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Journal of Virology, July 2003, p. 7281-7290, Vol. 77, No. 13
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.13.7281-7290.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

High-Frequency Genetic Recombination and Reactivation of Orthopoxviruses from DNA Fragments Transfected into Leporipoxvirus-Infected Cells

Xiao-Dan Yao and David H. Evans*

Department of Molecular Biology & Genetics, University of Guelph, Guelph, Ontario N1G 2W1, Canada

Received 3 December 2002/ Accepted 1 April 2003

Poxvirus DNA is not infectious because establishing an infection requires the activities of enzymes packaged in the virion. This barrier can be overcome by transfecting virus DNA into cells previously infected with another poxvirus, since the resident virus can provide the trans-acting systems needed to reactivate transfected DNA. In this study we show that cells infected with a leporipoxvirus, Shope fibroma virus (SFV), can reactivate vaccinia virus DNA. Similar heterologous packaging systems which used fowlpox-infected cells to reactivate vaccinia virus have been described, but SFV-infected cells promoted a far more efficient reaction that can produce virus titers exceeding 106 PFU/µg of transfected DNA. SFV-promoted reactions also exploit the hyperrecombinogenic systems previously characterized in SFV-infected cells, and these coupled recombination and reactivation reactions could be used to delete nonessential regions of the vaccinia virus genome and to reconstruct vaccinia virus from overlapping DNA fragments. SFV-catalyzed recombination reactions need only two 18- to 20-bp homologies to target PCR amplicons to restriction enzyme-cut vaccinia virus vectors, and this reaction feature was used to rapidly clone and express a gene encoding fluorescent green protein without the need for plaque purification or selectable markers. The ability of SFV-infected cells to reactivate fragments of vaccinia virus was ultimately limited by the number of recombinational exchanges required and one cannot reconstruct vaccinia virus from multiple PCR fragments spanning essential portions of the genome. These observations suggest that recombination is an integral part of poxvirus reactivation reactions and provide a useful new technique for altering the structure of poxvirus genomes.

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

Journal of Virology, July 2003, p. 7281-7290, Vol. 77, No. 13
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.13.7281-7290.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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