HIV-1 gp120 envelope protein modulates proliferation of human glomerular epithelial cells

Glomerular epithelial cells (GEC) have been demonstrated to undergo morphological alterations in human immunodeficiency virus (HIV)-associated focal glomerulosclerosis. In the present study, we evaluated the effect of HIV-1 gp120 envelope protein on the growth of cultured human (H) GEC. gp120 protein enhanced (P < 0.001) the proliferation of HGEC at lower concentrations. The mitogenic effect of gp120 protein on HGEC was further confirmed by enhanced accumulation of proliferating nuclear cell antigen (PCNA) by gp120 protein-treated cells, as compared with control cells. On the contrary, gp120 protein at higher concentrations suppressed (P < 0. 001) the growth of HGEC. To evaluate the mechanism of gp120 protein-induced HGEC growth suppression, we examined the effect of gp120 protein on HGEC apoptosis. gp120 protein at higher concentrations promoted the apoptosis of HGEC. At higher concentrations, gp120 protein also enhanced DNA fragmentation of HGEC. Anti-gp120 antibody attenuated the proliferative as well as the apoptotic effects of gp120 protein on HGEC. Because protein kinase C as well as tyrosine kinase inhibitors partially inhibited gp120-induced proliferation, gp120 appears to be activating both the protein kinase C and tyrosine kinase pathways. In addition, gp120 protein at lower concentrations enhanced mRNA expression of c-fos and at higher concentrations promoted mRNA expression of c-jun. We conclude that gp120 has a bimodal effect on proliferation of HGEC. This effect may be mediated through the activation of early growth genes.     

HIV/gp120 decreases adult neural progenitor cell proliferation via checkpoint kinase-mediated cell-cycle withdrawal and G1 arrest

Impaired adult neurogenesis has been observed in several neurodegenerative diseases, including human immunodeficiency virus (HIV-1)-associated dementia (HAD). Here we report that the HIV-envelope glycoprotein gp120, which is associated with HAD pathogenesis, inhibits proliferation of adult neural progenitor cells (aNPCs) in vitro and in vivo in the dentate gyrus of the hippocampus of HIV/gp120-transgenic mice. We demonstrate that HIV/gp120 arrests cell-cycle progression of aNPCs at the G1 phase via a cascade consisting of p38 mitogen-activated protein kinase (MAPK) --> MAPK-activated protein kinase 2 (a cell-cycle checkpoint kinase) --> Cdc25B/C. Our findings define a molecular mechanism that compromises adult neurogenesis in this neurodegenerative disorder.     

Downregulation of Map Kinase Activity Signalled by HIV-1-gp120 Coat Protein in Granular Neurons and Glial Cells from Rat Cerebellum

We have studied the effect of gp120 coat protein from HIV-1 on tyrosine phosphorylation processes in primary cultures of granular neurons or glial cells from the cerebellum of neonatal rats. The extracellular application of recombinant gp120 (200 pM) was able to reduce the phosphotyrosine content and the immunoreactivity for active form-specific antibodies of MAP kinase. Whereas in neurons MAP kinase appeared to be the only protein whose phosphotyrosine content was decreased, in glial cultures the inhibitory effect of gp120 on tyrosine phosphorylation processes appeared to be more widespread. In neuronal cultures, the effect of the viral protein was prevented by the concomitant treatment with depolarizing agents.     

p38 MAP kinase-mediated negative inotropic effect of HIV gp120 on cardiac myocytes

