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In vitro mutagenesis of a putative DNA binding domain of SV40 large-T

D Kalderon, A E Smith

    Virology
    |November 1, 1984
    PubMed

    Insights

    Mutations in the SV40 large-T gene affect viral DNA replication and cell transformation. Specific mutations impairing DNA binding also enhance transforming activity by disrupting autoregulation.

    Area of Science:

    • Molecular Biology
    • Virology
    • Genetics

    Background:

    • The SV40 large-T antigen is crucial for viral DNA replication and cellular transformation.
    • A specific region of large-T was hypothesized to contain a DNA-binding domain essential for its functions.

    Purpose of the Study:

    • To investigate the role of a specific region in the SV40 large-T gene in DNA binding, viral replication, and cell transformation.
    • To identify key amino acid residues responsible for large-T's autoregulation and transforming activities.

    Main Methods:

    • Introduction of numerous deletion and point mutations into a targeted region of the SV40 large-T gene.
    • Assays to evaluate the effects of mutant proteins on viral DNA replication and cell transformation (e.g., Rat-1 cells).
    • Analysis of mutant protein localization within the cell nucleus.

    Main Results:

    • Most mutants lost the ability to stimulate viral DNA replication but retained transforming activity.
    • Replication-defective mutants with lesions between amino acids 144-156 showed enhanced transforming activity, suggesting impaired autoregulation.
    • Two mutants (Glu 107----Lys, Ser 189----Asn) had reduced transforming activity but normal replication rates.
    • Phosphorylation site mutations and mutations outside the nuclear localization region did not significantly impair nuclear accumulation or transforming activity.

    Conclusions:

    • The studied region of SV40 large-T is critical for viral DNA replication, with specific residues involved in autoregulation.
    • Disruption of autoregulation can enhance the transforming potential of large-T.
    • Nuclear localization is separable from replication and transformation functions for certain large-T mutations.

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