Inhibition of tyrosine kinase activation blocks the down-regulation of CXC chemokine receptor 4 by HIV-1 gp120 in CD4+ T cells.

Because the binding of HIV-1 envelope to CD4 initiates a configurational change in glycoprotein 120 (gp120), enabling it to interact with fusion coreceptors, we investigated how this process interferes with the expression and function of CXC chemokine receptor 4 (CXCR4) in CD4+ T lymphocytes. A recombinant gp120 (MN), after preincubation with CD4+ T lymphocytes, significantly inhibited the binding and chemotaxis of the cells in response to the CXCR4 ligand stromal cell-derived factor-1alpha (SDF-1alpha), accompanied by a markedly reduced surface expression of CXCR4. gp120, but not SDF-1alpha, induced rapid tyrosine phosphorylation of src-like kinase p56lck in CD4+ T cells, whereas both gp120 and SDF-1alpha caused phosphorylation of the CXCR4. The tyrosine kinase inhibitor herbimycin A abolished the phosphorylation of p56lck and CXCR4 induced by gp120 in association with maintenance of normal expression of cell surface CXCR4 and a migratory response to SDF-1alpha. Thus, a CD4-associated signaling molecule(s) including p56lck is activated by gp120 and is required for the down-regulation of CXCR4.     

CXCR4/CCR5 down-modulation and chemotaxis are regulated by the proteasome pathway

Chemokines and their receptors play a critical role in host immune surveillance and are important mediators of human immunodeficiency virus (HIV) pathogenesis and inflammatory response. The chemokine receptors CCR5 and CXCR4, which act as co-receptors along with CD4 for HIV docking and entry, are down-modulated by their respective ligands, MIP-1beta/SDF-1alpha or by the HIV envelope protein, gp120. We have studied the role of the proteasome pathway in the down-regulation of these receptors. Using the yeast and mammalian two-hybrid systems, we observed that the CCR5 receptor is constitutively associated with the zeta subunit of proteasome. Immunoprecipitation studies in CCR5 L1.2 cells revealed that this association was increased with MIP-1beta stimulation. The proteasome inhibitors, lactacystin and epoxomicin, attenuated MIP-1beta induced CCR5 down-modulation as detected by fluorescence-activated cell sorter analysis and confocal microscopy. The proteasome inhibitors also inhibited the SDF-1alpha and gp120 protein-induced down-modulation of the CXCR4 receptor in Jurkat cells. However, the inhibitors had no significant effect on the gp120-induced internalization of the CD4 receptor. These inhibitors also blocked cognate ligand-mediated chemotaxis but had no effect on SDF-1alpha-induced p44/42 MAP kinase or MIP-1beta-induced p38 kinase activities, thus indicating differential effects of the inhibitors on signaling mediated by these receptors. These results indicate that the CCR5 and CXCR4 receptor down-modulation mechanism and chemotaxis mediated by these receptors are dependent upon proteasome activity.     

Comodulation of CXCR4 and CD26 in human lymphocytes

We provide convergent and multiple evidence for a CD26/CXCR4 interaction. Thus, CD26 codistributes with CXCR4, and both coimmunoprecipitate from membranes of T (CD4(+)) and B (CD4(-)) cell lines. Upon induction with stromal cell-derived factor 1alpha (SDF-1alpha), CD26 is cointernalized with CXCR4. CXCR4-mediated down-regulation of CD26 is not induced by antagonists or human immunodeficiency virus (HIV)-1 gp120. SDF-1alpha-mediated down-regulation of CD26 is not blocked by pertussis toxin but does not occur in cells expressing mutant CXCR4 receptors unable to internalize. Codistribution and cointernalization also occurs in peripheral blood lymphocytes. Since CD26 is a cell surface endopeptidase that has the capacity to cleave SDF-1alpha, the CXCR4.CD26 complex is likely a functional unit in which CD26 may directly modulate SDF-1alpha-induced chemotaxis and antiviral capacity. CD26 anchors adenosine deaminase (ADA) to the lymphocyte cell surface, and this interaction is blocked by HIV-1 gp120. Here we demonstrate that gp120 interacts with CD26 and that gp120-mediated disruption of ADA/CD26 interaction is a consequence of a first interaction of gp120 with a domain different from the ADA binding site. SDF-1alpha and gp120 induce the appearance of pseudopodia in which CD26 and CXCR4 colocalize and in which ADA is not present. The physical association of CXCR4 and CD26, direct or part of a supramolecular structure, suggests a role on the function of the immune system and the pathophysiology of HIV infection.     

