This project was funded by the NIH Intramural AIDS Targeted Antiviral Program for FY91 and FY92 only with support for Dr. Carrier until July, 1993 and for Dr. Bae FY93. It involves an investigation of the activation of HIV-1 gene expression by stresses such as DNA-damaging agents. Treatment of human cells with DNA-damaging agents, such as UV radiation, can lead to the activation of latent virus and to increased virus production. This activation is probably mediated by multiple mechanisms including the induction of a cellular Tat-like factor and the induction of at least one and probably more proteins that bind to specific elements in the HIV promoter. Current efforts focus on two areas. In the first, activation of the HIV-1 promoter by genotoxic stress is being studied primarily by the use of HIV-promoter CAT (chloramphenicol acetyltransferase) reporter gene constructs in various cell lines. We have confirmed the work of others that stably-integrated HIV-CAT constructs are much more strongly UV-inducible than in transient assays in HeLa cells; we have found that soluble cellular factor(s) can affect this response. Efforts are underway to identify HIV-1 and cellular factors that contribute to this DNA-damage responsiveness. In collaboration with D. Yarosh, activation of stably-integrated HIV-CAT has been found after exposure of cells to conditioned medium from UV-treated cells. Efforts are underway to identify the soluble factors that are required for this transactivation. The second area involves cellular TAR-binding proteins which may play a role in HIV activation in cells after genotoxic stress. UV-induction of HIV-CAT occurs in cells that lack Tat indicating that cellular proteins with Tat-like function may contribute to this activation. Using a novel northwestern blotting approach, we have shown for the first time that RNA-binding proteins (RBP) can often be induced in mammalian cells by DNA-damaging agents, such as UV radiation. Many RBP preferentially bound RNA probes with the TAR stem-loop structure. A major focus of future studies will be the characterization of these newly discovered inducible RBP and their role in HIV-1 regulation.
