Elmostafa Bahraoui

Hiv infection of monocytic cells: rôle of antibody-mediated virus binding to Fc-gamma receptors

We investigated whether human immunoglobulin G (IgG) directed to gp110 may serve as an attachment system to Fc-gamma receptors (Fc-gamma R), allowing eventual infection of cells of the macrophage lineage. An anti-HIV IgG preparation that prevented viral particles and soluble recombinant radiolabelled envelope precursor gp160 from binding to CD4 on CEM lymphoid cells, and that strongly inhibited infection of these cells by HIV, was selected. In contrast, anti-HIV IgG, whether or not previously complexed to viral particles, bound to monocytic U937 cells that express both high Fc-gamma RI and low affinity Fc-gamma RII receptors. Precoating these cells with anti-HIV IgG or complexing the antibodies with soluble 125I-gp160 resulted in increased fixation of gp160 to the cells, which was inhibited by aggregated human normal IgG. These data indicate that anti-HIV IgG-dependent attachment of gp160 to monocytic cells occurs through both types of Fc-gamma R. In addition, this method of attachment resulted in productive infection of U937 cells that, since it was blocked in the presence of Leu3a, still appeared to involve gp110-CD4 interaction. Only slight enhancement of infectivity, such as described for other enveloped viruses, was noted, even when antibody concentration was titrated down. This mechanism may be one of the explanations why the humoral response to HIV is not usually protective.

Effects of calcium ions on proteolytic processing of HIV-1 gp160 precursor and on cell fusion

Complete activation of human immunodeficiency virus type 1 (HIV‐1) requires the endoproteolytic cleavage by cellular protease of the envelope glycoprotein precursor (gp160) into the external glycoprotein gp120, and the transmembrane glycoprotein gp41. We report here the effect of depletion of cellular calcium ions on maturation of precursor gp160 and its concomitant effect on syncytium formation. We show that the cellular endoprotease activity responsible for gp160 maturation and the capacity for HIV‐1 to induce syncytium formation are calcium‐dependent. In addition, we show that endoproteolytic maturation is a key step in syncytium formation induced by HIV‐1.

Specificity of antipeptide antibodies produced against V2 and V3 regions of the external envelope of human immunodeficiency virus type 2

The V2 region of simian immunodeficiency virus (SIV) and V3 region of human immunodeficiency virus type 1 (HIV-1) have been reported to be neutralization epitopes. We analysed the corresponding regions in HIV-2. Synthetic peptides modeling the V2 (aa 149-168) and V3 (CV3: aa 298-315 and NV3: aa 306-324) regions of the HIV-2 external envelope glycoprotein were coupled to KLH and used as immunogens in rabbits. We characterized the resulting antiV2 and antiV3 antibodies for their ability to recognize native and deglycosylated HIV-2 envelope glycoprotein, to block gp-CD4 interaction and to inhibit syncytium formation in vitro. The three synthetic peptides induced antibodies able to recognize specifically the native HIV-2 envelope glycoprotein with a significant avidity (K0.5 between 6 x 10(-7) and 8 x 10(-9) M). Interestingly, the reactivity of antibodies produced against the V2 peptide, which contains two potential sites of N-glycosylation, was higher against the fully deglycosylated than glycosylated HIV-2 external envelope glycoprotein (gp105). The antipeptide antibodies were used to investigate the topography of these regions in the preformed gp-CD4 complex in indirect immunofluorescence assays. The V2 and V3 regions in the complex remained accessible to their respective antibodies. Moreover, preincubation of gp105 with anti V2 or anti V3 antibodies did not prevent gp-CD4 interaction. Thus the V2 and V3 regions are not directly involved in the gp105 binding site for the CD4 receptor. Finally, in contrast with results obtained with antibodies produced against the V3 region of HIV-1 gp120 and monoclonal antibodies produced against the V3 of SIV, antibodies produced against V2 and V3 of HIV-2 were unable to inhibit syncytium formation induced by HIV-2 in vitro.

