF-1alpha for 20 min before applying NMDA. By contrast, gp120(IIIB)-induced apoptosis was not affected by the inhibition of Src phosphorylation. Moreover, gp120(IIIB) enhanced NMDA calcium flux immediately, without modifying Src phosphorylation status. Finally, levels of phospho-JNK increased following exposure to gp120(IIIB) (by a factor of 1.46 +/- 0.4 at 120 min, P = 0.03), but not after exposure to SDF-1alpha. Thus, SDF-1alpha and gp120(IIIB) induced a similar level of neuronal apoptosis, but by activating different intracellular pathways. SDF-1alpha enhanced NMDA activity indirectly via Src phosphorylation, whereas gp120(IIIB) probably activated the NMDA receptor directly and phosphorylated JNK.     

Downregulation of CCR5 on activated CD4 T cells in HIV-infected Indians

BACKGROUND: HIV infection in India is unique as it occurs predominantly by CCR5-utilizing isolates that exhibit no co-receptor switch. OBJECTIVES: To study HIV-1 co-receptor dynamics on T cells and monocytes following viral infection. STUDY DESIGN: HIV co-receptor expression was evaluated by flow cytometry on various cell subsets in HIV-infected Indians and in vitro in human peripheral blood mononuclear cells infected with CCR5- or CXCR4-utilizing HIV-1. Transfection of the T cell line CEM-CCR5 (which expresses CD4, CCR5 and CXCR4) with HIV-1 Nef or Vpu expression vectors, or treatment with recombinant soluble gp120 from CCR5- and CXCR4-tropic HIV-1, was carried out to determine their effects on co-receptor expression. RESULTS: Indian HIV patients had fewer CD4(+)CCR5(+) T cells and CCR5-expressing activated CD4(+) T cells, but higher CXCR4-expressing activated CD4(+) T cells compared with controls. Expression of CCR5 was not different on monocytes in HIV patients as compared to controls. The CCR5 downregulation on T cells was HIV infection specific and was governed by the co-receptor-utilization phenotype of the virus. The Nef and soluble gp120 proteins induced CCR5 downregulation, the latter in a co-receptor-utilization phenotype specific manner. CONCLUSIONS: The HIV-1 co-receptor dynamics in Indian patients is distinct from western patients and depends upon the virus surface protein. We propose this to be a viral survival strategy.     

Synergistic interaction between ligands binding to the CD4 binding site and V3 domain of human immunodeficiency virus type I gp120.

We demonstrate that soluble CD4 (sCD4) or a monoclonal antibody (mAb), 39.13g, binding to a conformational epitope of gp120 involved in CD4 binding, and mAbs binding to the V3 domain of gp120, can synergistically neutralize human immunodeficiency virus type I (HIV-1). In contrast, a neutralizing mAb binding to a linear epitope within the CD4 binding domain was unable to exert a synergistic effect in combination with V3 mAbs, suggesting that synergism is dependent on ligands binding to the critical, discontinuous, gp120 residues constituting the CD4 binding site. A number of V3 mAbs showed increased binding to virion gp120 in the presence of sCD4, suggesting a mechanism for the synergistic neutralization. This effect was not observed with recombinant or detergent solubilized viral gp120, suggesting that the oligomeric structure of gp120 on viral particles affects V3 epitope exposure. This hypothesis is supported by the ability of two new V3 mAbs, 8/38c and 8/64b, to only neutralize HIV-1 in the presence of sCD4 or mAb 39.13g; binding studies demonstrate that these mAbs only bind to virion gp120 in the presence of sCD4. Thus, V3 epitope exposure is modulated by the interaction of virion gp120 with ligands specific for the CD4 binding domain and results in enhanced antibody-mediated neutralization.     

