1) By the end of 2003, approximately 2,000,000 HIV-positive individuals were estimated to be in the United States, representing a significant public health challenge. In patients undergoing antiretroviral therapy, elimination of HIV is confounded by a reservoir of latent virus blocked at the transcriptional level. Because of the latent reservoir, virus titers quickly rebound upon treatment cessation. Our laboratory has developed the J-Lat cell lines, a model system for the study of HIV latency in vitro. Jurkat cells were infected with recombinant HIV that expresses green fluorescence protein (GFP), and GFP fluorescence was used to isolate clonal populations of latently infected cells. HIV expression can be transiently and robustly reactivated in J-Lat cells to analyze the dynamic silencing and reactivation processes. A significant body of evidence indicates that chromatin structure is an important determinant of HIV transcriptional activity. We propose to use the J-Lat system to identify mechanisms by which chromatin structure regulates HIV transcription and latency. HIV transcription will be measured by GFP fluorescence and RT-PCR, and the role of heterochromatin in the regulation of transcription will be determined. First, chromatin immunoprecipitation (ChIP) will be used to assay the promoter of the HIV provirus for histone modifications involved in the formation of heterochromatin. Second, ChIP will be used to identify the transcriptional repressers that bind these modified histones and repress transcription, i.e. form heterochromatin, leading to latency. Third, the mechanism of latent HIV reactivation will be identified. Preliminary data indicates that histone methylation is involved in latency, and a limited number of processes that reverse this mark have been identified. These include histone demethylases, which will be tested by RNAi-mediated depletion, and histone replacement, which will be tested by ChIP. These studies will facilitate the development of treatments that interfere with latency and, in theory, permit the complete elimination of virus from patients. 2) While current antiretroviral drugs reduce HIV in patients to undetectable levels, virus is never eliminated due to a reservoir of latent virus. We will identify the molecular mechanism of latency, permitting reactivation of this reservoir and the elimination of virus from infected individuals.
