A novel human actin-binding protein homologue that binds to platelet glycoprotein Ibalpha

Glycoprotein (GP)Ib-IX-V is one of the major transmembrane complexes present on the platelet surface. Its extracellular domain binds von Willebrand factor (vWF) and thrombin, while its intracellular domain associates tightly with the cytoskeleton through the actin-binding protein (ABP)-280, also known as filamin. In the present study, a full-length cDNA coding for a human ABP homologue has been cloned and sequenced. This protein was identified by the yeast two-hybrid screening procedure via its interaction with the intracellular domain of GPIbalpha. Initially, a 1.3-kb partial cDNA was isolated from a megakaryocyte-like cell line (K562) cDNA library followed by a full-length cDNA of 9.4 kb that was identified in a human placenta library. The full-length cDNA encoded a protein of 2,578 amino acids with a calculated molecular weight of 276 kD (ABP-276). The amino terminal 248 amino acids contained an apparent actin binding domain followed by 24 tandem repeats each containing about 96 amino acids. The amino acid sequence of the protein shared a high degree of homology with human endothelial ABP-280 (70% identity) and chicken filamin (83% identity). However, the 32 amino acid Hinge I region in ABP-280 that contains a calpain cleavage site conferring flexibility on the molecule, was absent in the homologue. An isoform containing a 24 amino acid insertion with a unique sequence at the missing Hinge I region was also identified (ABP-278). This isoform resulted from alternative RNA splicing. ABP-276 and/or ABP-278 were present in all tissues examined, but the relative amount varied in that some tissue contained both forms, while other tissue contained predominately one or the other.     

Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein

A human immunodeficiency virus type 1 (HIV-1) mutant lacking the V1 and V2 variable loops in the gp120 exterior envelope glycoprotein replicated in Jurkat lymphocytes with only modest delays compared with the wild-type virus. Revertants that replicated with wild-type efficiency rapidly emerged and contained only a few amino acid changes in the envelope glycoproteins compared with the parent virus. Both the parent and revertant viruses exhibited increased sensitivity to neutralization by antibodies directed against the V3 loop or a CD4-induced epitope on gp120 but not by soluble CD4 or an antibody against the CD4 binding site. This result demonstrates the role of the gp120 V1 and V2 loops in protecting HIV-1 from some subsets of neutralizing antibodies.     

HIV-1 envelope protein gp120 triggers a Th2 response in mice that shifts to Th1 in the presence of human growth hormone

Immunization of mice with HIV-1-gp120 results in predominant activation of the Th2 lymphocyte subset, leading to enhanced IL-4 production. Administration of human growth hormone at the time of gp120 immunization provokes a change in the cytokine production pattern, with lower IL-4 and higher gamma-IFN and IL-2 synthesis levels, indicating a preferential switch in stimulation from Th2 to Th1 cells. A growth hormone would thus be of great use for pharmacological intervention in those cases in which an infectious microorganism evades immune defenses by provoking a Th2 response. In addition, the ability of growth hormone to induce a Th1-type response upon vaccination with an HIV-antigen should be examined in the development of new therapeutic strategies or in the design of novel vaccines against HIV infection.     

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody

The entry of human immunodeficiency virus (HIV) into cells requires the sequential interaction of the viral exterior envelope glycoprotein, gp120, with the CD4 glycoprotein and a chemokine receptor on the cell surface. These interactions initiate a fusion of the viral and cellular membranes. Although gp120 can elicit virus-neutralizing antibodies, HIV eludes the immune system. We have solved the X-ray crystal structure at 2.5 A resolution of an HIV-1 gp120 core complexed with a two-domain fragment of human CD4 and an antigen-binding fragment of a neutralizing antibody that blocks chemokine-receptor binding. The structure reveals a cavity-laden CD4-gp120 interface, a conserved binding site for the chemokine receptor, evidence for a conformational change upon CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for immune evasion. Our results provide a framework for understanding the complex biology of HIV entry into cells and should guide efforts to intervene.     

