The tatIII gene of human immunodeficiency virus (HIV), essential for viral replication, is involved in transactivation of HIV-LTR expression. We propose to introduce the HIV-LTR fused to the CAT gene into an adenovirus vector, and to use this system to analyze in detail the biochemical mechanisms involved in tatIII transactivation. Specifically, we intend to: 1. Use this recombinant HIV-LTR-adenovirus to infect HeLa cells expressing the tatIII gene (and control HeLa cells) and determine at what levels of regulation the tatIII protein acts on HIV-LTR gene expression. 2. Use this recombinant HIV-LTR-adenovirus to infect large numbers of cells in order to develop in vitro systems that mimic tatIII transactivation, allowing a dissection of the mechanisms at work. 3. Once the mode(s) of tatIII activation is known in this recombinant HIV-LTR-adenovirus system, to introduce the HIV- LTRCAT gene into pre-existing adenoviurses mutant in known functions (i.e., translational control, mRNA transport from the nucleus, transition from early to late phase of infection) in order to investigate the interplay of these well defined genes and the tatIII gene.
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