Myocardial dysfunction leading to dilated cardiomyopathy has been documented with surprisingly high frequency in human immunodeficiency virus (HIV)-infected individuals. p38 MAP kinase has been implicated as a mediator of myocardial dysfunction. We previously reported p38 MAP kinase activation by the HIV coat protein gp120 in neonatal rat cardiac myocytes. We now report the direct inotropic effects of HIV gp120 on adult rat ventricular myocytes (ARVM). ARVM were continuously superfused with gp120, and percent fractional shortening (FS) was determined by automated border detection and simultaneous intracellular ionized free Ca²⁺ concentration ([Ca²⁺]_i) measured by fura 2-AM fluorescence: gp120 alone increased FS and increased [Ca²⁺]_i within 5 min and then depressed FS without a decrease in [Ca²⁺]_i by 20–60 min, which persisted for at least 2 h. Exposure of ARVM to gp120 also resulted in the phosphorylation of the upstream regulator of p38 MAP kinase MKK3/6, p38 MAP kinase itself, and its downstream effector, ATF-2, over a similar time course. ERK (p44/42) and JNK stress signaling pathways were not similarly activated. The effects of the p38 MAP kinase inhibitor were concentration dependent. SB-203580 (10 µM) blocked both p38 MAP kinase phosphorylation and the delayed negative inotropic effect of gp120. SB-203580 (5 µM) selectively blocked phosphorylation of ATF-2 without blocking the phosphorylation of MKK3/6 or p38 MAP kinase itself. SB-203580 (5 µM) administered before, with, or after gp120 blocked the negative inotropic effect of gp120 in ARVM. p38 MAP kinase activation may be a common stress-response mechanism contributing to myocardial dysfunction in HIV and other nonischemic as well as ischemic cardiomyopathies. cardiomyopathy; cell signaling     

Long-term maintenance of gp120-specific immune responses by genetic vaccination with the HIV-1 envelope genes linked to the gene encoding Flt-3 ligand

DNA vaccines target dendritic cells (DC) to induce Ag-specific immune responses in animals. Potent HIV-specific immunity could be achieved by efficient priming of the immune system by DNA vaccines. We investigated a novel DNA vaccine approach based on the role of growth factors in DC expansion and differentiation. To this end, we constructed chimeric genes encoding the HIV envelope glycoproteins physically linked to the extracellular domain of Fms-like tyrosine kinase receptor-3 ligand (FLex; a DC growth factor; both mouse (m)FLex and human (h)FLex). These chimeric gene constructs synthesized biologically active, oligomeric FLex:gp120 fusion proteins and induced DC expansion (CD11c(+)CD11b(+)) when injected i.v. into mice. This DC expansion is comparable to that achieved by FLex DNA encoding native FLex protein. When delivered intramuscularly as DNA vaccines, hFLex:gp120 induced high frequencies of gp120-specific CD8(+) T cells in the presence or absence of FLex DNA-induced DC expansion, but gp120 and mFLex:gp120 elicited only low to moderate levels of Ag-specific CD8(+) T cells. In contrast, mFLex:gp120 induced high levels of anti-gp120 Abs under identical conditions of DNA vaccination. However, the Ab levels in mice immunized with DNA vaccines encoding hFLex:gp120 and gp120 proteins were low without DC expansion, but reached high levels comparable to that elicited by mFLex:gp120 only after the second boost in the presence of DC expansion. Importantly, the gp120-specific CD8(+) T cells persisted at high frequency for 114 days (16 wk) after a booster injection. These experiments provide insight into the importance of modulating DC function in vivo for effective genetic vaccination in animals.     

Screening HIV‐1 antigenic peptides as receptors for antibodies and CD4 in allosteric nanosensors

We have analyzed the suitability of six antigenic peptides from several HIV‐1 structural proteins (namely gp41, gp120, p17, and p24), as anti‐HIV‐1 antibody receptors in an allosteric enzymatic biosensor. These peptides were inserted in a solvent‐exposed surface of Escherichia coli (E. coli) beta‐galactosidase by means of conventional recombinant DNA technology. The resulting enzymes were tested to allosterically respond to sera from HIV‐1‐infected individuals. Only stretches from gp41 and gp120 envelope proteins were able to transduce the molecular contact signal in the presence of immunoreactive sera. Intriguingly, the enzyme displaying the CD4 binding site segment KQFINMWQEVGKAMYAPP was activated by soluble CD4, suggesting that it produces conformational modifications on the allosteric enzyme as those occurring during antibody‐promoted induced fit. This fact is discussed in the context of the design of smart protein drugs and markers targeted to CD4⁺ cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Prothoracicotropic hormone induces tyrosine phosphorylation in prothoracic glands of the silkworm,Bombyx mori