Desensitization of CXC chemokine receptor 4, mediated by IL-16/CD4, is independent of p56lck enzymatic activity

CCR5 and CXC chemokine receptor 4 (CXCR4) are coreceptors for CD4 as defined by HIV-1 glycoprotein (gp) 120 binding. Pretreatment of T cells with gp120 results in modulation of both CCR5 and CXCR4 responsiveness, which is dependent upon p56(lck) enzymatic activity. The recent findings that pretreatment of T cells with a natural CD4 ligand, IL-16, could alter cellular responsiveness to macrophage-inflammatory protein-1ss (MIP-1ss) stimulation, prompted us to investigate whether IL-16 could also alter CXCR4 signaling. These studies demonstrate that IL-16/CD4 signaling in T lymphocytes also results in loss of stromal derived factor-1alpha (SDF-1alpha)/CXCR4-induced chemotaxis; however, unlike MIP-1ss/CCR5, the effects were not reciprocal. There was no effect on eotaxin/CCR3-induced chemotaxis. Desensitization of CXCR4 by IL-16 required at least 10-15 min pretreatment; no modulation of CXCR4 expression was observed, nor was SDF-1alpha binding altered. Using murine T cell hybridomas transfected to express native or mutated forms of CD4, it was determined that IL-16/CD4 induces a p56(lck)-dependent inhibitory signal for CXCR4, which is independent of its tyrosine catalytic activity. By contrast, IL-16/CD4 desensitization of MIP-1ss/CCR5 responses requires p56(lck) enzymatic activity. IL-16/CD4 inhibition of SDF-1alpha/CXCR4 signals requires the presence of the Src homology 3 domain of p56(lck) and most likely involves activation of phosphatidylinositol-3 kinase. These studies indicate the mechanism of CXCR4 receptor desensitization induced by a natural ligand for CD4, IL-16, is distinct from the inhibitory effects induced by either gp120 or IL-16 on CCR5.     

CXCR4 function requires membrane cholesterol: implications for HIV infection

HIV requires cholesterol and lipid rafts on target cell membranes for infection. To elucidate a possible mechanism, we determined that cholesterol extraction by hydroxypropyl-beta-cyclodextrin (BCD) inhibits stromal cell-derived factor 1alpha (SDF-1alpha) binding to CXCR4 on T cell lines and PBMCs. Intracellular calcium responses to SDF-1alpha, as well as receptor internalization, were impaired in treated T cells. Loss in ligand binding is likely due to conformational changes in CXCR4 and not increased sensitivity to internalization. SDF-1alpha binding and calcium responses were effectively restored by reloading cholesterol. Immunofluorescence microscopy revealed that SDF-1alpha binding occurred in lipid raft microdomains that contained GM1. CXCR4 surface expression, on the other hand, only partially colocalized with GM1. HIV-1(IIIB) infection assays confirmed the functional loss of CXCR4 in the cell lines tested, Sup-T1 and CEM-NKR-CCR5. These data suggest that cholesterol is essential for CXCR4 conformation and function and that lipid rafts may play a regulatory role in SDF-1alpha signaling.     

The chemokine stromal cell-derived factor-1 alpha modulates alpha 4 beta 7 integrin-mediated lymphocyte adhesion to mucosal addressin cell adhesion molecule-1 and fibronectin

