Regions required for CD4 binding in the external glycoprotein gp120 of simian immunodeficiency virus

The external domain of the envelope glycoprotein, gp120, of simian immunodeficiency virus (SIV) has been expressed as a mature secreted product using recombinant baculoviruses and the expressed protein, which has an observed molecular mass of 110 kDa, was purified by monoclonal antibody (MAb) affinity chromatography. N-terminal sequence analysis showed a signal sequence cleavage identity similar to that of the gp120s of both human immunodeficiency virus type 1 (HIV-1) and HIV type 2. The expressed molecule bound to soluble CD4 with an affinity that was approximately 10-fold lower than that of gp120 from HIV-1. A screening of the ability of SIV envelope MAbs to inhibit CD4 binding revealed two groups of inhibitory MAbs. One group is dependent on conformation, while the second group maps to a discrete epitope near the amino terminus. The particular role of the V3 loop region of the molecule in CD4 binding was investigated by the construction of an SIV-HIV hybrid in which the V3 loop of SIV was precisely replaced with the equivalent domain from HIV-1 MN. The hybrid glycoprotein bound HIV-1 V3 loop MAbs and not SIV V3 MAbs but continued to bind conformational SIV MAbs and soluble CD4 as well as the parent molecule.     

lck-independent inhibition of T cell antigen response by the HIV gp120

Binding of the HIV envelope glycoprotein gp120 to CD4 inhibits T cell activation. We have used a murine T cell clone transfected with either wild-type human CD4 or mutated forms of CD4 to characterize the pathways involved in this inhibitory effect of gp120. Ag-induced proliferation of T cell clones transfected with human CD4 was completely inhibited in the presence of gp120, even though stimulation of this clone is independent of a CD4/MHC class II interaction. In addition, our results demonstrate that the inhibition by gp120 is not due to the sequestration of lck from TCR and does not require activation of lck by gp120. This suggests that CD4 can regulate the initiation of T cell activation independently of its interaction with lck. Moreover, we demonstrate that the nonresponsiveness induced by gp120 can be reversed by soluble CD4 when added early after onset of stimulation and that gp120 exerts its inhibitory effect when cells are in the G0 > or = 1 phase of the cell cycle.     

Monoclonal antibodies raised against covalently crosslinked complexes of human immunodeficiency virus type 1 gp120 and CD4 receptor identify a novel complex-dependent epitope on gp 120

The binding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120, to its cell surface receptor, CD4, represents a molecular interaction involving distinct alterations in protein structure. Consequently, the pattern of epitopes presented on the gp120-CD4 complex should differ from those on free gp120. To investigate this concept, mice were immunized with covalently crosslinked complexes of viral HIV-1IIIBgp120 and soluble CD4. Two monoclonal antibodies (MoAbs) obtained from the immunized mice exhibited a novel epitope specificity. The MoAbs were marginally reactive with HIV-1IIIBgp120, highly reactive with gp120-CD4 complexes, and unreactive with soluble CD4. The same pattern of reactivity was seen in solid-phase assays using HIV-1(451)gp120. A similar specificity for complexes was evident in flow cytometry experiments, in which MoAb reactivity was dependent upon the attachment of gp120 to CD4-positive cells. In addition, MoAb reactivity was detected upon the interaction of CD4 receptors with purified HIV-1IIIB virions. Notably, seroantibodies from HIV-positive individuals competed for MoAb binding, indicating that the epitope is immunogenic in humans. The results demonstrated that crosslinked gp120-CD4 complexes elicit antibodies to cryptic gp120 epitopes that are exposed during infection in response to receptor binding. These findings may have important implications for the consideration of HIV envelope-receptor complexes as targets for virus neutralization.     