In the present study, we investigated the tyrosine phosphorylation of Bombyx mori prothoracic glands using phosphotyrosine‐specific antibodies and Western blot analysis. Results showed that prothoracicotropic hormone (PTTH) stimulates a rapid increase in tyrosine phosphorylation of at least 2 proteins in prothoracic glands, one of which was identified as extracellular signal‐regulated kinase (ERK). The phosphorylation of another 120‐kDa protein showed dose‐ and time‐dependent stimulation by PTTH in vitro. In vitro activation of tyrosine phosphorylation was also verified by in vivo experiments: injection of PTTH into day‐6 last‐instar larvae greatly increased tyrosine phosphorylation. Treatment of prothoracic glands with the protein tyrosine phosphatase inhibitor, sodium orthovanadate, also resulted in tyrosine phosphorylation of several proteins and increased ecdysteroidogenesis. The PTTH‐stimulated phosphorylation of the 120‐kDa protein was markedly attenuated by genistein, a broad‐spectrum tyrosine kinase inhibitor, but not by HNMPA‐(AM)₃, a specific inhibitor of insulin receptor tyrosine kinase. PP2, a more‐selective inhibitor of the Src‐family tyrosine kinases, partially inhibited PTTH‐stimulated tyrosine phosphorylation, but not ecdysteroidogenesis. This result implies the possibility that in addition to ERK, the phosphorylation of the 120‐kDa protein, which is not Src‐family tyrosine kinase, is likely also involved in PTTH‐stimulated ecdysteroidogenesis in B. mori. © 2010 Wiley Periodicals, Inc.     

Crystal structure of a 3B3 variant—A broadly neutralizing HIV‐1 scFv antibody

We present the crystal structure determination of an anti‐HIV‐1 gp120 single‐chain variable fragment antibody variant, 3B3, at 2.5 Å resolution. This 3B3 variant was derived from the b12 antibody, using phage display and site‐directed mutagenesis of the variable heavy chain (V_H) complementary‐determining regions (CDRs). 3B3 exhibits enhanced binding affinity and neutralization activity against several cross‐clade primary isolates of HIV‐1 by interaction with the recessed CD4‐binding site on the gp120 envelope protein. Comparison with the structures of the unbound and bound forms of b12, the 3B3 structure closely resembles these structures with minimal differences with two notable exceptions. First, there is a reorientation of the CDR‐H3 of the V_H domain where the primary sequences evolved from b12 to 3B3. The structural changes in CDR‐H3 of 3B3, in light of the b12‐gp120 complex structure, allow for positioning an additional Trp side chain in the binding interface with gp120. Finally, the second region of structural change involves two peptide bond flips in CDR‐L3 of the variable light (V_L) domain triggered by a point mutation in CDR‐H3 of Q100eY resulting in changes in the intramolecular hydrogen bonding patterning between the V_L and V_H domains. Thus, the enhanced binding affinities and neutralization capabilities of 3B3 relative to b12 probably result from higher hydrophobic driving potential by burying more aromatic residues at the 3B3‐gp120 interface and by indirect stabilization of intramolecular contacts of the core framework residues between the V_L and V_H domains possibly through more favorable entropic effect through the expulsion of water.     

Intrastriatal administration of human immunodeficiency virus‐1 glycoprotein 120 reduces glial cell‐line derived neurotrophic factor levels and causes apoptosis in the substantia nigra

Uninfected neurons of the substantia nigra (SN) degenerate in human immunodeficiency virus (HIV)‐positive patients through an unknown etiology. The HIV envelope glycoprotein 120 (gp120) causes apoptotic neuronal cell death in the rodent striatum, but its primary neurotoxic mechanism is still under investigation. Previous studies have shown that gp120 causes neurotoxicity in the rat striatum by reducing brain‐derived neurotrophic factor (BDNF). Because glial cell line‐derived neurotrophic factor (GDNF) and BDNF are neurotrophic factors crucial for the survival of dopaminergic neurons of the SN, we investigated whether gp120 reduces GDNF and BDNF levels concomitantly to induce apoptosis. Rats received a microinjection of gp120 or vehicle into the striatum and were sacrificed at various time intervals. GDNF but not BDNF immunoreactivity was decreased in the SN by 4 days in gp120‐treated rats. In these animals, a significant increase in the number of caspase‐3‐ positive neurons, both tyrosine hydroxylase (TH)‐positive and ‐negative, was observed. Analysis of TH immunoreactivity revealed fewer TH‐positive neurons and fibers in a medial and lateral portion of cell group A9 of the SN, an area that projects to the striatum, suggesting that gp120 induces retrograde degeneration of nigrostriatal neurons. We propose that dysfunction of the nigrostriatal dopaminergic system associated with HIV may be caused by a reduction of neurotrophic factor expression by gp120. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Superantigen enhancement of specific immunity: antibody production and signaling pathways

