I propose to investigate the early proteins encoded by adenovirus type-2, and determine the mechanism by which expression of these proteins effect cellular transformation, viral DNA replication, and RNA processing. One approach is based on a strict genetic analysis, using methods previously developed in our laboratory, to introduce point-mutations into the exon domains of early region IA and IB (the coding sequences primarily responsible for viral-RNA regulation), and transformation of the adenovirus genome. Overall, it is believed that this particular approach will facilitate the creation of either cold-sensitive or ts adenovirus mutants. Rapid screening methods for the selection of these conditional adenovirus mutants will also be developed. Ultimately, the mutants will be used in dissecting early viral functions and in identifying proteins involved in the above aforementioned. A complementary approach to the genetic analysis of early viral proteins involves methods for obtaining two types of expression vectors, one which will allow for the synthesis of the viral proteins in a bacterial system and the other which will be genetically designed to allow expression of the viral proteins in eukaryotic cells. Viral proteins that will be synthesized in the bacterial system, by an expression vector containing the lac promoter, will be used primarily for the development of an immune assay. Construction of a eukaryotic vector containing the SV40 replication origin and early promoter will serve the purpose of synthesizing early viral proteins in their native form for subsequent enzymatic analysis. In additional experiments, we will continue our investigation of a new viral protein that appears to be encoded within an intron located in early region 4 of the viral chromosome. Thus, these experiments should broaden our understanding of mechanisms employed by the virus to exploit its host, as well as induce cellular transformation.
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