The proposed research is based on our recent observation that when HIV- infected transformed T-cells or monocytes are added to monolayers of epithelial cells derived from the human intestine or cervix they attached tightly to the epithelium. Adherence triggers directional release of HIV onto the epithelium. Virus is subsequently taken up by the epithelia which becomes productively infected. We theorize that this in vitro model could mimic the manner which HIV is sexually transmitted in vivo. We propose to develop systems that more accurately reflect the natural conditions which exist during sexual transmission of HIV and to employ these models to clarify the mechanisms involved in mononuclear cell-to-epithelia adhesion, directional secretion of HIV, and virus uptake. Infection by cell-free virus will also be studied. To date, we have used cell lines of mononuclear cells derived from human cancers. We propose to use HIV-infected primary mononuclear cells because they are more representative of the HIV-infected cells found in blood and genital tract secretions. In preliminary experiments we have been able to infect epithelia with HIV-infected primary mononuclear cells. As infection may occur by both cell-free and cell-to-cell mechanisms, work will also involve infection via cell-free virus. These studies will include the development of a quantitative blocking assay of cell-free virus infection of ME180 cells - a cervix-derived cell line which is similar to the human cervix and which we have found in preliminary experiments can be infected by cell-free virus. Research on both cell-free and cell-associated epithelial infection systems will involve virus strains believed to be involved in natural infection. Our preliminary studies suggest that rearrangement of microtubules and microfilaments may be involved in directional secretion of virus. We will use EM localization techniques to further investigate this phenomenon. Since cell adhesion molecules (CAM) are likely to be involved in adherence of mononuclear cells to epithelia we will carry out blocking experiments with antibodies to CAM, sugars, enzymes, and lectins. The role of cytokines in virus uptake and phagocytosis by cervix-derived epithelial cells will be elucidated with blocking experiments of cell-free virus and by phagocytosis of microspheres by cervix-derived epithelia. Lastly, blocking experiments will be carried out to determine whether antibodies to gpl2O can block cell-mediated infection of epithelia. We believe that the proposed studies will lead to a better understanding of the basic mechanisms of sexual transmission of HIV and to new AIDS prevention strategies.