Superantigens are microbial proteins that induce massive activation, proliferation, and cytokine production by CD4+ T cells via specific Vbeta elements on the TCR. In this study we examine superantigen enhancement of Ag-specific CD4+ T cell activity for humoral B cell responses to T-dependent Ags BSA and HIV gp120 envelope, type I T-independent Ag LPS, and type II T-independent Ag pneumococcal polysaccharides. Injection of BSA followed by a combination of superantigens staphylococcal enterotoxin A and staphylococcal enterotoxin B (SEB) 7 days later enhanced the anti-BSA Ab response in mice approximately 4-fold as compared with mice given BSA alone. The anti-gp120 response was enhanced approximately 3-fold by superantigens. The type II T-independent Ag pneumococcal polysaccharide response was enhanced approximately 2.3-fold by superantigens, whereas no effect was observed on the response to the type I T-independent Ag LPS. The superantigen effect was completely blocked by the CD4+ T cell inhibitory cytokine IL-10. SEB-stimulated human CD4+ T cells were examined to determine the role of the mitogen-activated protein (MAP) kinase signal transduction pathway in superantigen activation of T cells. Inhibitors of the mitogen pathway of MAP kinase blocked SEB-induced proliferation and IFN-gamma production, while an inhibitor of the p38 stress pathway had no effect. Consistent with this, SEB activated extracellular signal-regulated kinase/MAP kinase as well as MAP kinase-interacting kinase, a kinase that phosphorylates eIF4E, which is an important component of the eukaryotic protein synthesis initiation complex. Both kinases were inhibited by IL-10. Thus, superantigens enhance humoral immunity via Ag-specific CD4+ T cells involving the stress-independent pathway of MAP kinase.     

A ligand for the erbB-2 oncogene product (gp30) induces differentiation of human breast cancer cells.

The human erbB-2 oncogene encodes a tyrosine kinase receptor. A ligand for the erbB-2 receptor (gp30), with an apparent molecular weight of 30,000, was reported to modulate the growth of cells overexpressing erbB-2. Whereas low concentrations of gp30 induced proliferation of these cells, higher concentrations inhibited their growth. To elucidate the cellular mechanisms underlying cell growth inhibition by gp30, we tested the effect of this ligand on cell growth and differentiation of the human breast cancer cells AU-565 and MDA-MB-453 (which overexpress erbB-2) and MCF-7 cells (which express low levels of this protooncogene). Ligand concentrations that inhibited growth in cells overexpressing erbB-2 induced apparent differentiation of cells with a more mature phenotype, i.e., with characteristics such as inhibited cell growth, altered cytoplasmic and nuclear morphology, and increased synthesis of milk components (casein and lipids). No significant effect of the ligand was observed in the human breast cancer cell line MCF-7. Concomitant with the induction of differentiation in AU-565 and MDA-MB-453 cells, the erbB-2 protein was translocated from membrane to the cytoplasm and perinuclear sites. These findings indicate that ligand-induced growth inhibition in cells overexpressing erbB-2 is associated with an apparent induction of differentiation.     

