Mourad Bendjennat

UV or g irradiation mediated DNA damage activates p53 and induces cell cycle arrest. Induction of cyclin dependent kinase inhibitor p21WAF1 by p53 after DNA damage plays an important role in cell cycle arrest after gamma irradiation. The p53 mediated cell cycle arrest has been postulated to allow cells to repair the DNA damage. Repair of UV damaged DNA occurs primarily by the nucleotide excision pathway (NER). It is known that p21WAF1 binds PCNA and inhibits PCNA function in DNA replication. PCNA is also required for repair by NER but there have been conflicting reports on whether p21WAF1 can inhibit PCNA function in NER. It has therefore been difficult to integrate the UV induced cell cycle arrest by p21 in the context of repair of UV damaged DNA. A recent study reported that p21WAF1 protein is degraded after low but not high doses of UV irradiation, that cell cycle arrest after UV is p21 independent, and that at low dose UV irradiation p21WAF1 degradation is essential for optimal DNA repair. These findings shed new light on the role of p21 in the cellular response to UV and clarify some outstanding issues concerning p21WAF1 function.

Recently, we reported the purification to homogeneity and characterization of Ca(2+)- and Mg(2+)-dependent endonuclease P40 produced by Mycoplasma penetrans (M. Bendjennat, A. Blanchard, M. Loutfi, L. Montagnier, and E. Bahraoui, J. Bacteriol. 179; 2210-2220, 1997), a mycoplasma which was isolated for the first time from the urine of human immunodeficiency virus-infected patients. To evaluate how this nuclease could interact with host cells, we tested its effect on CEM and Molt-4 lymphocytic cell lines and on peripheral blood mononuclear cells. We observed that 10(-7) to 10(-9) M P40 is able to mediate a cytotoxic effect. We found that 100% of cells were killed after 24 h of incubation with 10(-7) M P40 while only 40% cytotoxicity was obtained after 72 h of incubation with 10(-9) M P40. Phase-contrast microscopy observations of P40-treated cells revealed morphological changes, including pronounced blebbing of the plasma membrane and cytoplasmic shrinkage characteristic of programmed cell death, which is in agreement with the internucleosomal fragmentation of P40-treated cell DNA as shown by agarose gel electrophoresis. We showed that (125)I-radiolabeled or fluorescein isothiocyanate-labeled P40 was able to bind specifically in a dose-dependent manner to the cell membrane of CEM cells, which suggested that the cytotoxicity of P40 endonuclease was mediated by its interaction with the cell surface receptor(s). The concentration of unlabeled P40 required to inhibit by 50% the formation of (125)I-P40-CEM complexes was about 3 x 10(-9) M, indicating a high-affinity interaction. Both P40 interaction and cytotoxicity are Ca(2+) dependent. Our results suggest that the cytotoxicity of M. penetrans observed in vitro is mediated at least partially by secreted P40, which, after interaction with host cells, can induce an apoptosis-like death. These results strongly suggest a major role of mycoplasmal nucleases as potential pathogenic determinants.

HIV virions assemble on the plasma membrane and bud out of infected cells using interactions with endosomal sorting complexes required for transport (ESCRTs). HIV protease activation is essential for maturation and infectivity of progeny virions, however, the precise timing of protease activation and its relationship to budding has not been well defined. We show that compromised interactions with ESCRTs result in delayed budding of virions from host cells. Specifically, we show that Gag mutants with compromised interactions with ALIX and Tsg101, two early ESCRT factors, have an average budding delay of ~75 minutes and ~10 hours, respectively. Virions with inactive proteases incorporated the full Gag-Pol and had ~60 minutes delay in budding. We demonstrate that during budding delay, activated proteases release critical HIV enzymes back to host cytosol leading to production of non-infectious progeny virions. To explain the molecular mechanism of the observed budding delay, we modulated the Pol size artificially and show that virion release delays are size-dependent and also show size-dependency in requirements for Tsg101 and ALIX. We highlight the sensitivity of HIV to budding "on-time" and suggest that budding delay is a potent mechanism for inhibition of infectious retroviral release.