INVOLVEMENT OF T ANTIGEN IN SV40 LATE GENE EXPRESSION

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The long-term objective of the proposed research is to understand at the molecular level the roles played by large T antigen in regulating late gene expression of SV40. During this grant, we will test the validity of a number of hypotheses relating to this general problem. Specifically, we will: (i) determine whether large T antigen is required for late strand RNA synthesis by looking for this RNA in cells infected with a mutant of SV40 that does not encode this protein; (ii) determine whether late strand RNA synthesis can occur in the absence of viral DNA replication by testing with appropriate restriction endonucleases whether replication-defective mutants that synthesize this RNA in monkey cells and Xenopus oocytes are truly unable to undergo even a single round of viral DNA replication; (iii) determine whether large T antigen directly enhances synthesis of late strand RNA via its interactions with promoter-regulatory region sequences on the viral genome by examining a variety of T antigen-defective and promoter-regulatory region mutants for their ability to make late strand RNA; (iv) determine by S1 mapping of the 5' ends of the RNAs made in WT- and tsA58-infected cells whether T antigen-induced changes occur during the lytic cycle in the transcription initiation sites used for late strand RNA synthesis; and (v) determine whether a late strand transcriptional "attenuator" exists in SV40 by looking by S1 mapping for the presence of small RNAs with appropriate 3' ends in (a) WT- and mutant-infected monkey cells at various times after infection; and (b) Xenopus oocytes injected with WT and mutant SV40 DNAs and, in later experiments, preparations of virus-coded proteins. These studies should help us to learn about mechanisms by which tumor antigens can alter gene expression in ways that may on occasion lead to the transformation of cells to an oncogenic state.