Human Glomerular Epithelial Cell Express CD4 and Interaction with gp120 Protein Promotes PYK2 Tyrosine Phosphorylation

Focal segmental glomerulosclerosis (FSGS) is the predominant glomerular lesion in patients with HIV infection. Visceral glomerular epithelial cell (vGEC) injury is a key feature of this glomerular lesion. However, the exact mechanism of HIV-1-induced vGEC injury is not clear. We studied the presence of CD4 (HIV-1 receptor) in vGECs. vGECs were cultured from human kidneys and used during the 5th to 10th passages. Immunocytochemical studies were carried out to visualize CD4 receptors in these cells. Protein and RNA were extracted from vGECs and renal cortical tissues. Western and Northern blots were generated and probed for the expression of CD4. To determine the downstream effect of ligand receptor interaction, vGECs were treated either with variable concentrations of HIV-1 gp120 protein (0.001 to 0.1 μg/ml) for 1 min or with a fixed dose of gp120 protein (0.01 μg/ml) for variable time periods (0 to 10 min), and at the end of the incubation period, tyrosine phosphorylation of pyk2 was studied. Immunocytochemical studies showed the presence of CD4 receptors in vGECs. Western and Northern blot studies confirmed the presence of CD4 expression in these cells. gp120 protein promoted vGEC tyrosine phosphorylation of pyk2 in a dose- and time-dependent manner. The present study provides a mechanistical insight for the role of HIV-1 in the development of glomerular injury in patients with HIV infection.     

Effect of human immunodeficiency virus gp120 glycoprotein on the association of the protein tyrosine kinase p56lck with CD4 in human T lymphocytes

The human immunodeficiency virus binds to CD4+ T lymphocytes through the interaction of its envelope glycoprotein (gp120) with the CD4 molecule. The src-related protein tyrosine kinase p56lck is physically associated with CD4 and is co-immunoprecipitated by CD4 monoclonal antibody (mAb). Activators of protein kinase C (PKC) cause the dissociation of p56lck from CD4. Here we report that gp120 mAb immunoprecipitated the p56lck.CD4.gp120 complex after short term treatment (20 min) of human T lymphocytes with gp120. The p56lck that was associated with the CD4.gp120 complex was dissociated by activators of PKC. This effect was abolished by pretreatment of cells with PKC inhibitors. Thus the p56lck.CD4.gp120 immune complex immunoprecipitated by gp120 mAb behaves in a similar manner, with respect to PKC activation or inhibition, to the p56lck.CD4 complex immunoprecipitated by CD4 mAb. Short term treatment of cells with gp120, followed by gp120 mAb, resulted in an increase in the tyrosine kinase activity of p56lck associated with CD4. However, the amount of enzyme associated with CD4 remained unchanged. Long term treatment (20 h) of human T lymphocytes with gp120 resulted in the down-regulation of cell surface CD4 molecules. A parallel decrease in CD4-associated gp120 was also observed. In addition, gp120 caused the dissociation of p56lck and CD4. However, the dissociation of the p56lck from CD4 occurred at much faster rate than the down-regulation of surface CD4 molecules. Such mechanisms may account for the down-regulation of cell surface CD4 molecules and the depletion of functional CD4+ T lymphocytes which are characteristic of human immunodeficiency virus infections and acquired immune deficiency syndrome pathogenesis.     

Innate immune dysfunction in HIV infection: effect of HIV envelope-NK cell interactions

We have previously described a number of NK cell dysfunctions in HIV-viremic individuals. In the present study, we performed DNA microarray analysis followed by phenotypic and functional characterization in an effort to investigate which HIV envelope glycoproteins (gp120) affect the physiologic functions of NK cells. Upon treatment of NK cells with HIV gp120, DNA microarray analyses indicated up-regulation of several categories of genes that are associated with apoptosis, suppression of both cellular proliferation and survival, as well as down-regulation of genes that play a vital role in cell proliferation, innate immune defense mechanism, and cell survival. Both subtypes of gp120 suppressed NK cell cytotoxicity, proliferation, and the ability to secrete IFN-gamma. NK cells exposed to X4-subtype HIV gp120 showed a significant decrease in the levels of CC chemokines, while exposure to R5-subtype HIV gp120 had minimal effect. Extended exposure to HIV gp120 resulted in apoptosis of NK cells, further validating the microarray data. Our data demonstrate that exposure of NK cells to HIV envelope proteins results in profound cellular abnormalities at the level of gene expression as well as generic cell functions. These findings are likely to be a consequence of a direct HIV gp120-mediated effect on NK cells. Identification of specific surface receptors on NK cells that interact with HIV envelope proteins might explain how HIV is capable of circumventing innate immune defense mechanisms and establishing infection in susceptible individuals.     

