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 may have important implications in disease progression both from environmental sources and during treatment of HIV-1-infected patients with cytotoxic agents. Our expertise in genotoxic stress responses has been applied to the study of responses in HIV-1. This project was funded by the NIH Intramural AIDS Targeted Antiviral Program for FY91 and FY92 with only partial salary support for a fellow in FY93: since that time, it has been supported by DTP funding. Initially, we focused on the induction by cytotoxic agents of stably-integrated HIV- promoter-CAT constructs. We have found that one or more cytokines in the medium can affect DNA-damage responsiveness; in addition, we have also found that a soluble factor(s) produced by UV-irradiated cells can activate stably-integrated HIV-CAT in trans. Since UV-irradiation can induce a cellular Tat-like factor, our search for cellular factors involved in HIV-1 regulation has focused primarily on proteins with Tat- like properties, and we have identified by a Northwestern blotting approach a variety of TAR RNA-binding proteins (TBP) in whole nuclear extracts. The levels of TBP were found to vary in different cell lines, and were DNA-damage inducible in many cases. With this approach, we have more than doubled the number of known TAR-binding proteins and have shown the first demonstration of a TBP response to stress. We plan to further characterize these TBP and their responses to genotoxic stress including the role of cytokines in their activation. Approaches will be developed to purify these DNA-damage-inducible RNA-binding proteins and to ultimately isolate cDNA clones based on partial protein sequence determinations. Identification of these proteins will probably provide important insights into the regulation of HIV-1 and the responses of HIV-1-infected cells to cytotoxic agents.