In vitro exposure to highly cytopathic HIV-1 X4 strains increases expression of mucosa-associated integrins on CD4(+) T cells

To determine whether infection with HIV-1 strains of different tropisms would influence expression of the mucosa-associated integrins alpha 4 beta 7 and alpha E beta 7 or the lymph node homing receptor L-selectin on peripheral T lymphocytes, cells were infected with the CXCR4-tropic (X4)/syncytium-inducing (SI) HIV-1(IIIB) strain or with X4/SI or CCR5-tropic (R5)/non-SI (NSI) primary human isolates. Flow cytometric analyses of CD4(+) T cells from cultures infected with HIV-1(IIIB) and one X4/SI primary HIV-1 isolate revealed a significant increase in surface expression of alpha 4 beta 7 and alpha E beta 7 12 days after infection. L-selectin expression was not significantly affected on CD4(+) T cells. However, infection with another X4/SI and two R5/NSI primary HIV-1 isolates did not significantly alter homing receptor expression on CD4(+) T cells. Since a higher degree of CD4 cytopathicity occurred in those cultures having increased integrin expression, these data suggest that significantly altered mucosal homing receptor expression on CD4(+) T cells may result as a "bystander" effect after infection with some cytopathic isolates of HIV-1.     

CXCR4 and CCR5 expression on CD4+ T cells in vivo and HIV-1 antigen beta-chemokine production in vitro after treatment with HIV-1 immunogen (REMUNE)

BACKGROUND: The chemokine receptors CXCR4 and CCR5 have been identified as the major coreceptors for HIV-1 on CD4+ cells and macrophages. The natural ligands for these receptors are SDF-1 and the beta-chemokines (MIP-1alpha, MIP-1beta, RANTES), respectively, and are the products of a variety of immune cells, including CD8+ T lymphocytes. STUDY DESIGN/METHODS: We hypothesized that the ability to stimulate the natural ligands for these receptors using an immune based therapy might influence in vivo chemokine receptor expression. RESULTS: In vivo CXCR4 expression remained stable after treatment with an HIV-1 Immunogen (REMUNE), whereas CCR5 expression on CD4+ T cells decreased (p < .05). Furthermore, HIV-1 antigen-specific production of beta-chemokines in vitro was also augmented (P < .05). CONCLUSIONS: These preliminary results suggest that this HIV-1-specific immune-based therapy can stimulate antigen-specific beta-chemokine production in vitro and downregulate CCR5 receptor expression on CD4 cells in vivo.     

HIV-1 envelope protein binds to and signals through integrin alpha4beta7, the gut mucosal homing receptor for peripheral T cells

Infection with human immunodeficiency virus 1 (HIV-1) results in the dissemination of virus to gut-associated lymphoid tissue. Subsequently, HIV-1 mediates massive depletion of gut CD4+ T cells, which contributes to HIV-1-induced immune dysfunction. The migration of lymphocytes to gut-associated lymphoid tissue is mediated by integrin alpha4beta7. We demonstrate here that the HIV-1 envelope protein gp120 bound to an activated form of alpha4beta7. This interaction was mediated by a tripeptide in the V2 loop of gp120, a peptide motif that mimics structures presented by the natural ligands of alpha4beta7. On CD4+ T cells, engagement of alpha4beta7 by gp120 resulted in rapid activation of LFA-1, the central integrin involved in the establishment of virological synapses, which facilitate efficient cell-to-cell spreading of HIV-1.     

Involvement of protein kinase C in HIV-1 gp120-induced apoptosis in primary endothelium

We previously showed that HIV-1 gp120-induced apoptosis in primary human umbilical vein endothelial cell cultures (HUVEC), through CCR5 and CXCR4. Here, we have found that agonists of protein kinase C (PKC), basic fibroblast growth factor (bFGF), and short exposure to low concentrations of phorbol esters were found to block gp120-induced apoptosis in HUVEC cultures. PKC antagonists, sphingosine, H7, and extended exposure of cultures to high concentrations of phorbol esters were also found to block gp120-induced apoptosis in HUVEC cultures. A significant increase in the total amount of cellular PKC enzymatic activity was observed on exposure of HUVEC to gp120. No increase in total PKC activity was observed on exposure of HUVECs to the natural ligands SDF-1alpha, or regulated-on-activation normal T-expressed and secreted (RANTES) cells, and gp120-induced PKC induction was found to be totally blocked by CXCR4 antibodies and partially blocked by the caspase 3 inhibitor, DEVD-CHO. Alternatively, CXCR4 antibodies and DEVD-CHO totally blocked apoptosis. Finally, gp120-induced effects were found to be insensitive to pertussis toxin. Accumulated evidence suggests PKC involvement at multiple points in the gp120-induced apoptotic pathway; also suggests involvement of the CXCR4 receptor internalization pathway, and potentially suggests different downstream effects of gp120-receptor interactions and natural ligand-receptor interactions.     