HIV-1 gp120-induced tubular epithelial cell apoptosis is mediated through p38-MAPK phosphorylation

BACKGROUND: HIV-associated nephropathy is accompanied by significant tubular alterations in the form of tubular cell proliferation, apoptosis, and microcystic dilatation. In the present study we evaluated the role of CD4 receptors in HIV-1-induced tubular cell injury. METHODS: To confirm the presence of CD4 receptors in tubular cells, immunocytochemical, Western and Northern blot studies were carried out. To determine the downstream effect of CD4 and gp120 interaction, we evaluated the effect of gp120 on tubular cell p38 mitogen-activated protein kinase (MAPK) activity and phosphorylation. To establish causal relationships between gp120, CD4, and p38 MAPK pathways, we studied the effect of anti-CD4 antibody and SB 202190 (an inhibitor of p38 MAPK) on gp120-induced tubular cell apoptosis. RESULTS: Proximal tubular cells in culture as well as in intact tissue showed expression of CD4 (immunocytochemical and Western blot studies). Cultured tubular cells also showed mRNA expression for CD4 (Northern blot studies). Gp120, at concentrations of 10-100 ng/ ml, triggered tubular cell apoptosis; however, this effect of gp120 was inhibited by anti-CD4 antibody. SB 202190 also inhibited gp120-induced tubular cell apoptosis. In addition, gp120 promoted tubular cell p38 MAPK phosphorylation in a time- and dose- dependent manner. CONCLUSION: Gp120 through interaction with CD4 triggers tubular cell apoptosis. This effect of gp120 on tubular cells is mediated through phosphorylation of p38 MAPK.     

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.

HIV gp120 enhances NO production by cardiac myocytes through p38 MAP kinase-mediated NF-kappaB activation

Human immunodeficiency virus (HIV) infection is associated with a surprisingly high frequency of myocardial dysfunction. Potential mechanisms include direct effects of HIV, indirect effects mediated by cytokines, or a combination. We have previously reported that interleukin-1beta (IL-1beta) (500 U/ml) alone induced nitric oxide (NO) production by neonatal rat cardiac myocytes (CM). Effects of the HIV-1 envelope, glycoprotein120 (gp120), on inducible NO synthase (iNOS) in CM have not been previously reported. Unlike IL-1beta, recombinant HIV-gp120 (1 microgram/ml) alone failed to enhance NO production in CM (0.5 +/- 0.4 vs. 0.4 +/- 0.5 micromol/1.25 x 10(5) cells/48 h, gp120 vs. control, respectively; n = 12, P = not significant). However, the addition of gp120 to IL-1beta significantly enhanced iNOS mRNA expression (70 +/- 1.5 vs. 26 +/- 2.4 optical units, IL-1beta + gp120 vs. IL-1beta, respectively; n = 3), iNOS protein synthesis (42 +/- 1.4 vs. 18 +/- 0.8 optical units, IL-1beta + gp120 vs. IL-1beta, respectively; n = 3), and NO production (NO(2)(-)) (6.6 +/- 0.6 vs. 4.1 +/- 0.8 micromol/1.25 x 10(5) cells/48 h, IL-1beta + gp120 vs. IL-1beta, respectively; n = 12, P 相似文献   

The HIV protein, GP120, activates nuclear protein kinase C in nuclei from lymphocytes and brain

Nuclear pool(s) of protein kinase C (PKC) may be a common target for hormones and growth factors which affect the trophic state of cells. The data presented demonstrate a time and dose-dependent activation of nuclear PKC by the HIV coat protein, gp120, in isolated nuclei from rat spleen and hippocampus. This gp120-stimulated PKC response was blocked by specific PKC inhibitors, a monoclonal antibody to PKC, and a monoclonal antibody directed against the murine T4 analog, L3T4. It is suggested that the gp120 interaction with the nuclear trophic factor-PKC system may impair normal gene expression, and thus result in the clinical symptoms associated in AIDS infection.     

Internalization of HIV glycoprotein gp120 is associated with down-modulation of membrane CD4 and p56lck together with impairment of T cell activation.

