The goal of the proposed research is to elucidate the mechanisms of gene regulation by the human immunodeficiency virus (HIV-1) encoded proteins tat and rev. A particular emphasis will be the establishment, characterization and analysis of reactions in vitro which duplicate the respective in vivo processes. Tat stimulates transcription of the proviral DNA by recognition of the transactivation response (TAR) element as RNA. Recent results suggest that tat action increases the efficiency of elongation of the polymerase. A reaction in vitro has been developed which responds to addition of tat protein p68 by increased transcription of the HIV-1 promoter. The role of a cellular protein 68 which specifically binds the TAR element will be studied in this system following purification of the protein. The mechanism of tat activation of transcription will be analyzed by: kinetic studies of transcription in vitro, definition of the activation domain of the protein, characterization of tat-TAR interactions under reaction conditions, and reconstitution of reactions using partially purified factors which duplicate the in vivo process. A possible relationship between the process inhibited by the drug 5,6-dichloro-1-beta- ribofuranosylbenzimidazole (DRB) and the process simulated by tat will be studied. The HIV-1 promoter may be uniquely configured to be responsive to tat stimulation. This possibility will be investigated by construction of mutant promoters. Rev stimulates the cytoplasmic appearance of unspliced or singly spliced viral RNA. In the absence of rev only multiply spliced RNA is transported to the cytoplasm while unspliced or singly spliced RNA is retained in the nucleus. We have proposed that rev functions by regulating the splicing of nuclear RNA containing the rev-responsive element (RRE). Potential regulation of splicing in vitro by purified rev protein will be investigated. A short peptide containing the basic region of rev will specifically repress the splicing of an RNA substrate containing RRE but not a control substrate in vitro. This reaction will be characterized and conditions will be sought to obtain regulation by the complete rev protein. The step in assembly of splicing complexes inhibited by rev protein will be determined. Experiments analyzing the possible role of rev in transport of RNA from nucleus to cytoplasm will also be tested. The two primary benefits of this research will be establishment of (1) a knowledge base for the future development of agents that inhibit tat and rev mediated process, and (2) the relationship between regulation of viral transcription and RNA processing by these viral proteins and regulation of cellular genes by cellular factors.
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