Maxime Moulard

Cytotoxic effect on lymphocytes of Tat from human immunodeficiency virus (HIV-1)

The human immunodeficiency virus type 1 (HIV‐1) genome codes for trans‐activator Tat, an 86‐residue protein whose expression is critical for viral replication. Full‐length Tat and Tat peptides from HIV‐1 were chemically synthesized using optimized solid phase technique. Synthetic Tat 2 in86, was found not only to inhibit antigen‐induced human peripheral blood lymphocyte (PBL) proliferation in vitro, as described by Viscidi et al. [1989, Science 246, 1606‐1608], but also mitogen‐induced PBL proliferation, with 50% inhibition obtained at 0.9 and 8 μ;M, respectively. To assess the mechanism by which Tat exert its inhibitory effect, we analysed its interaction and effect on CD4+‐cells. Direct fluorescence and indirect immunofluorescence assays analysed by flow cytometry showed that fluorescein isothiocyanate‐labeled and ‐unlabeled Tat interact (>0.2 μ;M) with CD4‐expressing lymphoid cells (CEM cell line). Experiments of chromium‐51 release and Trypan blue exclusion on these tumor cells in vitro have demonstrated the capacity of Tat to modify cellular membrane permeability and cell viability, in a dose‐dependent manner. The use of Tat peptides revealed that those containing the Tat basic region from 49 to 57 were able to bind to the cell membrane and to exhibit a cytotoxic activity on lymphocytes. Together, the data suggest that the potential cytotoxicity of Tat on lymphocytes could be directly implicated in virus‐induced immune dysfunction observed in HIV‐1 infected patients.

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.

Crosslinking of Dam methyltransferase with S-adenosyl-methionine

Highly purified DNA‐adenine methyltransferase was irradiated in the presence of different concentrations of radiolabelled S‐adenosyl‐methionine (AdoMet) with a conventional Mineralight UV‐lamp from several minutes up to 1 h while incubating in ice. Incorporation of radioactivity was monitored by electrophoresis of the crosslink between S‐adenosyl‐methionine and Dam methylase on SDS‐polyacrylamide gels followed by fluorography. Crosslinking reached a maximum in presence of 10 μM S‐adenosyl‐methionine; it was inhibited in the presence of substates which competitively inhibit methylation of DNA by Dam methylase, like sinefungin or S‐adenosyl‐homocysteine, but not in the presence of non‐inhibitors like ATP or S‐isobutyl‐adenosine. The crosslink obtained was resistant against a wide range of even drastic conditions commonly used in protein and peptide chemistry. Proteins which do not bind S‐adenosyl‐methionine, as well as heat activated Dam methylase were not photolabelled. After limited proteolysis the radioactive label appeared only in certain of the peptides obtained. From Western blots carried out with polyclonal antibodies produced against a synthetic peptide corresponding in its sequence to amino acids 92‐106 of the Dam methylase, the crosslinking of AdoMet could be tentatively mapped at a position after amino acid 106.

Liposomal encapsulation enhances antiviral efficacy of SPC3 against human immunodeficiency virus type-1 infection in human lymphocytes

Because encapsulation of antiviral drugs in liposomes resulted generally in improved activity against retroviral replication in vivo, the antiviral effects of free-SPC3 and liposome-associated SPC3 were compared in cultured human lymphocytes infected with HIV-1. SPC3 was entrapped in various liposomal formulations, either different in size (mean diameter of 100 and 250 nm), SPC3 concentration or cholesterol content. Liposome-associated SPC3 were tested for both inhibition of cell-cell fusion and infection with HIV-1 clones. SPC3 inhibited HIV-1-induced fusion at a micromolar concentration range. When associated with liposomes, SPC3 was found to be about 10-fold more potent than free SPC3 in inhibiting syncytium formation. Continuous treatment with free SPC3 also inhibited virus production in a dose-dependent manner, with inhibition of HIV infection of C8166 T-cells or human peripheral blood lymphocytes (PBLs) at micromolar concentrations. Liposomal entrapment was found to increase the antiviral efficacy of SPC3 by more than 10- and 5-fold in C8166 and PBLs, respectively. These data suggest that the liposome approach may be used to improve SPC3 antiviral efficacy.

Further studies on the human pancreatic binary complexes involving procarboxypeptidase A.

In contrast to procarboxypeptidase B which has always been reported to be secreted by the pancreas as a monomer, procarboxypeptidase A occurs as a monomer and/or associated to one or two functionally different proteins, depending on the species. Recent studies showed that, in the human pancreatic secretion, procarboxypeptidase A is mainly secreted as a 44 kDa protein involved in at least three different binary complexes. As previously reported, two of these complexes associated procarboxypeptidase A to either a glycosylated truncated protease E or zymogen E. In this paper, we identified proelastase 2 as the partner of procarboxypeptidase A in the third complex, thus reporting for the first time the occurrence of a proelastase 2/procarboxypeptidase A binary complex in vertebrates. Moreover, from N‐terminal sequence analyses, the 44 kDa procarboxypeptidase A involved in these complexes was identified as being of the A1 type. Only one type of procarboxypeptidase B, the B1 type, has been detected in the analyzed pancreatic juices, thus emphasizing the previously observed genetic differences between individuals.

Identification of a procarboxypeptidase A-truncated protease E binary complex in human pancreatic juice

The characterization, in human pancreatic juice, of a binary complex associating procarboxypeptidase A with a 32 kDa inactive glycoprotein (G32) is reported in this paper. Free G32 was isolated after dissociation of the binary complex. N‐terminal sequence analysis revealed a complete homology between this protein and human protease E (HPE 1), except for the two strongly hydrophobic N‐terminal residues (Val‐Val) which are missing in G32. This protein might be a truncated protease E highly analogous to the subunit III of the ruminant procarboxypeptidase A‐S6 ternary complex. The analogy with bovine subunit III is further supported by interspecies reassociation experiments showing that bovine procarboxypeptidase A can specifically bind human G32.

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.

A multiple branch peptide construction derived from a conserved sequence of the envelope glycoprotein gp41 inhibits human immunodeficiency virus infection

We have recently reported some anti-HIV properties of SPC3 [1-3], a gp120 V3 loopderived synthetic polymer construction [4, 5] which is currently under phase II clinical trials in HIV-1-infected humans. Due to the importance of both gp120 and gp41 envelope glycoproteins in the virus-cell and cell-cell fusion processes [6], we have chemically synthesized a series of other multiple branch peptides (SPCs) derived from the conserved sequences of gp41 [7, unpublished results]. The gp41-derived SPCs have been tested in vitro for their ability to inhibit i) syncytium formation induced either by a recombinant vaccinia virus-expressing HIVLai Env or various HIV strains, and ii) infection of human peripheral blood lymphocytes by laboratory strains and clinical isolates of both HIV-1 and HIV-2.