HIV-1 envelope gp120 inhibits the monocyte response to chemokines through CD4 signal-dependent chemokine receptor down-regulation

Since HIV-1 infection results in severe immunosuppression, and the envelope protein gp120 has been reported to interact with some of the chemokine receptors on human T lymphocytes, we postulated that gp120 may also affect monocyte activation by a variety of chemokines. This study shows that human peripheral blood monocytes when preincubated with gp120 either purified from laboratory-adapted strains or as recombinant proteins exhibited markedly reduced binding, calcium mobilization, and chemotactic response to chemokines. The gp-120-pretreated monocytes also showed a decreased response to FMLP. This broad inhibition of monocyte activation by chemoattractants required interaction of gp120 with CD4, since the effect of gp120 was only observed in CD4+ monocytes and in HEK 293 cells only if cotransfected with both chemokine receptors and an intact CD4, but not a CD4 lacking its cytoplasmic domain. Anti-CD4 mAbs mimicked the effect of gp120, and both anti-CD4 Ab and gp120 caused internalization of CXCR4 in HEK 293 cells provided they also expressed CD4. Staurosporine blocked the inhibitory effect of gp120 on monocytes, suggesting that cellular signaling was required for gp120 to inhibit the response of CD4+ cells to chemoattractants. Our study demonstrates a broad suppressive effect of gp120 on monocyte activation by chemoattractants through the down-regulation of cell surface receptors. Thus, gp120 may be used by HIV-1 to disarm the monocyte response to inflammatory stimulation.     

CD4 is a critical component of the receptor for human herpesvirus 7: interference with human immunodeficiency virus

In this study, we demonstrate that the glycoprotein CD4, a member of the immunoglobulin superfamily, is a critical component of the receptor for human herpesvirus 7 (HHV-7), a recently discovered T-lymphotropic human herpesvirus. A selective and progressive downregulation of the surface membrane expression of CD4 was observed in human CD4+ T cells in the course of HHV-7 infection. Various murine monoclonal antibodies to CD4 and the recombinant soluble form of human CD4 caused a dose-dependent inhibition of HHV-7 infection in primary CD4+ T lymphocytes. Moreover, radiolabeled HHV-7 specifically bound to cervical carcinoma cells (HeLa) expressing human CD4. A marked carcinoma cells (HeLa) expressing human CD4. A marked reciprocal interference was observed between HHV-7 and human immunodeficiency virus (HIV), the retrovirus that causes the acquired immunodeficiency syndrome and also uses CD4 as a receptor. Previous exposure of CD4+ T cells to HHV-7 dramatically interfered with infection by both primary and in vitro-passaged HIV-1 isolates. Reciprocally, persistent infection with HIV-1 or treatment with the soluble form of gp120, the CD4-binding envelope glycoprotein of HIV-1, rendered CD4+ T cells resistant to HHV-7 infection. These data indicate that CD4 is critically involved in the receptor mechanism for HHV-7. The antagonistic effect between HHV-7 and HIV could be exploited to devise therapeutic approaches to AIDS.     

On-farm monitoring of mouse-invasive Salmonella enterica serovar enteritidis and a model for its association with the production of contaminated eggs

CD4 molecules are the primary receptors for human immunodeficiency virus (HIV) and bind the envelope glycoprotein gp120 of HIV with high-affinity. We have previously shown that cross-linking of CD4 molecules (CD4XL) in normal peripheral blood mononuclear cells (PBMC) results in secretion of cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), but not of interleukin-2 (IL-2) or IL-4. To investigate the intracellular signaling events associated with CD4-gp120 interaction, we incubated CD4+ T cells from peripheral blood of HIV-negative healthy donors with HIV envelope protein gp160 alone or performed CD4XL with gp160 and anti-gp160 antibody. This procedure resulted in tyrosine phosphorylation of intracellular substrates p59fyn, zap 70, and p95vav and also led to ras activation, as assessed by conversion of rasGDP to rasGTP. The role of ras in CD4 signaling was further investigated using CD4+ Jurkat cells transfected with a dominant negative ras mutant. CD4+ T cells expressing dn-ras secreted significantly reduced levels of TNF-alpha in response to CD4XL. These studies indicate that interaction of HIV gp160 with CD4 molecules activates the ras pathway in T cells, which may result in the cells becoming unresponsive to subsequent stimulation.     