Differential effects of stromal derived factor-1 alpha (SDF-1 alpha) on early and late stages of human megakaryocytic development

Stromal derived factor-1 alpha (SDF-1 alpha), the high-affinity ligand of CXC-chemokine receptor 4 (CXCR4), was added to human CD34(+) hematopoietic progenitor cells that can be induced to differentiate along the monocytic or megakaryocytic lineages. In control liquid cell cultures supplemented with two different cytokine cocktails: stem cell factor (SCF), interleukin-3 (IL-3), macrophage-colony stimulating factor (M-CSF), and 10% fetal calf serum (FCS), or, SCF and thrombopoietin (TPO), the expression of surface CXCR4 progressively increased in both the CD14(+) monocytic and CD41(+) megakaryocytic lineages. While SDF-1 alpha caused only modest effects on cells of the monocytic lineage, it induced profound down-regulation of CXCR4 in megakaryocytic cells at all stages of differentiation. Moreover, while SDF-1 alpha initially up-regulated the early megakaryocytic antigen CD41, at later time points (days 12-16) it induced down-regulation of the late megakaryocytic antigen CD42b. Consistently, at day 16, the number of mature megakaryocytes was significantly decreased in cultures supplemented with SDF-1 alpha. These findings indicate that, besides its primary role in regulating the retention of precursor cells in hematopoietic tissues, the SDF-1 alpha/CXCR4 system participates in the regulation of megakaryocytic development by stimulating the formation of immature megakaryoblasts and inhibiting the formation of mature megakaryocytes.     

Antigen stimulation induces HIV envelope gp120-specific CD4(+) T cells to secrete CCR5 ligands and suppress HIV infection

CD4(+) T cells are critical for effective immune responses against HIV, but they are also the main cell type targeted by the virus. To investigate the key factors that could protect these cells from infection, we evaluated the capacity of HIV gp120-specific human CD4(+) T cells to produce chemokines that inhibit HIV and determined their contribution in suppressing infection in the cells. Antigen stimulation of the CD4(+) T cells elicited production of high amounts of CCR5 chemokines MIP-1alpha (CCL3), MIP-1beta (CCL4), and RANTES (CCL5). Production of these CCR5 ligands was more readily and reproducibly detected than that of IFN-gamma or IL-2. Importantly, in association with secretion of the CCR5 ligands, antigen stimulation made these CD4(+) T cells more resistant to CCR5-tropic HIV-1. Conversely, in the absence of antigen stimulation, the cells were readily infected by the virus, and after infection, their capacity to produce MIP-1beta and IFN-gamma rapidly declined. Thus, vaccines that trigger HIV-specific CD4(+) T cells to elicit robust and rapid production of anti-viral chemokines would be advantageous. Such responses would protect virus-specific CD4(+) T cells from HIV infection and preserve their critical functions in mounting and maintaining long-lasting immunity against the virus.     

Selective up-regulation of functional CXCR4 expression in erythroid cells by HIV-1 Tat protein

CXCR4 is the high affinity receptor for the SDF-1 alpha chemokine and represents the main coreceptor for HIV-1 T-tropic strains. The surface expression of CXCR4 was analysed in CD34+ haematopoietic progenitors, induced to differentiate along the erythroid or granulocytic lineages, in liquid cultures supplemented or not with HIV-1 Tat protein. At concentrations as low as 1-10 ng/ml, synthetic Tat protein significantly increased the surface expression of CXCR4 in erythroid but not in granulocytic cells. The Tat-mediated up-regulation of surface CXCR4 was accompanied by a concomitant increase of CXCR4 mRNA and total CXCR4 protein content in cells developing along the erythroid lineage after 6-10 days of culture. Moreover, addition of SDF-1 alpha (200 ng/ml) induced a significant higher rate of apoptosis in Tat-treated erythroid cells in comparison with control cells. These results demonstrated for the first time a direct positive role in haematopoietic gene regulation of Tat protein, and suggest the possible involvement of Tat in HIV-1-induced anaemia.