The interaction between the integrin alpha(4)beta(7) and its ligand, mucosal addressin cell adhesion molecule-1, on high endothelial venules represents a key adhesion event during lymphocyte homing to secondary lymphoid tissue. Stromal cell-derived factor-1alpha (SDF-1alpha) is a chemokine that attracts T and B lymphocytes and has been hypothesized to be involved in lymphocyte homing. In this work we show that alpha(4)beta(7)-mediated adhesion of CD4(+) T lymphocytes and the RPMI 8866 cell line to mucosal addressin cell adhesion molecule-1 was up-regulated by SDF-1alpha in both static adhesion and cell detachment under shear stress assays. Both naive and memory phenotype CD4(+) T cells were targets of SDF-1alpha-triggered increased adhesion. In addition, SDF-1alpha augmented alpha(4)beta(7)-dependent adhesion of RPMI 8866 cells to connecting segment-1 of fibronectin. While pertussis toxin totally blocked chemotaxis of CD4(+) and RPMI 8866 cells to SDF-1alpha, enhanced alpha(4)beta(7)-dependent adhesion triggered by this chemokine was partially inhibited, indicating the participation of Galpha(i)-dependent as well as Galpha(i)-independent signaling. Accordingly, we show that SDF-1alpha induced a rapid and transient association between its receptor CXCR4 and Galpha(i), whereas association of pertussis toxin-insensitive Galpha(13) with CXCR4 was slower and of a lesser extent. SDF-1alpha also activated the small GTPases RhoA and Rac1, and inhibition of RhoA activation reduced the up-regulation of alpha(4)beta(7)-mediated lymphocyte adhesion in response to SDF-1alpha, suggesting that activation of RhoA could play an important role in the enhanced adhesion. These data indicate that up-regulation by SDF-1alpha of lymphocyte adhesion mediated by alpha(4)beta(7) could contribute to lymphocyte homing to secondary lymphoid tissues.     

Dynamic reorganization of chemokine receptors, cholesterol, lipid rafts, and adhesion molecules to sites of CD4 engagement

T cell polarization and redistribution of cellular components are critical to processes such as activation, migration, and potentially HIV infection. Here, we investigate the effects of CD4 engagement on the redistribution and localization of chemokine receptors, CXCR4 and CCR5, adhesion molecules, and lipid raft components including cholesterol, GM1, and glycosyl-phosphatidylinositol (GPI)-anchored proteins. We demonstrate that anti-CD4-coated beads (alpha CD4-B) rapidly induce co-capping of chemokine receptors as well as GPI-anchored proteins and adhesion molecules with membrane cholesterol and lipid rafts on human T cell lines and primary T cells to the area of bead-cell contact. This process was dependent on the presence of cellular cholesterol, cytoskeletal reorganization, and lck signaling. Lck-deficient JCaM 1.6 cells failed to cap CXCR4 or lipid rafts to alpha CD4-B. Biochemical analysis reveals that CXCR4 and LFA-1 are recruited to lipid rafts upon CD4 but not CD45 engagement. Furthermore, we also demonstrate T cell capping of both lipid rafts and chemokine receptors at sites of contact with HIV-infected cells, despite the binding of an HIV inhibitory mAb to CXCR4. We conclude that cell surface rearrangements in response to CD4 engagement may serve as a means to enhance cell-to-cell signaling at the immunological synapse and modulate chemokine responsiveness, as well as facilitate HIV entry and expansion by synaptic transmission.     

Possible role of stromal-cell-derived factor-1/CXCR4 signaling on lymph node metastasis of oral squamous cell carcinoma

We examined the role of chemokine signaling on the lymph node metastasis of oral squamous cell carcinoma (SCC) using lymph node metastatic (HNt and B88) and nonmetastatic oral SCC cells. Of 13 kinds of chemokine receptors examined, only CXCR4 expression was up-regulated in HNt and B88 cells. CXCR4 ligand, stromal-cell-derived factor-1alpha (SDF-1alpha; CXCL12), induced characteristic calcium fluxes and chemotaxis only in CXCR4-expressing cells. CXCR4 expression in metastatic cancer tissue was significantly higher than that in nonmetastatic cancer tissue or normal gingiva. Although SDF-1alpha was undetectable in either oral SCC or normal epithelial cells, submandibular lymph nodes expressed the SDF-1alpha protein, mainly in the stromal cells, but occasionally in metastatic cancer cells. The conditioned medium from lymphatic stromal cells promoted the chemotaxis of B88 cells, which was blocked by the CXCR4 neutralization. SDF-1alpha rapidly activated extracellular signal-regulated kinase (ERK)1/2 and Akt/protein kinase B (PKB), and their synthetic inhibitors attenuated the chemotaxis by SDF-1alpha. SDF-1alpha also activated Src family kinases (SFKs), and its inhibitor PP1 diminished the SDF-1alpha-induced chemotaxis and activation of both ERK1/2 and Akt/PKB. These results indicate that SDF-1/CXCR4 signaling may be involved in the establishment of lymph node metastasis in oral SCC via activation of both ERK1/2 and Akt/PKB induced by SFKs.     

