The long-term objective of the proposed studies is a better understanding of the mechanisms by which viruses exploit the cellular transcriptional machinery to achieve orderly and efficient expression of viral genetic information. The proposed research will focus on activation of transcription of human adenovirus late genes by viral DNA synthesis and the viral IVa2 protein, a sequence-specific activator of transcription. In previous in vitro studies, we have identified a cellular factor that represses IVa2 transcription. The most immediate aim will be to use genetic methods to test the role of this cellular repressor (IVa2-RF) in regulation of IVa2 transcription in infected cells, and hence progression through the infectious cycle. These experiments are designed to test the hypothesis that the relative concentrations of IVa2-RF and its binding site in the IVa2 promoter control the activity of the IVa2 promoter. Because of its pivotal role in the adenoviral transcriptional program, the human gene(s) encoding IVa2-RF will be cloned, so that the normal function of this transcriptionaI regulator and the mechanism by which it represses transcription can be investigated. It is well established that the IVa2 protein is one (DEF-B) of two sequence-specific activators responsible for late phase-specific stimulation of ML protein. Additional functions of this viral protein in regulation of transcription, including autoregulation of ML transcription and regulation of the processivity of ML transcriptions will be investigation. Genetic and biochemical methods will he used to identify the protein(s) with which the IVa2 protein interacts to form the second of these late phase-specific activators of ML transcription, DEF-A. These studies are designed to lay the groundwork for more detailed analysis of the IVa2 protein. Molecular genetic methods, in conjunction with in vitro assays for specific functions of the IVa2 protein, will be used to obtain a more detailed understanding of the molecular organization of protein, its mechanism of action and its relationship to cellular activators of transcription. The amenability of human adenoviruses to genetic analysis and the particular properties of the Iva2 protein will then be exploited to perform a similar analysis of the IVa2 protein within infected cells. A major goal of the latter experiments is to assess the relevance of mechanisms of transcriptional activation inferred from results obtained in standard (but artificial) systems to the conditions that pertain within cells. These studies will improve our understanding of the mechanisms that control progression through the infectious cycle of human adenovirus, a virus currently receiving considerable attention as a potential vector for gene therapy, as well as of mechanisms of transcriptional regulation in eukaryotes.
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