CD4-Induced conformational changes in the human immunodeficiency virus type 1 gp120 glycoprotein: consequences for virus entry and neutralization

Human immunodeficiency virus type 1 (HIV-1) entry into target cells involves sequential binding of the gp120 exterior envelope glycoprotein to CD4 and to specific chemokine receptors. Soluble CD4 (sCD4) is thought to mimic membrane-anchored CD4, and its binding alters the conformation of the HIV-1 envelope glycoproteins. Two cross-competing monoclonal antibodies, 17b and CG10, that recognize CD4-inducible gp120 epitopes and that block gp120-chemokine receptor binding were used to investigate the nature and functional significance of gp120 conformational changes initiated by CD4 binding. Envelope glycoproteins derived from both T-cell line-adapted and primary HIV-1 isolates exhibited increased binding of the 17b antibody in the presence of sCD4. CD4-induced exposure of the 17b epitope on the oligomeric envelope glycoprotein complex occurred over a wide range of temperatures and involved movement of the gp120 V1/V2 variable loops. Amino acid changes that reduced the efficiency of 17b epitope exposure following CD4 binding invariably compromised the ability of the HIV-1 envelope glycoproteins to form syncytia or to support virus entry. Comparison of the CD4 dependence and neutralization efficiencies of the 17b and CG10 antibodies suggested that the epitopes for these antibodies are minimally accessible following attachment of gp120 to cell surface CD4. These results underscore the functional importance of these CD4-induced changes in gp120 conformation and illustrate viral strategies for sequestering chemokine receptor-binding regions from the humoral immune response.     

Epithelial uptake and transport of cell-free human immunodeficiency virus type 1 and gp120-coated microparticles

Cell-free human immunodeficiency virus type 1 (HIV-1) can be taken up and released by a monolayer of primary human gingival cells and remain infectious for CD4+ cells. Virus-sized latex particles covalently coated with purified native HIV-1 envelope glycoprotein gp120 are also transported through the primary epithelial cells. This process is significantly stimulated by increasing the intracellular cyclic AMP (cAMP) concentration. Inhibition experiments with mannan and alpha-methyl-mannopyranoside indicated that mannosyl groups are involved in the interaction between gp120 and gingival cells. An increase of cellular oligomannosyl receptors by incubation with the mannosidase inhibitor deoxymannojirimycin augmented transcellular transport of the gp120-coated particles. The results suggest that infectious HIV can penetrate gingival epithelia by a cAMP-dependent transport mechanism involving interaction of the lectin-like domain of gp120 and mannosyl residues on glycoproteins on the mucosal surface. Penetration of HIV could be inhibited by soluble glycoconjugates present in oral mucins.     

Determinants of human immunodeficiency virus type 1 envelope glycoprotein activation by soluble CD4 and monoclonal antibodies

Infection by some human immunodeficiency virus type 1 (HIV-1) isolates is enhanced by the binding of subneutralizing concentrations of soluble receptor, soluble CD4 (sCD4), or monoclonal antibodies directed against the viral envelope glycoproteins. In this work, we studied the abilities of different antibodies to mediate activation of the envelope glycoproteins of a primary HIV-1 isolate, YU2, and identified the regions of gp120 envelope glycoprotein contributing to activation. Binding of antibodies to a variety of epitopes on gp120, including the CD4 binding site, the third variable (V3) loop, and CD4-induced epitopes, enhanced the entry of viruses containing YU2 envelope glycoproteins. Fab fragments of antibodies directed against either the CD4 binding site or V3 loop also activated YU2 virus infection. The activation phenotype was conferred on the envelope glycoproteins of a laboratory-adapted HIV-1 isolate (HXBc2) by replacing the gp120 V3 loop or V1/V2 and V3 loops with those of the YU2 virus. Infection by the YU2 virus in the presence of activating antibodies remained inhibitable by macrophage inhibitory protein 1beta, indicating dependence on the CCR5 coreceptor on the target cells. Thus, antibody enhancement of YU2 entry involves neither Fc receptor binding nor envelope glycoprotein cross-linking, is determined by the same variable loops that dictate enhancement by sCD4, and probably proceeds by a process fundamentally similar to the receptor-activated virus entry pathway.     

