CELLULAR GENES REQUIRED FOR HIV1 INFECTION AS TARGETS FOR ANTIVIRAL THERAPY

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The goals of this project are: (1) To identify cellular genes which are required for optimal HIV infection (2) To identify cellular genes which are involved in the pathogenesis of HIV-1 infection (e.g enhanced programmed cell death ) (3) To design molecular approaches to suppress the expression of these cellular genes in HIV infection. (4) To study HIV-1 infection and pathogenesis in SCID-Hu animal model. We generated a panel of HIV-1 resistant T cell clones derived from the HIV-susceptible line CEM. We analyzed these clones by cellular and molecular approaches, and identified a n NF-kB p50 defficient clone s that are resistant to HIV-1 entry and reactivation. One of these clones were shwon to have reduced surface expression of the newly discovered hiv co-receptor CXCR4. In studies with SCID/Hu model we found that intrathymic HIV-1 infection results in a productive infection in a small fraction of mature CD3hiCD8+ thymocytes. These cells got ifected at an earlier differentiation stage expressing both CD4 and CD8 (DP). These cells die intrathymically and do not seed the periphery. This study explains the gradual loss of peripheral "naive" CD8 cells observed in HIV infected individual, especially children. Significant progress was done in studies of HIV-1 coreceptors, CXCR4 and CCR5, on primary cells: (a) Very high expression of CXCr4 was found on immature human thymocytes (from infants) including the CD34+ pintrathymic recursors. Thymocytes responded to the alpha chemokine SDF1 (but not to any beta chemokines) by chemotaxis and Ca++ mobilization. Thymocytes fused readily with T-tropic envelope and the fusion was blocked with rabbit IgG against CXCR4 (generated in our laboratory). In addition we found evidence that thymocytes express additional chemokine receptor (I309R) that also behave as a n HIV-1 co-receptor and support fusion with T-tropic envelopes. This study sheds light on the exquisite sensistivity of neonatal thymuse to infection with T-tropic HIV strains that often results in sever thymic depletion. (b) Extensive studies are under way to understand the correlation between surface expression and function of the hiv-1 coreceptors on a variety of cells. Especially monocytes Vs. macrophages, and several cell lines. Both biochemical and biological approaches are used in these studies. In addition we examine the effects of proinflamatory cytokines (IFNgamma, TNFalpha, IL10, IL-1beta) on coreceptor expression and function in macrophages, dendritic cells, Langerhans cells, and T cells. Assays developed will be used in the evaluation of the potency of anti-viral agents directed at blocking of HIV-1 cell entry and reactivation. Knowledge accumulated will be directly applied to evaluation of Cytokine-based, Ribozyme-based, and anti-sense-based therapeutic approaches which are already in early phases of clinical trials.