LRRC4 inhibits human glioblastoma cells proliferation, invasion, and proMMP-2 activation by reducing SDF-1 alpha/CXCR4-mediated ERK1/2 and Akt signaling pathways

Gliomas take a number of different genetic routes in the progression to glioblastoma multiforme, a highly invasive variant that is mostly unresponsive to current therapies. The alpha-chemokine stromal cell-derived factor (SDF)-1 alpha binds to the seven transmembrane G-protein-coupled CXCR-4 receptor and acts to modulate cell migration and proliferation by activating multiple signal transduction pathways. Leucine-rich repeats containing 4 (LRRC4), a putative glioma suppressive gene, inhibits glioblastoma cells tumorigenesis in vivo and cell proliferation and invasion in vitro. We also previously demonstrated that LRRC4 controlled glioblastoma cells proliferation by ERK/AKT/NF-kappa B signaling pathway. In the present study, we demonstrate that CXC chemokine receptor 4 (CXCR4) is expressed in human glioblastoma U251 cell line, and that SDF-1 alpha increases the proliferation, chemotaxis, and invasion in CXCR4+ glioblastoma U251 cells through the activation of ERK1/2 and Akt. The reintroduction of LRRC4 in U251 cells inhibits the expression of CXCR4 and SDF-1 alpha/CXCR4 axis-mediated downstream intracellular pathways such as ERK1/2 and Akt leading to proliferate, chemotactic and invasive effects. Furthermore, we provide evidence for proMMP-2 activation involvement in the SDF-1 alpha/CXCR4 axis-mediated signaling pathway. LRRC4 significantly inhibits proMMP-2 activation by SDF-1 alpha/CXCR4 axis-mediated ERK1/2 and Akt signaling pathway. Collectively, these results suggest a possible important "cross-talk" between LRRC4 and SDF-1 alpha/CXCR4 axis-mediated intracellular pathways that can link signals of cell proliferation, chemotaxis and invasion in glioblastoma, and may represent a new target for development of new therapeutic strategies in glioma.     

HIV glycoprotein gp120 enhances mesenchymal stem cell migration by upregulating CXCR4 expression

Background

HIV infection and/or the direct pathogenic effects of circulating HIV proteins impairs the physiological function of mesenchymal stem cells (MSCs), and contribute to the pathogenesis of age-related clinical comorbidities in people living with HIV. The SDF-1/CXCR4 pathway is vital for modulating MSC proliferation, migration and differentiation. HIV glycoprotein gp120 inhibits SDF-1 induced chemotaxis by downregulating the expression and function of CXCR4 in monocytes, B and T cells. The influence of gp120 on CXCR4 expression and migration in MSCs is unknown.

SDF-1α Degrades whereas Glycoprotein 120 Upregulates Bcl-2 Interacting Mediator of Death Extralong Isoform: Implications for the Development of T Cell Memory

After a primary immune response, T cell memory occurs when a subset of Ag-specific T cells resists peripheral selection by acquiring resistance to TCR-induced death. Recent data have implicated Bcl-2 interacting mediator of death (Bim) as an essential mediator of the contraction phase of T cell immunity. In this article, we describe that stromal-derived factor-1α (SDF-1α) ligation of CXCR4 on activated T cells promotes two parallel processes that favor survival, phospho-inactivation of Foxo3A, as well as Bim extralong isoform (Bim(EL)) degradation, both in an Akt- and Erk-dependent manner. Activated primary CD4 T cells treated with SDF-1α therefore become resistant to the proapoptotic effects of TCR ligation or IL-2 deprivation and accumulate cells of a memory phenotype. Unlike SDF-1α, gp120 ligation of CXCR4 has the opposite effect because it causes p38-dependent Bim(EL) upregulation. However, when activated CD4 T cells are treated with both gp120 and SDF-1α, the SDF-1α-driven effects of Bim(EL) degradation and acquired resistance to TCR-induced death predominate. These results provide a novel causal link between SDF-1α-induced chemotaxis, degradation of Bim(EL), and the development of CD4 T cell memory.     