Fas ligand-mediated depletion of CD4 and CD8 lymphocytes by monomeric HIV-1-gp120

In order to determine in what condition and by what mechanism gp 120 can deplete not only CD4 but also CD8 T cells, an in vitro system was established in which peripheral blood lymphocytes from healthy donors were treated with recombinant gp 120. We found that gp 120 can deplete both CD4 and CD8 T cells when they have recently been activated and are exposed to IL-2-deficient conditions. Bioassay of the Fas ligand (FasL) demonstrated augmented expression and release of soluble FasL by CD4 T cells in the supernatant of this culture. The administration of anti-FasL mAb and anti-Fas mAb, both of which exhibit neutralizing activity, completely abolished the depletion of these two T cell populations in culture. Based on these findings, we concluded that FasL depletes Fas antigen expressing CD4 and CD8 T cells by programmed cell death.     

gp130, the cytokine common signal-transducer of interleukin-6 cytokine family, is downregulated in T cells in vivo by interleukin-6

gp130 is a common signal-transducing receptor component for the interleukin-6 (IL-6) family of cytokines. To investigate the expression of gp130 in T-cell subsets and its regulation, anti-murine gp130 monoclonal antibody (MoAb) was used for flow cytometric analysis. In normal mice, gp130 was differentially expressed in thymocyte and splenic T-cell subpopulations defined by CD4/CD8 expression. In aged MRL/lpr mice, although gp130 expression was detectable in splenic CD4(+) or CD8(+) T cells, gp130 expression was significantly downregulated. Because serum levels of IL-6 and soluble IL-6 receptor (sIL-6R) are elevated in these mice, we examined the possibility that the downregulation of gp130 expression on splenic T cells might be produced in response to continuous activation of gp130 by high levels of serum IL-6. In transgenic mice overexpressing IL-6, gp130 expression in the splenic T cells was significantly decreased. After stimulation with IL-6 in vitro, the level of gp130 on CD4(+) or CD8(+) splenic T cells from normal mice was significantly decreased. These results suggest that the expression of gp130 in splenic T cells could be downregulated by the IL-6 stimulation under physiological or pathological circumstances.     

Human interleukin-10 can directly inhibit T-cell growth

Human interleukin-10 (IL-10) inhibits T-cell proliferation and cytokine production in the presence of monocytes. In this study, we have investigated whether IL-10 can directly inhibit T cells. Highly purified peripheral blood T cells containing less than 0.1% CD14+ cells and unresponsive to phytohemagglutinin (PHA), were growth-inhibited by IL-10 when stimulated with immobilized OKT3 monoclonal antibody (MoAb; 55.4% inhibition). This effect was neutralized by the murine MoAb 19F1 directed against human IL-10. In addition, IL-10 inhibited by 52.5% the proliferation of a human tetanus toxoid-specific T-cell clone (TM11) induced by immobilized OKT3 MoAb in the absence of antigen-presenting function. T-cell growth inhibition by IL-10 did not reflect a cytokine-induced change in the kinetics of T-cell response to immobilized OKT3 MoAb, and was observed over a wide range of cell and OKT3 MoAb concentrations. Addition of 1% to 5% monocytes to highly purified peripheral blood T cells resulted in the emergence of proliferation to PHA and to soluble OKT3 MoAb, but did not significantly affect levels of growth inhibition by IL-10 in the presence of immobilized OKT3 MoAb. Similarly, addition of 10% monocytes to the TM11 T-cell clone resulted in the emergence of proliferation in response to tetanus toxoid, but did not significantly influence growth inhibition by IL-10 in the presence of immobilized OKT3 MoAb. When stimulated with immobilized OKT3 MoAb in the absence of accessory cells, T cells secreted IL-2. Secretion of IL-2 under these conditions was inhibited by IL-10 (51.5% inhibition). Thus, IL-10 can directly inhibit growth and IL-2 production in T cells triggered by immobilized OKT3 MoAb in the absence of monocytes.     

