The overall aims of these studies are to define the funcitons performed by Simian Virus 40 (SV40) large T antigen and to determine the domains of the polypeptide involved in different functions. During the next project period, studies will be focused on two major questions: 1. What is the nature of the host range/helper function (hr/hf) activity of T antigen? This function is provide by the C-terminal 35 amino acids of large T. Hr/hf mutants are most defective in CV-lP and grow best in Vero monkey kidney cells. Mutants lacking this function are positive for viral DNA replication but have defects in viral late gene expression. Translation of viral mRNAs is most affected, but other steps in the infection cycle are also affected. Mutants are also defective for adenovirus helper function. Proposed studies will investigate the possibility that the hr/hf defect affects an SV40 response to interferon or to activation of enogenous P1/eIF2-alpha kinase. Patterns of macromolecular synthesis in mutant-infected cells will be examined further, particularly the structures of mutant late mRNAs and the levels of phosphorylation of capsid protein VP1. Other studies will determine whether various adenovirus or polymavirus functions can compensate for the hr/hf defect and whether SV40 gene products other than the C-terminus of T can enhance adenovirus growth in CV-1P monkey cells. Second-site revertants of hr/hf mutants will be isolated and characterized. 2. What is the relationship between T antigen's ability to stimulate host DNA replication and other activities of T antigen? The ability of mutants of SV40 to induce host cell DNA replication will be determined by microinjection of mutant genome into quiescent primary cultures of rodent cells and established rodent cell lines. Initially, mutants with small deletions and linker insertions will be used. Subsequent studies will use point mutants. Additional studies will be performed to determine whether T antigen's ability to induce host DNA synthesis co-segregates with its activities in viral DNA replication, transactivation of gene expression from the SV40 late and RSV LTR promoters, immortalizaton of primary rodent cells, and malignant transformation of established cell lines.
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