CELLULAR GENES REQUIRED FOR HIV-1 INFECTION AS TARGETS FOR ANTI-VIRAL 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 study the mechanism by which intrathymic HIV-1 infection affects the development of different T cell subsets. - To understand how HIV-1 infection leads to high levels of programmed cell in both CD4 and CD8 cells. - 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. - Hu/Scid mice were infected intrathymically with primary isolate of HIV. Thymocytes were separated into various subsets by 3 color stainings and the presence of proviral DNA was determined by quantitative PCR. -The role of the negative kinase lsk in fas-mediated apoptosis was determined using a panel of human cell lines lacking lsk or expressing wild type or mutated lsk. (3) Major Findings: - 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). The reduced levels of NF-kB proteins does not effect cell growth only HIV-1 susceptibility. These genes could be the target of anti-sense suppression to maintain patients' PBL at a resting state, preventing reactivation of integrated virus. Publication: J. Qian, V. Bours, J. Manischewitz, and H. Golding. 1994. J. Immunol. 152: 4183-4191. - The Hu/Scid model demonstrated that in the infected thymus, CD8+ mature cells are also infected with the virus. It may occur during the development of CD4+CD8+ infected cell into single positive CD4 and CD8 cells. - The lsk kinase was found to have strong inhibitory effect on FAS-mediated apoptosis. It is postuLated that in HIV-infected patients the intracellular of lsk may be reduced, leading to enhanced susceptibility to apoptosis.