Regulatory mutants of simian virus 40: Effect of mutations at a T antigen binding site on DNA replication and expression of viral genes

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Abstract

We have characterized a series of simian virus 40 (SV40) mutants with defined deletions or base-pair substitutions in the viral regulatory region near the origin of DNA replication. This segment of DNA is thought to contain three binding sites of differing affinity for the SV40 early protein, large T antigen, which is involved in initiation of viral DNA replication and autoregulation of early gene expression. Cells infected with viruses containing mutations in the high affinity binding site I were found to have a moderate, generally cold-sensitive defect in viral DNA replication and late protein synthesis that correlates with the temperature-dependence of their plaque size. In addition, the mutants overproduce early RNA and T antigen at various temperatures, suggesting a defect in autoregulation of early gene transcription. These changes appear to be related to T antigen functions at the binding site, since a second-site mutation in the T antigen gene restores viral DNA replication and gene expression to normal. Moreover, regulatory segments from mutant DNAs show decreased in vitro binding of T antigen to the high affinity site I. We conclude that T antigen binding to site I is involved in autoregulation of early gene transcription but is not essential for viral DNA replication. These properties contrast with previously reported properties of site II mutants, some of which show marked defects in viral DNA replication.

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    This research was supported by grant 5 PO1 CA16519 from the National Cancer Institute.

    Present address: Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA, U.S.A.

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