CELLULAR GENES REQUIRED FOR HIV-1 INFECTION AS TARGETS FOR THERAPY

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(1) Goals of project: - To identify cellular genes required for optimal HIV-1 infection, or involved in HIV-1 pathogenesis. - To apply the knowledge gained towards development of anti-viral therapy which target both viral and cellular genes. (2) Experimental approach: A panel of mutants were derived from the human T cell line clone CEM, demonstrating different susceptibilities to HIV-1 infection. Multiple assays were developed to analyze these clones: (a) Quantitative flow cytometry analysis of surface receptors and binding of soluble gp120. (b) Quantitative PCR to measure viral entry and proviral DNA formation. (c) Vaccinia-gp160-vector based syncytia assay. (d) Gel retardation assays to study the levels of DNA binding proteins required for optimal HIV-1 provira reactivation. (e) Protein Kinase C enzymatic activity assay. (f) Chloramphenicol acetyl transferase (CAT) assay to measure activation of the HIV-1 LTR. (3) Major Findings: - No direct correlation exist between the number of surface CD4 receptors and the kinetics of HIV-1 infection. - Two CEM subclones were isolated with dramatically reduced susceptibility to infection by multiple strains of HIV-1. These mutants were found to have a markedly reduced level of DNA binding protein belonging to the NFk-B family (p50). Other DNA binding proteins are normally expressed. The reduce levels of NF-kB proteins does not effect cell growth only HIV-1 susceptibility. These genes could be the target of anti-sense suppression t maintain patients' PBL at a resting state, preventing reactivation of integrated virus.