We have demonstrated that CD4-negative cell lines derived from the human cervix and vagina, and primary foreskin keratinocytes can become productively infected following co-culture with HIV-infected PBMCs or free virus. In this revised renewal application we propose to use assays for cell-free and cell-mediated HIV-1 infection to determine the susceptibility of transformed epithelial cells and primary epithelial cells to HIV-1 infection. We will determine the ability of the progeny virus from infected epithelial cells to infect other cells and if epithelial cells are differentially infected by well characterized T- tropic and M-tropic viruses and clinical isolates of different clades. We will explore the entry mechanisms of HIV-1 by determining the cellular receptor(s) and co-receptor(s) for HIV-1 in these CD4-negative cells from the reproductive tract. Blocking experiments with a panel of monoclonal antibodies with various epitope specificities will be carried out to identify the regions of HIV-1 envelope glycoprotein that mediate virus binding and entry. We also propose to study the effect of seminal fluids in the viral infection of epithelial cells. To do this seminal plasma will be depleted of immunosuppressive factors and subsequently fractionated by HPLC to determine the active components in HIV-1 transmission. Lastly we have proposed to study cell trafficking from the vaginal vault. Experiments will be based on our recent observation that mononuclear cells placed in the vagina of mice can traffic across reproductive tract epithelia into the lamina propria and inguinal lymph nodes. We will determine the identity of the mononuclear cells that migrate between epithelial cells, using immunocytochemistry and confocal microscopic techniques. Time course of the migration will be determined. We will determine whether mononuclear cells inoculated in intravaginally will migrate to the spleen and distant lymph nodes. We also propose to identify possible associations between donor mononuclear blood cells and cells of the host immune system. The work proposed in our 5 aims should lead to a clearer understanding of how HIV crosses genital tract epithelia and more informed strategies to prevent sexual transmission of HIV.