The objective of this project is to define the regulatory signals that control the expression of HIV-1 in infected cells. We are interested in the molecular mechanisms of HIV-1 transcriptional control since they ultimately determine the amount of viral mRNA, proteins and virons produced. Transcriptional control of HIV operates at several distinct levels including the 5'LTR, where the promotor is located, the 3'LTR where transcription is terminated, and by a region in the pol gene where we have identified a new enhancer element. Since the regions of genes associated with regulatory functions generally present an increased sensitivity to digestion with nucleases in vivo, we have probed purified nuclei from chronically infected cells with several nucleases. These studies have identified several domains hypersensitive to digestion with nucleases associated with the 5'and 3'LTRs and the pol intragenic enhancer. We are now examining the chromatin structure of HIV in several cell lines of lymphoid or monocytic/macrophage origin that we have chronically infected with molecularly cloned viruses. The nuclease-hypersensitive regions defined as above are also studied at high resolution by genomic footprinting after DNase I or dimethyl sulfate (DMS) treatment. In parallel, we are characterizing by transfections, band shift assays, methylation interference and DNAse I footprinting in vitro, the DNA and protein factors responsible for the enhancer activity of the pol enhancer. The significance of this work lies in a better understanding of the mechanisms controlling the rate of replication of HIV and with the long-term potential for finding ways to suppress replication.
