The objective of this project is to define the molecular mechanisms controlling the expression of HIV~1 transcriptional regulation in vivo. Because of the emerging role of chromatin in modulating transcriptional regulatory mechanisms, we have analyzed the chromatin organization of the promoter of HIV~1 integrated in chronically infected cell lines. We have found that 2 regions, localized in the promoter~enhancer (U3 region) and immediately downstream of the 5'LTR, respectively, are nucleosome free. The DNA separating these two domains is incorporated into a nucleosome (called nuc~1) in basal conditions, when no viral expression is noted. Following TPA or TNF~alpha treatment, two agents known to induce viral expression at the transcriptional level in our cell lines, this nucleosome is displaced or disrupted. Our efforts in this last year have been directed toward understanding the mechanism of disruption of this nucleosome following activation of viral expression. Since most chromatin remodelling takes place during DNA replication, we have examined the time course of disruption of nuc~1 following TPA or TNF~alpha treatment and found it to be essentially completed in 20 min, which is inconsistent with a requirement for DNA replication. Since this nucleosome is on the path of the transcribing polymerase, we have also examined the effect of transcription on the disruption of nuc~1. Pretreatment of the cells with alpha~amanitin had no effect on the disruption of nuc~1, indicating that this phenomenon is independent of transcription. A nucleosome~free region was also noted downstream of nuc~1, possibly indicating that DNA~binding factors are present in this region in vivo. We have examined this region using in vitro and in vivo footprinting and have identified several binding sites for transcription factors including Sp1, AP3, AP1 and for a new factor that we have called DBF~1. We have generated mutations in each of these sites, and have reengineered them back into infectious molecular clones of HIV~1. Current studies are examining the effect of each of these mutations on viral replication in cell lines and human primary cultures (PBMCs). The significance of these studies lies in their potential relevance for HIV~1 latency and reactivation.
