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Journal of Virology, September 2009, p. 8781-8788, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00621-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

A Structure-Guided Mutational Analysis of Simian Virus 40 Large T Antigen: Identification of Surface Residues Required for Viral Replication and Transformation {triangledown} ,{dagger}

Deepika Ahuja,1,{ddagger} Abhilasha V. Rathi,1 Amy E. Greer,1,§ Xiaojiang S. Chen,2 and James M. Pipas1*

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260,1 Molecular and Computational Biology, University of Southern California, Los Angeles, California 900892

Received 25 March 2009/ Accepted 18 June 2009

Simian virus 40 large T antigen (TAg) transforms cells in culture and induces tumors in rodents. Genetic studies suggest that TAg interaction with the chaperone hsp70 and tumor suppressors pRb and p53 may not be sufficient to elicit complete transformation of cells. In order to identify additional cellular factors important for transformation, we designed mutations on the solvent-exposed surface of TAg. We hypothesized that surface residues would interact directly with cellular targets and that the mutation of these residues might disrupt this interaction without perturbing TAg's global structure. Using structural data, we identified 61 amino acids on the surface of TAg. Each surface amino acid was changed to an alanine. Furthermore, five patches containing clusters of charged amino acids on the surface of TAg were identified. Within these patches, we selectively mutated three to four charged amino acids and thus generated five mutants (patch mutants 1 to 5). We observed that while patch mutants 3 and 4 induced foci in REF52 cells, patch mutants 1 and 2 were deficient in focus formation. We determined that the patch 1 mutant is defective in p53 binding, thus explaining its defect in transformation. The patch 2 mutant can interact with the Rb family members and p53 like wild-type TAg but is unable to transform cells, suggesting that it is defective for action on an unknown cellular target essential for transformation. Our results suggest that the histone acetyltransferase CBP/p300 is one of the potential targets affected by the mutations in patch 2.

* Corresponding author. Mailing address: Department of Biological Sciences, University of Pittsburgh, 559 Crawford Hall, Pittsburgh, PA 15260. Phone: (412) 624-4691. Fax: (412) 624-4759. E-mail: pipas{at}pitt.edu

{triangledown} Published ahead of print on 24 June 2009.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

{ddagger} Present address: Department of Medicine, University of California—San Francisco, San Francisco, CA 94143.

§ Present address: W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205.

Journal of Virology, September 2009, p. 8781-8788, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00621-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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