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.     

Three-dimensional structure-activity analysis of a series of porphyrin derivatives with anti-HIV-1 activity targeted to the V3 loop of the gp120 envelope glycoprotein of the human immunodeficiency virus type 1

Using comparative molecular field analysis (CoMFA), a 3D-QSAR model was developed for 21 porphyrin derivatives which have anti-HIV-1 activity and bind to the V3 loop of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1. A significant PLS cross-validated r2cv (0.590) was obtained, indicating that the model could be used as a predictive tool for further design of porphyrin analogs. The model revealed at least three important sites for favorable electrostatic interactions and indicated favorable and unfavorable steric interaction sites. It was found that the occurrence of at least three positively charged and several hydrophobic amino acid residues is highly conserved at fixed positions of gp120 V3 loop sequences. This may support the validity of the proposed model and the hypothesis that porphyrins containing anionic and hydrophobic groups may interact with some of the highly conserved positively charged and hydrophobic sites, respectively, of the V3 loop. These interactions may induce conformational changes in the gp120 envelope glycoprotein leading to inhibition of virus entry into cells and of syncytium formation (cell-to-cell fusion) and thus to inhibition of virus replication.     

Demonstration of peripheral fucose units in N-linked glycans of human immunodeficiency virus type 1 gp 120: effects on glycoprotein conformation

Fucosylated N-linked glycans are important constituents of membrane glycoproteins, owing to their significance as biologically active ligands for several selectins and their role in modulating protein conformation of viral glycoproteins. The human immunodeficiency virus type 1 (HIV-1) glycoprotein contains more than 30 different glycan structures but so far fucose was found associated solely with the innermost GlcNAc of N-linked glycans. In the present report we determined whether fucose units also were linked to the distal GlcNAc via alpha(1-3) or alpha(1-4) linkages in N-linked glycans of gp 120. [3H]-fucose labelled gp 120 was subjected to endoglycosidase F digestion, releasing diantennary complex type N-linked glycans, but leaving the inner polypeptide-bound carbohydrates, GlcNAc and possibly associated fucose units, intact. Gel filtration of the digested material revealed that [3H]-fucose label was released from gp 120 by this treatment, indicating presence of peripheral fucose units. Furthermore, [3H]-focuse label was also released by treatment of the labelled gp 120 with an alpha-L-fucosidase specifically removing fucose in alpha(1-3) and alpha(1-4) linkages. Altogether the results indicated presence of fucose units linked to peripheral GlcNAc of gp 120 N-linked glycans. We have earlier shown that other peripheral carbohydrate determinants, i.e. beta(1-4)-galactose on N-linked glycans, maintain a correct antigenic conformation of gp 120. Using a coupled ELISA system, where changes in antigenic behaviour of a viral glycoprotein were correlated to stepwise elimination of peripheral monosaccharides from N-linked glycans, we found that treatment of gp 120 with a pan-specific alpha-fucosidase as well as an enzyme specific for alpha(1-3)- or alpha(1-4)-linked fucose disclosed a hidden linear epitope situated in the gp 120 C2 region. The effects of the general fucosidase on epitope exposure was more prominent than those obtained with the enzyme with narrow specificity, suggesting that peripheral and inner fucose units co-operate in the maintenance of gp 120 conformation.     

