Our long term goals are to determine the mechanism of viral mRNA synthesis and its regulation during the early phase of Adenovirus infection, and the mechanism of cell transformation by Adenovirus. In the work proposed here, our first specific aim is to determine viral DNA sequence required for initiation of transcription, splicing, polyadenylation, and transport of mRNAs encoded in the transforming region. This will be done by constructing specific mutants with deletions, insertions, and base-pair changes and analyzing the impact of these mutations on these steps in mRNA synthesis. Second, we will determine the requirement of each of the several closely related, overlapping proteins encoded in the transforming region for the process of tranformation by constructing viral mutants with specific base-pair changes in sequences required for splicing specific mRNAs. Because of genetic code degeneracy, base-pair changes can be designed which do not alter the coding capacity of overlapping genes, or make conservative amino acid replacements in the encoded proteins. Thrid, we will analyze the mechanism by which viral proteins encoded in early region IA catalyze the conversion of the viral genome from a poor to an efficient template for transcription. To do this we will search for differences in the nucleoprotein structure of intracellular viral DNA in cells infected with wild-type virus and early region IA mutants by analyzing the products of viral DNA following digestion of isolated nuclei with micrococcal nuclease and DNase I. We will purify early region IA proteins and study their interaction with specific viral DNA and RNA sequences in vitro. To facilitate purification, recombinant viruses will be constructed to greatly overproduce these proteins. Finally, we will isolate mutants with ts lesions in these proteins by selection of ts second site revertants after directed mutagenesis. Studies with such mutants will indicate if these proteins are continuously required for expression of viral mRNAs, and maintenance of the transformed phenotypes of transformed cells.
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