[Effect of regulator protein Nef on the viral activity of human immunodeficiency virus]. / Effet de la protéine régulatrice Nef sur le pouvoir infectieux du virus de l'immunodéficience humaine.

Despite 25 years of research on human immunodeficiency virus (HIV), the functions of some viral proteins are not fully understood. The role of Nef in the evolution of HIV-1 infection towards immunodeficiency is undeniable; however, the mechanisms involved in this function of Nef remain elusive. The interaction of Nef with a large number of cellular partners disrupts the metabolism of infected cells, including the endocytic pathway, in favor of viral spread. Down-regulation of cell-surface major histocompatibility complex (MHC) molecules by Nef enables infected cells to escape immune surveillance. Nef-induced down-regulation of CD4 increases the infectivity of virions by increasing the incorporation of the envelope glycoproteins into the viral membrane. In addition, Nef increases viral infectivity through a mechanism that is independent of MHC and CD4 down-regulation that nonetheless requires the ability of Nef to interfere with the endocytic process. Overall, these properties promote viral spread in the infected host.

Mapping the CD4 binding domain of gp17, a glycoprotein secreted from seminal vesicles and breast carcinomas.

gp17, a secretory CD4-binding factor isolated from the human seminal plasma, is identical to the gross cystic disease fluid protein-15, a specific marker for primary and metastatic breast tumors. We previously demonstrated that gp17 binds to CD4 with high affinity and strongly inhibits T lymphocyte apoptosis induced by sequential cross-linking of CD4 and T cell receptor (TCR). To further characterize the gp17/CD4 interaction and map the gp17 binding site, we produced a secreted form of recombinant gp17 fused to human IgG1 Fc, gp17-Ig. We showed that gp17-Ig exhibits a binding affinity for CD4 similar to that of native gp17. As no information about gp17 structure is presently available, 99 overlapping gp17 peptides were synthesized by the Spot method, which allowed the mapping of two CD4 binding regions. Alanine scanning of CD4-reactive peptides identified critical residues, selected for site-directed mutagenesis. Nine gp17-Ig mutants were generated and characterized. Three residues within the carboxy-terminal region were identified as the major binding domain to CD4. The Spot method combined with mutagenesis represents a refined approach to distinguish the contact residues from the ones contributing to the conformation of the CD4-binding domain.

Abnormal restriction pattern of PIP gene associated with human primary prostate cancers.

The PIP gene, localized in the 7q34 region that contains a number of fragile sites such as FRA 7H and FRA TI, codes for gp17/PIP, a protein secreted by breast apocrine tumors. We analyzed the integrity of this gene in 20 tumors of the urogenital tract. We found rearranged EcoRI fragments in 5 of 15 primary prostate carcinomas. No rearrangement was found in normal prostates derived from five patients undergoing prostatocystectomy during treatment of bladder cancers. By Southern blot hybridization with PIP gene exon-specific probes, the rearrangements were mapped at or near the 3' end of the gene. These abnormalities were found, not only in the neoplastic cells invading the prostatic tissues, but also in seminal vesicles without histologic tumoral features. These data suggest a critical role of the PIP gene or neighboring genes in prostate cancer.

Potent inhibition of CD4/TCR-mediated T cell apoptosis by a CD4-binding glycoprotein secreted from breast tumor and seminal vesicle cells.

We previously isolated a CD4 ligand glycoprotein, gp17, from human seminal plasma; this glycoprotein is identical with gross cystic disease fluid protein-15 (GCDFP-15), a factor specifically secreted from primary and secondary breast tumors. The function of gp17/GCDFP-15 in physiological as well as in pathological conditions has remained elusive thus far. As a follow up to our previous findings that gp17 binds to CD4 with high affinity and interferes with both HIV-1 gp120 binding to CD4 and syncytium formation, we investigated whether gp17 could affect the T lymphocyte apoptosis induced by a separate ligation of CD4 and TCR. We show here that gp17/GCDFP-15 is in fact a strong and specific inhibitor of the T lymphocyte programmed cell death induced by CD4 cross-linking and subsequent TCR activation. The antiapoptotic effect observed in the presence of gp17 correlates with a moderate up-regulation of Bcl-2 expression in treated cells. The presence of gp17 also prevents the down-modulation of Bcl-2 expression in Bcl-2bright CD4+ T cells that is caused by the triggering of apoptosis. Our results suggest that gp17 may represent a new immunomodulatory CD4 binding factor playing a role in host defense against infections and tumors.

Differential antibody reactivity and CD4 binding of the mammary tumor marker protein GCDFP-15 from breast cyst and its counterparts from exocrine epithelia.

Analysis of biopsies from breast cancer patients demonstrated that GCDFP-15 (gross cystic disease fluid protein-15) is a specific immunocytochemical marker of primary and secondary apocrine breast tumors. The protein has an amino acid sequence identical to SABP (secretory actin-binding protein), to PIP (prolactin-inducible protein) and to gp17, a protein isolated from human seminal plasma. The latter was found to bind to CD4, a T-cell co-receptor involved in antigen recognition, thereby inhibiting the ability of the receptor to interact with the HIV-1 envelope protein gp120. We compare here the ability of independently purified GCDFP-15, SABP and gp17 and of recombinant PIP both to cross-react with a panel of monoclonal antibodies (MAbs) raised against GCDFP-15 or gp17, respectively, and to bind to CD4. We show that, although the various factors share the ability to bind to the panel of antibodies used, differences in the pattern of MAb recognition can be demonstrated. By comparing the kinetic constants for binding of GCDFP-5 and gp17 to CD4 by biosensor technology, significant differences in binding affinities were observed between the 2 factors, thus reflecting structural differences. Surface plasmon resonance analysis also showed that anti-GCDFP-15 and anti-gp17 antibodies inhibit the binding of CD4 to GCDFP-15 and gp17, respectively, to different extents. Our data thus indicate that, while the various forms of the protein are encoded by the same cDNA, tissue specificities due to post-translational modifications exist. This information may be relevant for developing more sensitive and accurate tests for the use of GCDFP-15 as a diagnostic mammary tumor marker and, most importantly, raises the possibility that GCDFP-15 may constitute a breast tumor-specific antigen.

A plasmid-mediated CMY-2 beta-lactamase from an Algerian clinical isolate of Salmonella senftenberg.

Multiresistance to antibiotics including beta-lactams, e.g. cefoxitin, was transferred by conjugation to Escherichia coli strain C1a from a clinical isolate of Salmonella senftenberg recovered from stools of an Algerian child. The susceptibility pattern to beta-lactams was similar to the profile mediated by an AmpC-type beta-lactamase. By biochemical analysis, typical AmpC-type enzyme substrate and inhibition profiles were obtained. Finally, an ampC plasmid-encoded beta-lactamase gene was cloned and sequenced. Its deduced amino acid sequence confirmed its identity as a class C beta-lactamase. It showed 99.5% sequence identity with the plasmid-mediated beta-lactamase CMY-2. The differences in the amino acid sequences of the two enzymes were located in the signal peptide.

Molecular basis of T lymphocyte CD4 antigen functions.

The T cell surface antigen CD4 plays a pivotal role in the MHC class II-restricted response of specific T lymphocytes and serves as the major receptor of human immunodeficiency viruses (HIV). Recent studies have shown the high complexity of CD4 functions in physiological and pathological conditions. We report here a short review of recent developments in the field and discuss the structural features which regulate the functions mediated by the CD4 coreceptor in mature T lymphocytes.