The long-range goal of this project is to understand the transforming interactions between DNA tumor virus and target cells. The approach has been to define molecular interactions and biological functions expressed by the SV40 transforming protein, large tumor antigen (T-ag). There is evidence for multiple effects exerted by distinct T-ag subpopulations on transformed cells, including possible role for plasma-membrane-associated T-ag (pmT-ag). This renewal application focuses on questions fundamental to understanding the molecular mechanisms of SV40 carcinogenesis. This program will correlate T-ag structure and function with transformation events. The following specific aims are present. (1) The structure and function of SV40 T-ag containing, membrane-associated protein complexes will be analyzed. We have developed a butane extraction method that selectively recovers membrane protein complexes that contain pmT-ag and p53-related protein (p56). Other cellular components in the complexes (60K, 50K, 45K, 35K) will be identified; it is hypothesized they are members of a receptor complex. the composition modifications, and biochemical activities of pmT-ag-containing complexes will be investigated . Possible involvement of T-ag in a growth-factor-like pathway will be examined. (2) Transformation phenotypes of new in-phase deletion mutants will be related to specific interactions of mutant T-89 with cellular proteins and the plasma membrane. Different assays will distinguish T-ag domain required for cell immortalization and phenotypic transformation. Complementation transformation tests may permit assignment of T-ag functions to categories defined by known oncogenes. Mutant T-ag polypeptides will be characterized structurally and biochemically, including complex formation with membrane proteins, and those analyses correlated with transformation by the mutants. (3)Trafficking of SV40 T- ag to the cell surface will be studied. Deletion mutants will reveal possible effects of the short hydrophobic sequence in T-ag on transport and membrane interaction. The potential involvement of cellular heat- shock protein p73 (that bind T-ag) in pmT-ag transport will be addressed. A role for O-glycosylation o T-ag in trafficking or membrane association will be considered. (4) Cellular and molecular events involved in muoti- stage carcinogenesis in livers of SV40 transgenic mice will be defined. We have found that mice with the WT T-ag gene controlled by the alpha-1- antitrypsin promoter develop liver tumors. New transgenic mice carrying the p53 or mutants T-ag genes will be made. Various transgenic lines will be crossed and pathology in progeny animal determined. The expression of viral (pmT-ag and cellular (oncogenes) events will be correlated with different stages of neoplastic progression. Hyperplastic liver cells will be cultured to determine in vitro growth properties that correspond to preneoplasia in vivo.
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