Marked structural and functional heterogeneity in CXCR4: separation of HIV-1 and SDF-1alpha responses

CXCR4, the chemotactic cell receptor for SDF-1alpha, is essential for immune trafficking and HIV infection. CXCR4 is remarkably heterogeneous and the purpose of this study was to better identify the isoforms expressed by cells and compare their structure and function. We found that cells express either a predominant isoform or multiple isoforms. These were best resolved on SDS-PAGE using sucrose-gradient-fractionated, triton-insoluble, membrane extracts. We hypothesized that glycosyl modification may underpin some of this heterogeneity and that cell isoform(s) differences may underscore CXCR4's multiple cell functions. A comparison of wild-type (WT) and dual N-linked glycosylation site, N11A/N176A, mutant CXCR4 expressed in 3T3 and HEK-293 cells served to implicate variabilities in glycosylation and oligomerization in almost half of the isoforms. Immunoprecipitation of CXCR4 revealed monomer and dimer non-glycosylated forms of 34 kDa and 68 kDa from the N11A/N176A mutant, compared with glycosylated 40 kDa and 47 kDa and 73 kDa and 80 kDa forms from WT. The functional specificity of isoform action was also implicated because, despite CEMT4 cells expressing high levels of CXCR4 and 11 different isoforms, a single 83 kDa form was found to bind gp120 for HIV-1 IIIB infection. Furthermore, comparative studies found that in contrast to SDF-1alpha-responsive Nalm-6 cells that expressed similar levels of a single isoform, CEMT4 cells did not show a Ca(++) flux or a chemotactic response to SDF-1alpha. Thus, CXCR4 can differ both structurally and functionally between cells, with HIV-1 infection and chemotaxis apparently mediated by different isoforms. This separation of structure and function has implications for understanding HIV-1 entry and SDF-1alpha responses and may indicate therapeutic possibilities.     

Role of N-glycans and SDF-1alpha on the coassociation of CD4 with CXCR4 at the plasma membrane of monocytic cells and blood lymphocytes

CXCR4 is a coreceptor, along with CD4, for human immunodeficiency virus type 1 (HIV-1). Trimolecular complexes between HIV-1 glycoprotein (gp)120, CD4 and CXCR4 constitute a prerequisite for HIV entry. We studied whether CD4 is associated with CXCR4 on CD4+ CXCR4+ cells. Using the conformation-dependent anti-CXCR4 mAb 12G5, CD4 was coimmunoprecipitated with CXCR4 from the membrane of U937 cells which support HIV-1(LAI) efficient infection, and from that of peripheral blood lymphocytes (PBL). CD4 association with CXCR4 increased upon PBL coculture for 5 days with autologous monocytes, decreased upon treatment of the cells or the CD4-CXCR4 complex with either N-glycanase or stromal cell derived factor-1alpha (SDF-1alpha) and was abolished by incubation of the cells with both, N-glycanase and SDF-1alpha. This indicates that glycans are partly involved in CD4 association with CXCR4 and may partly explain the inhibitory effect of SDF-1alpha on HIV infection.     

T cell-tropic HIV gp120 mediates CD4 and CD8 cell chemotaxis through CXCR4 independent of CD4: implications for HIV pathogenesis

HIV entry is determined by one or more chemokine receptors. T cell-tropic viruses bind CXCR4, whereas macrophage-tropic viruses use CCR5 and other CCRs. Infection with CXCR4 and CCR5-tropic HIV requires initial binding to CD4, and chemotaxis induced by the CCR5-tropic envelope has been reported to be strictly dependent on CD4 binding. We demonstrate that, in contrast to CD4-dependent gp120 signaling via CCR5, envelope signaling through CXCR4 is CD4 independent, inducing chemotaxis of both CD4 and CD8 T cells. Signaling by virus or soluble envelope through CXCR4 may affect pathogenesis by attracting and activating target and effector cells.     

