This project will examine the activation of HIV-1 gene expression by DNA-damaging agents and other stresses. 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. We plan to apply our expertise with DNA-damage-inducible (DDI) genes and their promoters to study the mechanisms involved in the induction of the HIV-1 promoter. One portion of this project will involve the use of various HIV promoter constructs fused to reporter genes such as chloramphenicol acetyltransferase (CAT). These constructs will be stably integrated in human cells and the effect of various deletions and mutations in the promoter will be analyzed. The effect of various inhibitors such as kinase inhibitors will be employed to delineate the role of different kinases and other proteins. Experiments such as band-shift assays will be employed to identify binding-factor elements; competition experiments with various sequences, which bind transcription factors, including novel DDI elements, which we have recently identified, will be undertaken to tentatively identify common factors. The effect of various types of DNA-damaging agents on induction will be studied: this may provide evidence for the mechanisms involved in induction and may also be of clinical importance when planning therapy for HIV-infected patients with malignancies.