Envelope glycoprotein gp120 of human immunodeficiency virus type 1 alters ion transport in astrocytes: implications for AIDS dementia complex

Infection by human immunodeficiency virus type 1 (HIV-1) is often complicated by a variety of neurological abnormalities. The most common clinical syndrome, termed acquired immunodeficiency syndrome (AIDS) dementia complex, presents as a subcortical dementia with cognitive, motor, and behavioral disturbances and is unique to HIV-1 infection. The pathogenesis of this syndrome is poorly understood but is believed to involve interactions among virally infected macrophages/microglia, astrocytes, and neurons. In this study, we show that exposure of primary rat and human astrocytes to heat-activated HIV-1 virions, or to eukaryotically expressed HIV-1 and HIV-2 envelope glycoproteins (gp120) stimulates amiloride-sensitive Na+/H+ antiport, potassium conductance, and glutamate efflux. These effects are blocked specifically by amiloride, an inhibitor of Na+/H+ antiport and by the selective removal of gp120 with immobilized monoclonal antibody. As a result of modulation of astrocytic function by gp120, the ensuing neuronal depolarization and glutamate exposure could activate both voltage-gated and N-methyl-D-aspartate-regulated Ca2+ channels, leading to increases in intraneuronal Ca2+ and neuronal death. These findings implicate the astrocyte directly in the pathogenesis of AIDS dementia complex.     

Conserved N-linked oligosaccharides of the C-terminal portion of human immunodeficiency virus type 1 gp120 and viral susceptibility to neutralizing antibodies

We have constructed a mutated infectious HIV variant lacking the signals for addition of three N-linked glycans situated in the V4, C4 and V5 regions of HIV gp120. When comparing mutated virus with wildtype virus we found essentially no differences in the phenotypic characteristics of the two viruses except for the expected electrophoretic mobility shift of radioimmuno-precipitated mutated gp120, resulting from the missing N-glycans. Thus, the infectivity titer and the capacity to induce syncytia were similar for the two viruses. The sensitivity of mutant and wildtype virus to a number of neutralizing agents was determined. As expected, the mutant virus was significantly less sensitive to neutralization by Con A, with affinity for the N-glycans eliminated. We found, however, that antibodies to the V3 loop and sCD4 neutralized wild-type virus as efficiently as mutant virus, whereas 2G12, a monoclonal antibody, binding to a discontinuous neutralization epitope, and GP13, binding to the CD4-binding domain, neutralized wildtype virus better than mutant virus. Altogether the data suggest that the three conserved N-linked glycans, despite their location in immediate association with the CD4-binding domain, which is an important neutralization epitope, are not essential for virus replication in cell culture and they are not engaged in shielding neutralization epitopes of gp120 from neutralizing antibodies. However, the glycans evidently influence the three-dimensional conformation of gp120, since their presence increases the availability of the neutralization epitope of 2G12.     

Identification of determinants on a dualtropic human immunodeficiency virus type 1 envelope glycoprotein that confer usage of CXCR4

The chemokine receptors CCR5 and CXCR4, in combination with CD4, mediate cellular entry of macrophage-tropic (M-tropic) and T-cell-tropic strains of human immunodeficiency virus type 1 (HIV-1), respectively, while dualtropic viruses can use either receptor. We have constructed a panel of chimeric viruses and envelope glycoproteins in which various domains of the dualtropic HIV-1(DH12) gp160 were introduced into the genetic background of an M-tropic HIV-1 isolate, HIV-1(AD8). These constructs were employed in cell fusion and virus infectivity assays using peripheral blood mononuclear cells, MT4 T cells, primary monocyte-derived macrophages, or HOS-CD4 cell lines, expressing various chemokine receptors, to assess the contributions of different gp120 subdomains in coreceptor usage and cellular tropism. As expected, the dualtropic HIV-1(DH12) gp120 utilized either CCR3, CCR5, or CXCR4, whereas HIV-1(AD8) gp120 was able to use only CCR3 or CCR5. We found that either the V1/V2 or the V3 region of HIV-1(DH12) gp120 individually conferred on HIV-1(AD8) the ability to use CXCR4, while the combination of both the V1/V2 and V3 regions increased the efficiency of CXCR4 use. In addition, while the V4 or the V5 region of HIV-1(DH12) gp120 failed to confer the capacity to utilize CXCR4 on HIV-1(AD8), these regions were required in conjunction with regions V1 to V3 of HIV-1(DH12) gp120 for efficient utilization of CXCR4. Comparison of virus infectivity analyses with various cell types and cell fusion assays revealed assay-dependent discrepancies and indicated that events occurring at the cell surface during infection are complex and cannot always be predicted by any one assay.     

