This proposal concerns the gene products of polyoma virus required for viral growth and neoplastic transformation. Our goal is to understand how the large T and middle T antigens act in both processes. This includes their interactions with elements of other cellular pathways which are, regulatory for cell growth. We emphasize phosphorylation, because phosphorylation appears central to their function. Our approach is both biochemical and genetic. Large T antigen, which is important for DNA replication and RNA transcription, has a complicated pattern of phosphorylation that mutant analysis relates to function. We will continue identification of the phosphorylation sites. As each site is identified, it will be subjected to oligonucleotide mutagenesis. We will express and study the properties of the C-terminal domain of the protein. We will probe the architecture of the large T molecule. Labeling experiments will be carried out to determine the key contact points between the N- and C-terminal domains, between large T in oligomers and between large T and DNA. Second-site revertants will be sought as a genetic way to determine important interactions. For the biochemical studies, we will express important mutants and the C-terminal domains using baculovirus vectors. For middle T we will complete the analysis and mutagenesis of the serine/threonine phosphorylation sites. Phosphatidylinositol-3-kinase is the target of middle T most closely linked with transformation. It has wide general significance because of its association with other oncogenes and growth factors. Our analysis of this enzyme will continue. Besides working out the details of its association with middle T, we will assess whether this enzyme is sufficient to regulate cell growth. Because a mutation in an NPXY sequence that has been associated with localization to coated pits renders middle T non-transforming, experiments on the endosomal targeting of middle T will be carried out. The effects of middle T on internalization and endosomal trafficking will also be tested. The interactions of polyoma T antigens with other pathways which are regulatory for cell growth win also be tested. It appears that a functioning ras pathway is required for middle T transformation, so elements of that pathway will be examined. Wild type p53 is a negative regulator of cell growth inactivated by SV40 transformation; we will determine whether polyoma transformation of cells is affected by p53 and whether p53 is in turn affected by polyoma virus.
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