Optimal inhibition of X4 HIV isolates by the CXC chemokine stromal cell-derived factor 1 alpha requires interaction with cell surface heparan sulfate proteoglycans

The chemokine stromal cell-derived factor 1 (SDF-1) is the natural ligand for CXC chemokine receptor 4 (CXCR4). SDF-1 inhibits infection of CD4+ cells by X4 (CXCR4-dependent) human immunodeficiency virus (HIV) strains. We previously showed that SDF-1 alpha interacts specifically with heparin or heparan sulfates (HSs). Herein, we delimited the boundaries of the HS-binding domain located in the first beta-strand of SDF-1 alpha as the critical residues. We also provide evidence that binding to cell surface heparan sulfate proteoglycans (HSPGs) determines the capacity of SDF-1 alpha to prevent the fusogenic activity of HIV-1 X4 isolates in leukocytes. Indeed, SDF-1 alpha mutants lacking the capacity to interact with HSPGs showed a substantially reduced capacity to prevent cell-to-cell fusion mediated by X4 HIV envelope glycoproteins. Moreover, the enzymatic removal of cell surface HS diminishes the HIV-inhibitory capacity of the chemokine to the levels shown by the HS-binding-disabled mutant counterparts. The mechanisms underlying the optimal HIV-inhibitory activity of SDF-1 alpha when attached to HSPGs were investigated. Combining fluorescence resonance energy transfer and laser confocal microscopy, we demonstrate the concomitant binding of SDF-1 alpha to CXCR4 and HSPGs at the cell membrane. Using FRET between a Texas Red-labeled SDF-1 alpha and an enhanced green fluorescent protein-tagged CXCR4, we show that binding of SDF-1 alpha to cell surface HSPGs modifies neither the kinetics of occupancy nor activation in real time of CXCR4 by the chemokine. Moreover, attachment to HSPGs does not modify the potency of the chemokine to promote internalization of CXCR4. Attachment to cellular HSPGs may co-operate in the optimal anti-HIV activity of SDF-1 alpha by increasing the local concentration of the chemokine in the surrounding environment of CXCR4, thus facilitating sustained occupancy and down-regulation of the HIV coreceptor.     

Extracellular Nef protein targets CD4+ T cells for apoptosis by interacting with CXCR4 surface receptors

The effects of soluble Nef protein on CD4(+) T cells were examined. CD4(+)-T-cell cultures exposed to soluble Nef were analyzed for apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and hallmarks of apoptosis including cytoplasmic shrinkage, nuclear fragmentation, DNA laddering, and caspase activation. We observed dose- and time-dependent inductions of apoptosis. DNA laddering and activated caspase 3 were also evident. Cells treated with Nef/protein kinase inhibitor complexes were protected from Nef-induced apoptosis, suggesting possible roles for protein kinases in the apoptosis pathway. Similarly, cells treated with Nef/anti-Nef antibody complexes were protected from Nef-induced apoptosis. The cellular receptor responsible for Nef-induced apoptosis was identified through antibody- and ligand-blocking experiments as a receptor commonly involved in viral entry. CXCR4 antibodies, as well as the endogenous ligand SDF-1alpha, were effective in blocking Nef-induced apoptosis, while CCR5 and CD4 antibodies were ineffective. Moreover, a CXCR4-deficient cell line, MDA-MB-468, which was resistant to Nef-induced apoptosis, became sensitive upon transfection with a CXCR4-expressing vector. This study suggests that extracellular Nef protein could contribute to the decline of CD4 counts prior to and during the onset of AIDS in patients with human immunodeficiency virus type 1 infections.     

CXCR7 mediated Giα independent activation of ERK and Akt promotes cell survival and chemotaxis in T cells

Chemokine receptors CXCR7 and CXCR4 bind to the same ligand stromal cell derived factor-1alpha (SDF-1α/CXCL12). We assessed the downstream signaling pathways mediated by CXCL12-CXCR7 interaction in Jurkat T cells. All experiments were carried out after functionally blocking the CXCR4 receptor. CXCL12, on binding CXCR7, induced phosphorylation of extra cellular regulated protein kinases (ERK 1/2) and Akt. Selective inhibition of each signal demonstrated that phosphorylated ERK 1/2 is essential for chemotaxis and survival of T cells whereas activation of Akt promotes only cell survival. Another interesting finding of this study is that CXCL12-CXCR7 interaction under normal physiological conditions does not activate the p38 pathway. Furthermore, we observed that the CXCL12 signaling via CXCR7 is Giα independent. Our findings suggest that CXCR7 promotes cell survival and does not induce cell death in T cells. The CXCL12 signaling via CXCR7 may be crucial in determining the fate of the activated T cells.     

