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Journal of Virology, May 2009, p. 4236-4250, Vol. 83, No. 9
0022-538X/09/$08.00+0     doi:10.1128/JVI.02255-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The 3'-to-5' Exonuclease Activity of Vaccinia Virus DNA Polymerase Is Essential and Plays a Role in Promoting Virus Genetic Recombination{triangledown}

Don B. Gammon and David H. Evans*

Department of Medical Microbiology and Immunology, 1-41 Medical Sciences Building, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

Received 27 October 2008/ Accepted 5 February 2009

Poxviruses are subjected to extraordinarily high levels of genetic recombination during infection, although the enzymes catalyzing these reactions have never been identified. However, it is clear that virus-encoded DNA polymerases play some unknown yet critical role in virus recombination. Using a novel, antiviral-drug-based strategy to dissect recombination and replication reactions, we now show that the 3'-to-5' proofreading exonuclease activity of the viral DNA polymerase plays a key role in promoting recombination reactions. Linear DNA substrates were prepared containing the dCMP analog cidofovir (CDV) incorporated into the 3' ends of the molecules. The drug blocked the formation of concatemeric recombinant molecules in vitro in a process that was catalyzed by the proofreading activity of vaccinia virus DNA polymerase. Recombinant formation was also blocked when CDV-containing recombination substrates were transfected into cells infected with wild-type vaccinia virus. These inhibitory effects could be overcome if CDV-containing substrates were transfected into cells infected with CDV-resistant (CDVr) viruses, but only when resistance was linked to an A314T substitution mutation mapping within the 3'-to-5' exonuclease domain of the viral polymerase. Viruses encoding a CDVr mutation in the polymerase domain still exhibited a CDV-induced recombination deficiency. The A314T substitution also enhanced the enzyme's capacity to excise CDV molecules from the 3' ends of duplex DNA and to recombine these DNAs in vitro, as judged from experiments using purified mutant DNA polymerase. The 3'-to-5' exonuclease activity appears to be an essential virus function, and our results suggest that this might be because poxviruses use it to promote genetic exchange.

* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, 1-41 Medical Sciences Building, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Phone: (780) 492-2308. Fax: (780) 492-7521. E-mail: devans{at}ualberta.ca

{triangledown} Published ahead of print on 18 February 2009.

Journal of Virology, May 2009, p. 4236-4250, Vol. 83, No. 9
0022-538X/09/$08.00+0     doi:10.1128/JVI.02255-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Senkevich, T. G., Koonin, E. V., Moss, B. (2009). Predicted poxvirus FEN1-like nuclease required for homologous recombination, double-strand break repair and full-size genome formation. Proc. Natl. Acad. Sci. USA 106: 17921-17926 [Abstract] [Full Text]  


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