Proposed atomic structure of a truncated human immunodeficiency virus glycoprotein gp120 derived by molecular modeling: target CD4 recognition and docking mechanism

The atomic structure of a truncated glycoprotein gp120 from human immunodeficiency virus 1 (HIV-1) that contains the principal neutralizing antigenic sites and the CD4 binding domain has been derived by molecular dynamics and calculation of potential energy using the DREIDING force field. The resultant N-glycosylated molecular model is consistent with known properties of gp120 and docks with CD4 with a substantial reduction in the sum of the internal potential energies of the individual proteins (delta E = -200 kcal/mol). The primary mechanism of recognition and binding is the insertion of the solvent-accessible Phe-43 of CD4 into a gp120 solvent-accessible acceptor pit formed by Trp-427, Tyr-435, and the high-mannose oligosaccharide N-linked to Asn-230. delta E for the nonglycosylated complex is reduced significantly (-75 kcal/mol). Binding is by pi-pi* interactions of the aromatic groups forming a hydrophobic, thermodynamically stable environment for these functional noncovalent bonding participants. This model for gp120 provides a theoretical basis for the evaluation of HIV molecular pathogenesis involving the env proteins, the analysis of conformation on functional immune response of the host, and the design of nonproteinaceous inhibitors specific for the CD4 binding site on gp120.     

Micro-sequencing strategies for the human A33 antigen, a novel surface glycoprotein of human gastrointestinal epithelium

At the end of the XIXth Century the attitude towards malaria changed dramatically from fatalism and resignation to an active policy that made the eradication of the disease a possible objective. This dramatic change in the scientific political and cultural attitudes towards malaria was the result of two main phenomena: i) the impact of the scientific medicine and Pasteurian revolution on medicine and health policies, and ii) the discovery of the theoretical simplicity of the cycle of malaria transmission and of the possibility to interrupt it, by avoiding the contacts between people and the Anopheles mosquitoes. However, scientifically based strategies against malaria were in place before the discovery of the real causative agents and of the transmission cycle at the end of the XIXth century, as the origin of the scientific medicine had already produced a 'rationale' for local and national campaigns against malaria. According to Tommasi-Crudeli, for example, the cause of malaria was not a 'chemical compound', a 'miasma', but a 'living ferment', specific and autonomous. As a consequence, the aim of antimalarial measures was to eliminate the conditions indispensable to the multiplication of the specific ferment contained in the soil. The theory of malaria aetiology changed after the discovery of the transmission cycle by Ross and Grassi, but the general strategy remained the same: to eliminate one of the factors indispensable to the multiplication and diffusion of the agent. The detailed knowledge of the malaria transmission cycle made it possible to define the exact conditions which were alone responsible for the propagation of the disease and its persistence in the endemic areas. The theoretical linearity and the specificity of the 'Grassi's law' was decisive and produced a fundamental paradigmatic shift in the antimalarial policies. The essential point for the epidemiology and prophylaxis of malaria became to clarify the conditions which contribute to facilitate or to prevent the infection of the Anopheles.     

Gads is a novel SH2 and SH3 domain-containing adaptor protein that binds to tyrosine-phosphorylated Shc