Infection of monocytic cells by HIV1: combined role of FcR and CD4

Human immunodeficiency virus (HIV) complexed with human anti-HIV IgG can attach to Fc gamma receptors (Fch) of mononuclear phagocytes. To determine whether the FcR-mediated infection that results also requires interaction between HIV gp120 and cell membrane CD4, monocytic cells of the U937 line were transiently treated with phorbol 12,13-dibutyrate (PDB) so that they temporarily presented a CD4-FcR+ phenotype at the time of HIV infection. HIV production was not abolished, but only significantly delayed after infection of these cells with free virus. Leu3a monoclonal antibody or soluble recombinant CD4 completely blocked this delayed infection. This indicates that enough CD4 still remained at the membrane to allow infection of a reduced cell number. Infection of PDB-treated cells with virus preincubated with high anti-HIV IgG concentrations was inhibited, contrasting with what was observed with control cells infected under the same conditions. Inhibition of infection was also observed when HIV became attached to untreated U937 cells through the binding of CD4-IgG hybrid molecules to FcR. Thus, the binding of IgG-coated virus to FcR is not sufficient in itself to elicit productive infection of monocytic cells, which still requires the interaction of viral gp120 and membrane CD4.

Specificity of anti-peptide antibodies elicited against synthetic peptides mimicking conserved regions of HIV1 envelope glycoprotein.

Comparison of HIV1Bru and HIV2Rod external envelope glycoprotein sequences enabled us to select ten highly conserved peptide sequences. The corresponding peptides were chemically synthesized, then coupled to bovine serum albumin before injection in rabbits. Although all peptides were immunogenic, only antibodies directed against peptides P1 (amino acid residues 33-55), P22 (418-462), P8 (487-508) and P21 (487-534) were able to interact with significant affinity (K0.5 about 10(-6) to 10(-8) M) with the native glycoprotein by radioimmunoassay. Noteworthy was the capacity of anti-P1 antibodies to also recognize the glycoprotein of HIV2. Anti-peptide antibodies were tested for their ability to interfere with the gp120-CD4 interaction, membrane fusion and virus replication. Preincubation of gp120 with antibodies directed to the region previously described as the putative CD4-binding site, P22 (418-462), did not abolish gp120 binding to CD4-positive cells.

Evaluation of structure-antigenicity relationship of peptides from human immunodeficiency virus type 1 (HIV-1) p18 protein by circular dichroism

The antigenicity of Human Immunodeficiency Virus type 1 (HIV-1) matrix p18 protein was evaluated by analyzing the specificity of anti-p18 antibodies elicited either in HIV-1 infected humans, or in HIV-1 infected or immunized chimpanzees, against a panel of long and short overlapping synthetic peptides [from 12 to 46 amino acid (aa) residues] covering the entire sequence of p18. The relationship between peptide structure and antigenicity was further investigated by probing the secondary structures of the peptides by circular dichroism. The results obtained clearly showed the immunodominance of the N-terminal region mimicked by peptide P1 (aa 2-45), which reacted with 52 and 100% of human and chimpanzee anti-p18 sera, respectively. In contrast smaller 15 aa long peptides C1, C2, C3, C4 and P3 which cover the entire sequence of immunodominant peptide P1, showed only weak or no reactivity. In contrast to widely accepted hypotheses, circular dichroism analysis of both small and large peptides secondary structures did not show any obvious correlation between antigenicity and the ability of peptides to adopt an ordered conformation.

T4-lymphocyte endoprotease responsible for the proteolytic processing of HIV-1 GP160, like Kex2p endoprotease, is a calcium-dependent enzyme

In the present study we show that precursor gp160 is cleaved in the HIV-1 infected CEM (CD4+) cell line preferentially in the presence of calcium ions demonstrating that the responsible cellular endoprotease is a calcium-dependent enzyme. Taking into account this similarity, a synthetic peptide modelling the cleavage site of HIV-1 envelope glycoprotein precursor was used as substrate for Kex2p. Results obtained clearly showed that the processing enzyme Kex2p (EC 3.4.21.61), a subtilisin-like serine protease that is encoded by the KEX2 gene of yeast Saccharomyces cerevisiae is able to cleave correctly this peptide at the potential cleavage site.