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Effects of mutations within the SV40 large T antigen ATPase/p53 binding domain on viral replication and
K W Peden1, A Srinivasan, J V Vartikar
1Laboratory of Retrovirus Research, FDA, Bethesda, Maryland 20892, USA.
Abstract:
The simian virus 40 (SV40) large T antigen is a 708 amino-acid protein possessing multiple biochemical activities that play distinct roles in productive infection or virus-induced cell transformation. The carboxy-terminal portion of T antigen includes a domain that carries the nucleotide binding and ATPase activities of the protein, as well as sequences required for T antigen to associate with the cellular tumor suppressor p53. Consequently this domain functions both in viral DNA replication and cellular transformation. We have generated a collection of SV40 mutants with amino-acid deletions, insertions or substitutions in specific domains of the protein. Here we report the properties of nine mutants with single or multiple substitutions between amino acids 402 and 430, a region thought to be important for both the p53 binding and ATPase functions. The mutants were examined for the ability to produce infectious progeny virions, replicate viral DNA in vivo, perform in trans complementation tests, and transform established cell lines. Two of the mutants exhibited a wild-type phenotype in all these tests. The remaining seven mutants were defective for plaque formation and viral DNA replication, but in each case these defects could be complemented by a wild-type T antigen supplied in trans. One of these replication-defective mutants efficiently transformed the REF52 and C3H10T1/2 cell lines as assessed by the dense-focus assay. The remaining six mutants were defective for transforming REF52 cells and transformed the C3H10T1/2 line with a reduced efficiency. The ability of mutant T antigen to transform REF52 cells correlated with their ability to induce increased levels of p53.
Insights
Simian virus 40 (SV40) large T antigen mutants affecting p53 binding and ATPase activity were studied. Some mutants showed defects in viral DNA replication and cell transformation, with varying efficiencies depending on cell type and p53 interaction.
Area of Science:
- Virology
- Molecular Biology
- Oncogenesis
Background:
- Simian virus 40 (SV40) large T antigen (T-ag) is crucial for viral replication and cell transformation.
- A specific domain of T-ag mediates nucleotide binding, ATPase activity, and interaction with the tumor suppressor p53.
- This domain is essential for both viral DNA replication and cellular transformation processes.
Purpose of the Study:
- To investigate the functional roles of the SV40 T-ag domain (amino acids 402-430) in viral DNA replication and cell transformation.
- To characterize SV40 mutants with alterations in this critical T-ag region.
- To assess the correlation between p53 binding, ATPase activity, and the transforming potential of T-ag mutants.
Main Methods:
- Generation of SV40 mutants with amino acid substitutions between residues 402 and 430.
- Assays for viral DNA replication, plaque formation, and in trans complementation.
- Evaluation of cell transformation efficiency using dense-focus assays in REF52 and C3H10T1/2 cell lines.
- Measurement of p53 levels in transformed cells.
Main Results:
- Two mutants retained wild-type phenotypes for all tested functions.
- Seven mutants were defective in plaque formation and viral DNA replication, but complementable in trans.
- One replication-defective mutant efficiently transformed both cell lines; others showed reduced transformation efficiency, particularly in REF52 cells.
- Transformation of REF52 cells correlated with the induction of elevated p53 levels.
Conclusions:
- The region between amino acids 402 and 430 of SV40 T-ag is critical for viral DNA replication and efficient cell transformation.
- Mutations in this region differentially affect transformation depending on the cell type and interaction with p53.
- SV40 T-ag's ability to transform REF52 cells is linked to its capacity to modulate p53 levels.