Shc proteins are important substrates of receptor and cytoplasmic tyrosine kinases that couple activated receptors to downstream signaling enzymes. Phosphorylation of Shc tyrosine residues 239 and 317 leads to recruitment of the Grb2-Sos complex, thus linking Shc phosphorylation to Ras activation. We have used phosphorylated peptides corresponding to the regions spanning tyrosine 239/240 and 317 of Shc in an expression library screen to identify additional downstream targets of Shc. Here we report the identification of Gads, a novel adaptor protein most similar to Grb2 and Grap that contains amino and carboxy terminal SH3 domains flanking a central SH2 domain and a 120 amino acid unique region. Gads is most highly expressed in the thymus and spleen of adult animals and in human leukemic cell lines. The binding specificity of the Gads SH2 domain is similar to Grb2 and mediates the interaction of Gads with Shc, Bcr-Abl and c-kit. Gads does not interact with Sos, Cbl or Sam68, although the isolated carboxy terminal Gads SH3 domain is able to bind these molecules in vitro. Our results suggest that the unique structure of Gads regulates its interaction with downstream SH3 domain-binding proteins and that Gads may function to couple tyrosine-phosphorylated proteins such as Shc, Bcr-Abl and activated receptor tyrosine kinases to downstream effectors distinct from Sos and Ras.     

Production of uninfectious human immunodeficiency virus type 1 containing viral protein R fused to a single-chain antibody against viral integrase

A single-chain antibody (scAb) against human immunodeficiency virus type 1 (HIV-1) integrase was expressed as a fusion protein of scAb and HIV-1 viral protein R (Vpr), together with the HIV-1 genome, in human 293T cells. The expression did not affect virion production much but markedly reduced the infectivity of progeny virions. The fusion protein was found to be incorporated into the virions. The incorporation appears to account for the reduced infectivity.     

Induction of interleukin-12 (IL-12) by recombinant glycoprotein gp120 of human immunodeficiency virus type 1 in human monocytes/macrophages: requirement of gamma interferon for IL-12 secretion

We studied the effects of the gp120 glycoprotein of human immunodeficiency virus type 1 on the expression of interleukin-12 (IL-12) in human monocytes and in monocyte-derived macrophages. Induction of the mRNA for both the p35 and p40 subunits of IL-12 was observed in both cell types after gp120 treatment. We then evaluated cytokine secretion by using an enzyme-linked immunosorbent assay which recognizes only the IL-12 heterodimer. No IL-12 was detected in monocytes/macrophages treated with gp120 alone. A consistent IL-12 secretion was found in macrophages primed with gamma interferon (IFN-gamma) and subsequently treated with gp120. Low levels of IL-12 were occasionally observed in IFN-gamma-primed monocytes stimulated with gp120. The greater response of macrophages than of monocytes to the priming effect of IFN-gamma was consistent with the finding that IFN-gamma induced a much stronger antiviral state to vesicular stomatitis virus in macrophages than in monocytes. These data indicate that gp120 is an inducer of IL-12 expression in monocytes/macrophages and that IFN-gamma is an essential cofactor for IL-12 secretion, especially in differentiated macrophages.     

A novel antibody-dependent cellular cytotoxicity epitope in gp120 is identified by two monoclonal antibodies isolated from a long-term survivor of human immunodeficiency virus type 1 infection

Two monoclonal antibodies (MAbs), 42F and 43F, were isolated some 14 months apart from a single long-term survivor of human immunodeficiency virus type 1 (HIV-1) infection. These MAbs were found to be indistinguishable in terms of their isotypes, specificities, affinities, and biological activities. Both 42F and 43F directed substantial antibody-dependent cellular cytotoxicity (ADCC) against cells infected with four divergent lab-adapted strains of HIV-1, but no neutralizing activity against these strains was detectable. The ability of MAbs 42F and 43F, as well as that of MAbs against two other gp120 epitopes, to direct ADCC against uninfected CD4+ cells to which recombinant gp120SF2 had been adsorbed (i.e., "innocent bystanders") was demonstrated to be less efficient by at least an order of magnitude than their ability to direct ADCC against HIV-1-infected cells. Flow cytometry analyses showed that 42F and 43F also bind to native primary isolate Envs from clades B and E expressed on cell surfaces. By direct binding and competition assays, it was demonstrated that the 42F/43F epitope lies in a domain of gp120 outside the previously described CD4-binding site and V3 loop ADCC epitope clusters. Immunoblot analysis revealed that the 42F/43F epitope is not dependent on disulfide bonds or N-linked glycans in gp120. Epitope mapping of 42F and 43F by binding to linear peptides demonstrated specificity of these MAbs for a sequence of 10 amino acids in the C5 domain comprising residues 491 to 500 (Los Alamos National Laboratory numbering for the HXB2 strain). Thus, 42F and 43F define a new ADCC epitope in gp120. Because of the relative conservation of this epitope and the fact that it appears to have been significantly immunogenic in the individual from which these MAbs were derived, it may prove to be a useful component of HIV vaccines. Furthermore, these MAbs may be used as tools to probe the potential importance of ADCC as an antiviral activity in HIV-1 infection.     

