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Journal of Virology, June 2007, p. 6099-6105, Vol. 81, No. 11
0022-538X/07/$08.00+0     doi:10.1128/JVI.02195-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Importance of Calcium-Binding Site 2 in Simian Virus 40 Infection{triangledown}

Peggy P. Li,1 Albert P. Nguyen,1 Qiumin Qu,1 Qumber H. Jafri,1 Saharat Aungsumart,2 R. Holland Cheng,2 and Harumi Kasamatsu1*

Department of Molecular, Cell and Developmental Biology and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095,1 Department of Molecular and Cellular Biology, University of California, Davis, California 956162

Received 6 October 2006/ Accepted 8 March 2007

The exposure of molecular signals for simian virus 40 (SV40) cell entry and nuclear entry has been postulated to involve calcium coordination at two sites on the capsid made of Vp1. The role of calcium-binding site 2 in SV40 infection was examined by analyzing four single mutants of site 2, the Glu160Lys, Glu160Arg, Glu157Lys (E157K), and Glu157Arg mutants, and an E157K-E330K combination mutant. The last three mutants were nonviable. All mutants replicated viral DNA normally, and all except the last two produced particles containing all three capsid proteins and viral DNA. The defect of the site 1-site 2 E157K-E330K double mutant implies that at least one of the sites is required for particle assembly in vivo. The nonviable E157K particles, about 10% larger in diameter than the wild type, were able to enter cells but did not lead to T-antigen expression. Cell-internalized E157K DNA effectively coimmunoprecipitated with anti-Vp1 antibody, but little of the DNA did so with anti-Vp3 antibody, and none was detected in anti-importin immunoprecipitate. Yet, a substantial amount of Vp3 was present in anti-Vp1 immune complexes, suggesting that internalized E157K particles are ineffective at exposing Vp3. Our data show that E157K mutant infection is blocked at a stage prior to the interaction of the Vp3 nuclear localization signal with importins, consistent with a role for calcium-binding site 2 in postentry steps leading to the nuclear import of the infecting SV40.

* Corresponding author. Mailing address: Molecular Biology Institute, 456 Boyer Hall, University of California at Los Angeles, 611 East Charles E. Young Dr., Box 951570, Los Angeles, CA 90095-1570. Phone: (310) 825-3048. Fax: (310) 206-7286. E-mail: harumi_K{at}mbi.ucla.edu

{triangledown} Published ahead of print on 14 March 2007.

Journal of Virology, June 2007, p. 6099-6105, Vol. 81, No. 11
0022-538X/07/$08.00+0     doi:10.1128/JVI.02195-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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