Nef proteins from diverse groups of primate lentiviruses downmodulate CXCR4 to inhibit migration to the chemokine stromal derived factor 1

Nef proteins of primate lentiviruses promote viral replication, virion infectivity, and evasion of antiviral immune responses by modulating signal transduction pathways and downregulating expression of receptors at the cell surface that are important for efficient antigen-specific responses, such as CD4, CD28, T-cell antigen receptor, and class I and class II major histocompatibility complex. Here we show that Nef proteins from diverse groups of primate lentiviruses which do not require the chemokine receptor CXCR4 for entry into target cells strongly downmodulate the cell surface expression of CXCR4. In contrast, all human immunodeficiency virus type 1 (HIV-1) and the majority of HIV-2 Nef proteins tested did not have such strong effects. SIVmac239 Nef strongly inhibited lymphocyte migration to CXCR4 ligand, the chemokine stromal derived factor 1 (SDF-1). SIVmac239 Nef downregulated CXCR4 by accelerating the rate of its endocytosis. Downmodulation of CXCR4 was abolished by mutations that disrupt the constitutively strong AP-2 clathrin adaptor binding element located in the N-terminal region of the Nef molecule, suggesting that Nef accelerates CXCR4 endocytosis via an AP-2-dependent pathway. Together, these results point to CXCR4 as playing an important role in simian immunodeficiency virus and possibly also HIV-2 persistence in vivo that is unrelated to viral entry into target cells. We speculate that Nef targets CXCR4 to disrupt ordered trafficking of infected leukocytes between local microenvironments in order to facilitate their dissemination and/or impair the antiviral immune response.     

Expression of Nef downregulates CXCR4, the major coreceptor of human immunodeficiency virus, from the surfaces of target cells and thereby enhances resistance to superinfection

Lentiviral Nef proteins are key factors for pathogenesis and are known to downregulate functionally important molecules, including CD4 and major histocompatibility complex class I (MHC-I), from the surfaces of infected cells. Recently, we demonstrated that Nef reduces cell surface levels of the human immunodeficiency virus type 1 (HIV-1) entry coreceptor CCR5 (N. Michel, I. Allespach, S. Venzke, O. T. Fackler, and O. T. Keppler, Curr. Biol. 15:714-723, 2005). Here, we report that Nef downregulates the second major HIV-1 coreceptor, CXCR4, from the surfaces of HIV-infected primary CD4 T lymphocytes with efficiencies comparable to those of the natural CXCR4 ligand, stromal cell-derived factor-1 alpha. Analysis of a panel of mutants of HIV-1(SF2) Nef revealed that the viral protein utilized the same signature motifs for downmodulation of CXCR4 and MHC-I, including the proline-rich motif P(73)P(76)P(79)P(82) and the acidic cluster motif E(66)E(67)E(68)E(69.) Expression of wild-type Nef, but not of specific Nef mutants, resulted in a perinuclear accumulation of the coreceptor. Remarkably, the carboxy terminus of CXCR4, which harbors the classical motifs critical for basal and ligand-induced receptor endocytosis, was dispensable for the Nef-mediated reduction of surface exposure. Functionally, the ability of Nef to simultaneously downmodulate CXCR4 and CD4 correlated with maximum-level protection of Nef-expressing target cells from fusion with cells exposing X4 HIV-1 envelopes. Furthermore, the Nef-mediated downregulation of CXCR4 alone on target T lymphocytes was sufficient to diminish cells' susceptibility to X4 HIV-1 virions at the entry step. The downregulation of chemokine coreceptors is a conserved activity of Nef to modulate infected cells, an important functional consequence of which is an enhanced resistance to HIV superinfection.