Simple enzyme immunoassay for titration of antibodies to the CD4-binding site of human immunodeficiency virus type 1 gp120

We report the development of an immunoassay for the titration of antibody to the CD4-binding site (CD4BS) of the human immunodeficiency virus type 1 (HIV-1) surface glycoprotein gp120. This assay is a competitive enzyme-linked immunosorbent assay in which serum antibodies compete with labeled F105, a human monoclonal antibody whose corresponding epitope overlaps the conformation-dependent CD4BS, for binding to purified recombinant gp120 coated on a solid phase. Ninety-nine percent (109 of 110) of HIV-1-positive French patients and 91% (51 of 56) of HIV-1-positive African patients had CD4BS antibodies, indicating that the conformational CD4BS epitope is well conserved among different subtypes of HIV-1. Titers of CD4BS antibodies according to clinical status appeared to be not statistically different. A longitudinal study in 21 seroconverters showed that, for the majority of individuals, CD4BS antibodies appeared early and persisted at relatively high titers for several years. None of 21 HIV-2-seropositive patients had CD4BS antibodies in our assay, suggesting that the antibodies produced during HIV-2 infection are not cross-reactive with the CD4BS of HIV-1 gp120.     

Interactions among HIV gp120, CD4, and CXCR4: dependence on CD4 expression level, gp120 viral origin, conservation of the gp120 COOH- and NH2-termini and V1/V2 and V3 loops, and sensitivity to neutralizing antibodies

The binding of HIV-derived recombinant soluble (s)gp120 to the CD4(+)/CXCR4(+) A3.01 T cell line inhibits the binding of the CXCR4-specific monoclonal antibodies 12G5, which interacts with the second extracellular loop, and 6H8, which binds the NH2 terminus. We have used this as an assay to analyse the interaction of recombinant sgp120 from diverse viral origins with CXCR4. The strength of the interaction between sgp120 and CXCR4 correlated with sgp120 affinity for the CD4-CXCR4 complex, and the interaction of sgp120MN and sgp120IIIB with CXCR4 was highly dependent on the level of CD4 expressed on a variety of different T cell lines. sgp120 from X4, R5X4, and R5 viruses interacted with CXCR4, although the R5 sgp120-CXCR4 interactions were weaker than those of the other gp120s. The interaction of sgp120IIIB or sgp120MN with CXCR4 was inhibited by neutralizing monoclonal antibodies that prevent the sgp120-CD4 interaction but also by antibodies specific for the gp120 V2 and V3 loops, the CD4-induced epitope and the 2G12 epitope, which interfere weakly or not at all with CD4-sgp120 binding. The binding to A3.01 cells of wild-type sgp120HxB2, but not of sgp120 deleted in the COOH and NH2 termini, interfered with 12G5 binding in a dose-dependent manner. Further deletion of the V1 and V2 loops restored CXCR4 binding activity, but additional removal of the V3 loop eliminated the gp120-CXCR4 interaction, without decreasing the affinity between mutated sgp120 and CD4. Taken together, these results demonstrate that the interactions between sgp120 and CXCR4 are globally similar to those previously observed between sgp120 and CCR5, with some apparent differences in the strength of the sgp120-CXCR4 interactions and their dependence on CD4.