We previously demonstrated that long term treatment of the Ag-specific CD4+ T cell clone P28D with soluble HIV envelope glycoprotein gp120 results in a marked impairment of CD3/TCR-mediated responses. In this report, to further understand these inhibitory effects, the binding properties and internalization of gp120 have been investigated, in parallel with functional studies, in long term incubations of P28D cells with gp120. Immunofluorescence studies show that surface-bound gp120 level is maximal within 1 h of incubation at 37 degrees C and then gradually decreases. This decrease is accompanied by a progressive down-modulation of membrane CD4 (30-35% loss over a 18-h incubation period) without concomitant alteration of the CD4 mRNA steady-state level. Similar experiments performed with 125I-labeled gp120 demonstrate that the glycoprotein is progressively internalized (up to 35% internalized material after 18 h) and that it accumulates inside the cells. Confocal microscopy studies show that internalized gp120 is concentrated in localized intracellular compartments. CD4 also accumulates in compartments with a similar localization and is stained with mAb OKT4 but not with mAb OKT4a. Concomitantly to internalization of gp120 and disappearance of membrane CD4, a correlated loss of the CD4-associated tyrosine kinase p56lck is evidenced. Interestingly, a progressive impairment of the P28D responses to specific Ag or to anti-CD3 mAb is also observed. Inhibitions of T cell proliferation increase with the degree of both CD4 and p56lck down-modulation. Removal of exogenous gp120 results in a rapid and spontaneous release of internalized gp120 into a degraded form. A progressive restoration of CD4 and p56lck levels is also noticed. In parallel, CD3/TCR-mediated responses of clone P28D are fully recovered. Altogether, our results suggest that HIV-1 glycoprotein gp120 is able to down-modulate membrane CD4 presumably by a cointernalization process and to further down-modulate the associated p56lck. This dual phenomenon is presumably involved in the direct immunosuppressive effect of gp120 on the CD3/TCR-mediated activation pathway.     

Human immunodeficiency virus envelope (gp120) binding to DC-SIGN and primary dendritic cells is carbohydrate dependent but does not involve 2G12 or cyanovirin binding sites: implications for structural analyses of gp120-DC-SIGN binding

The calcium-dependent lectin, DC-SIGN, binds to human immunodeficiency virus (HIV) (and simian immunodeficiency virus) gp120 and mediates the binding and transfer of HIV from monocyte-derived dendritic cells (MDDCs) to permissive T cells. However, it has been recently reported that DC-SIGN binding to HIV gp120 may be carbohydrate independent. Here, we formally demonstrate that gp120 binding to DC-SIGN and MDDCs is largely if not wholly carbohydrate dependent. Endo-beta-N-glucosaminidase H (EndoH) treatment of gp120-Fc under conditions that maintained wild-type CD4 binding-and the full complement of complex glycans-significantly decreased (>90%) binding to DC-SIGN expressing cell lines, as well as to MDDCs. Any residual binding of EndoH-treated gp120-Fc to DC-SIGN was completely competed off with mannan. Mutational analysis indicated that no single glycosylation site affected the ability of gp120-Fc to bind DC-SIGN. To further guide our efforts in mapping the DC-SIGN binding sites on gp120, we used two well-characterized HIV inhibitory agents (2G12 monoclonal antibody and cyanovirin) that bind to high-mannose sugars on gp120. We showed that 2G12 and DC-SIGN bound to nonoverlapping sites in gp120 because (i) 2G12 did not block soluble gp120 or virion binding to DC-SIGN, (ii) 2G12 bound to gp120-Fc that was prebound to cell surface DC-SIGN, and (iii) gp120-Fc mutants that lack glycosylation sites involved in 2G12's epitope were also fully capable of binding DC-SIGN. These data were substantiated by the inability of cyanovirin to block gp120-Fc binding to DC-SIGN. Cyanovirin has been shown to effectively compete for 2G12 binding to gp120. Indeed, high concentrations of cyanovirin dramatically enhanced gp120-Fc binding to cell surfaces in the presence or absence of DC-SIGN. We provide evidence that this enhancement may be due to cyanovirin's ability to bridge gp120 to mannosylated cell surface proteins. These results have implications for antiviral therapeutics and for ongoing efforts to finely map the glycan structures on gp120 responsible for DC-SIGN binding.