The interactions of polyomavirus tumor (T) antigens with proteins of host origin appear to regulate specific functions related to virus replication and cell transformation. Recent studies have demonstrated that polyomavirus middle T antigen forms a stable physical association with src family tyrosine kinases as well as components of the phosphoserine/threonine-specific Type 2A protein phosphatase (PP2A). The sequence of events which leads to formation of this protein complex is presently unknown. Moreover, it is unclear whether association of both kinase and phosphatase activities are necessary for the transforming properties of middle T antigen, or for its function which is required during virus maturation. Here it is postulated that the host factors are recruited by middle T antigen to directly influence key events in virus replication and cell transformation which are regulated by phosphorylation. The goals of this program are to determine how middle T antigen interacts with these host factors and to elucidate how these protein complexes might function during polyomavirus replication and cell transformation. individual protein complexes containing middle T antigen and associated kinase and/or phosphatase will be isolated and characterized with regard to their phosphorylation status and temporal appearance. Sites in small T and middle T antigens which mediate binding to pp2A will be defined by both structural and genetic approaches, and mutants with lesions in potential PP2A and src kinase binding domains will be generated. Biochemical and molecular approaches will be used to identify potential functions PP2A may have in regulating the phosphorylation of middle T antigen and its association with src family kinases. The capacity to which PP2A influences the activity of src family kinases may illuminate its role in cell transformation. Finally middle T antigen-associated kinase and phosphatase activities will be evaluated for their effect on modification of the major capsid protein VP1. Experiments to address mechanisms by which middle T antigen-associated proteins influence VP1 modification may ultimately define a function for this viral T antigen during polyomavirus maturation.