The largest (70 kDa) product of the bacteriophage T4 DNA terminase gene 17 binds to single-stranded DNA segments and digests them towards junctions with double-stranded DNA

Bacteriophage terminases are oligomeric multifunctional proteins that bind to vegetative DNA, cut it and, together with portal proteins, translocate the DNA into preformed heads. Most terminases are encoded by two partially overlapping genes. In phage T4 they are genes 16 and 17. We have shown before that the larger of these, gene 17, can yield, in addition to a full-length 70 kDa product, several shorter peptides. At least two of these, gene product (gp) 17' and gp17", are initiated in the same reading frame as the 70 kDa gp17 from internal ribosome binding sites. Most of the shorter gp17 s contain predicted ATPase motifs, but only the largest (70 kDa) peptide has a predicted single-stranded DNA binding domain. Here we describe the DNA binding and cutting properties of the purified 70 kDa protein, expressed from two different clones containing gene 17 but no other T4 gene. Epitope-specific antibodies, which recognize several different gene 17 products in extracts of induced clones or of T4-infected cells, precipitate the purified 70 kDa gp17. When Mg2+ is chelated by EDTA this 70 kDa protein binds to single-stranded DNA, preferentially to junctions of single- and double-stranded DNA segments. It does not bind to blunt-ended double-stranded DNA. When Mg2+ is present the purified 70 kDa gp17 digests single-stranded segments preferentially up to junctions with double-stranded DNA. A 70 kDa gp17 from a P379L temperature sensitive (ts) mutant, which has lost the nuclease and ATPase activities, retains the single-stranded DNA binding activity. Taken together with earlier findings these results support a model for packaging of T4 DNA from single-stranded regions in recombinational or replicative intermediates, which occur at nearly random positions of the genome. This mechanism may be an alternative to site-specific initiation of packaging proposed by other investigators.     

2B4, the natural killer and T cell immunoglobulin superfamily surface protein, is a ligand for CD48

2B4 is a cell surface glycoprotein related to CD2 and implicated in the regulation of natural killer and T lymphocyte function. A recombinant protein containing the extracellular region of mouse (m)2B4 attached to avidin-coated fluorescent beads bound to rodent cells, and binding was completely blocked by CD48 monoclonal antibodies (mAbs). Using surface plasmon resonance, we showed that purified soluble mCD48 bound m2B4 with a six- to ninefold higher affinity (Kd approximately 16 microM at 37 degreesC) than its other ligand, CD2. Human CD48 bound human 2B4 with a similar affinity (Kd approximately 8 microM). The finding of an additional ligand for CD48 provides an explanation for distinct functional effects observed on perturbing CD2 and CD48 with mAbs or by genetic manipulation.     

Expression of gross cystic disease fluid protein-15/Prolactin-inducible protein in rat salivary glands

Gross cystic disease fluid protein-15 (GCDFP-15)/prolactin-inducible protein (PIP) is present at moderate levels in human submandibular and sublingual glands and is barely detectable in human parotid gland. The rodent homologue, PIP, has previously been identified in adult submandibular and lacrimal glands. Here we present the molecular characterization of rat PIP and show that this protein is a product of neonatal and adult rat submandibular, sublingual, and parotid glands. cDNA clones encoding rat PIP were isolated and sequenced. The deduced amino acid sequence of rat PIP shows 56% overall identity and 80% similarity with mouse PIP. By SDS-PAGE, secreted rat PIP has an apparent Mr of 17,000, with a minor proportion present as Mr 20-22,000 N-glycosylated forms. PIP was localized in rat salivary glands by immunogold silver staining. PIP was identified in acinar cells of developing and mature submandibular and parotid glands and at very low levels in sublingual gland serous demilunes. Typically, rat submandibular gland secretory proteins are produced by either acinar cell progenitors (Type III cells) or mature acinar cells. The expression pattern observed for PIP is similar to that previously reported for salivary peroxidase, an important component of nonimmune mucosal defense.     

