The regulation of human immunodeficiency virus (HIV) gene expression is dependent on specific regulatory sequences in the long terminal repeat. Elements that appear to be important for regulation of viral gene expression include IL-2-like enhancer sequences, negative regulatory elements, an enhancer containing two NF-kB motifs, three SP1 binding sites, the TATA element, and the TAR region. These regulatory sequences serve as the binding sites for cellular transcription factors. A number of cellular proteins that bind to these DNA sites have been characterized including NFAT-1, which binds to the IL-2 like enhancer elements; EBP-1 and NF-kB, which bind to the enhancer, SP-1, which binds to the three SP1 binding sites; UBP-1 or LBP, UBP-2, and CTF which bind to the TAR region. In addition to these regulatory sequences, the viral regulatory protein, tat, acts to enhance viral gene expression. The goal of this grant is to understand how these regulatory elements and their binding proteins function in activating HIV gene expression. Several approaches will be undertaken to address these questions. First, infectious viral constructs containing mutations in various LTR regulatory domains will be studied to determine the effects of these mutations on viral growth. Second, the role of the TAR region on tat activation of the HIV LTR will be determined by nuclear run-on experiments using the HIV LTR, a variety of heterologous TAR constructs, and DNA and RNA competition experiments. Third, we will investigate the role of cloned cellular factors that bind to IL-2 like enhancer sequences, the enhancer, and UBP-L binding sites in the TAR region on the regulation of HIV gene expression. Finally, the role of mutant tat proteins on regulating viral gene expression will be investigated. These studies should further define cellular and viral regulatory mechanisms that mediate HIV gene expression.