Dissociation of the CD4 and CXCR4 binding properties of human immunodeficiency virus type 1 gp120 by deletion of the first putative alpha-helical conserved structure

To evaluate conserved structures of the surface gp120 subunit (SU) of the human immunodeficiency virus type 1 (HIV-1) envelope in gp120-cell interactions, we designed and produced an HIV-1 IIIB (HXB2R) gp120 carrying a deletion of amino acids E61 to S85. This sequence corresponds to a highly conserved predicted amphipathic alpha-helical structure located in the gp120 C1 region. The resultant soluble mutant with a deleted alpha helix 1 (gp120 DeltaalphaHX1) exhibited a strong interaction with CXCR4, although CD4 binding was undetectable. The former interaction was specific since it inhibited the binding of the anti-CXCR4 monoclonal antibody (12G5), as well as SDF1alpha, the natural ligand of CXCR4. Additionally, the mutant gp120 was able to bind to CXCR4(+)/CD4(-) cells but not to CXCR4(-)/CD4(-) cells. Although efficiently expressed on cell surface, HIV envelope harboring the deleted gp120 DeltaalphaHX1 associated with wild-type transmembrane gp41 was unable to induce cell-to-cell fusion with HeLa CD4(+) cells. Nevertheless, the soluble gp120 DeltaalphaHX1 efficiently inhibited a single round of HIV-1 LAI infection in HeLa P4 cells, with a 50% inhibitory concentration of 100 nM. Our data demonstrate that interaction with the CXCR4 coreceptor was maintained in a SUgp120 HIV envelope lacking alphaHX1. Moreover, in the absence of CD4 binding, the interaction of gp120 DeltaalphaHX1 with CXCR4 was sufficient to inhibit HIV-1 infection.     

Tumor immunity and autoimmunity induced by immunization with homologous DNA

The immune system can recognize self antigens expressed by cancer cells. Differentiation antigens are prototypes of these self antigens, being expressed by cancer cells and their normal cell counterparts. The tyrosinase family proteins are well characterized differentiation antigens recognized by antibodies and T cells of patients with melanoma. However, immune tolerance may prevent immunity directed against these antigens. Immunity to the brown locus protein, gp75/ tyrosinase-related protein-1, was investigated in a syngeneic mouse model. C57BL/6 mice, which are tolerant to gp75, generated autoantibodies against gp75 after immunization with DNA encoding human gp75 but not syngeneic mouse gp75. Priming with human gp75 DNA broke tolerance to mouse gp75. Immunity against mouse gp75 provided significant tumor protection. Manifestations of autoimmunity were observed, characterized by coat depigmentation. Rejection of tumor challenge required CD4(+) and NK1.1(+) cells and Fc receptor gamma-chain, but depigmentation did not require these components. Thus, immunization with homologous DNA broke tolerance against mouse gp75, possibly by providing help from CD4(+) T cells. Mechanisms required for tumor protection were not necessary for autoimmunity, demonstrating that tumor immunity can be uncoupled from autoimmune manifestations.     

The role of complement and gp120-specific antibodies in virus lysis and CD4+ T cell depletion in HIV-1-infected patients

The substantial virus lysis was induced by HIV-1-infected patient serum and normal human complement serum in the presence of purified patient IgG. Non-infected CD4+ T cells coated with the whole virus or with a recombinant HIV-1 envelope gp120 and sensitised with patient IgG were also shown to be susceptible to complement-dependent lysis. The serum level of complement regulatory protein in a fluid phase, the C1-esterase inhibitor, was significantly correlated with serum concentration of C1q-circulating immune complexes (P=0.0062), but inversely with CD4+ T cell count (P < 0.0001). Accordingly, the disease progression in HIV-1-infected patients was significantly correlated with the level of complement activation as determined by serum level of C1-esterase inhibitor (P=0.0001), and inversely correlated with CD4+ cell count (P < 0. 0001) and gp120-specific antibody titre (P=0.0086). These results strongly suggest that the complement activation by gp120-specific antibodies play a very important role in virus clearance, but also in depletion of infected as well as gp120-coated non-infected CD4+ bystander T cells during the course of HIV-1 infection.