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Journal of Virology, July 1999, p. 5473-5480, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Replication, Integration, and Packaging of Plasmid DNA following Cotransfection with Baculovirus Viral DNA

Yuntao Wu,dagger Ge Liu,Dagger and Eric B. Carstens*

Department of Microbiology and Immunology, Queen's University, Kingston, Ontario K7L 3N6, Canada

Received 10 February 1999/Accepted 13 April 1999

Infection-dependent replication assays have been used to identify numerous putative origins of baculovirus replication. However, plasmid DNA, when cotransfected into insect cells with Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) DNA, replicates independently of any viral sequence in cis (11). Cotransfection of transfer plasmids and baculovirus DNA is a common procedure used in generating recombinant viruses and in measuring the level of gene expression in transient-expression assays. We have examined the fate of a series of vector plasmids in cotransfection experiments. The data reveal that these plasmids replicate following cotransfection and the replication of plasmid DNA is not due to acquisition of viral putative origin sequences. The conformation of plasmid DNA replicating in the cotransfected cells was analyzed and found to exist as high-molecular-weight concatemers. Ten to 25% of the replicated plasmid DNA was integrated into multiple locations on the viral genome and was present in progeny virions following serial passage. Sequence analysis of plasmid-viral DNA junction sites revealed no homologous or conserved sequences in the proximity of the integration sites, suggesting that nonhomologous recombination was involved during the integration process. These data suggest that while a rolling-circle mechanism could be used for baculovirus DNA replication, recombination may also be involved in this process. Plasmid integration may generate large deletions of the viral genome, suggesting that the process of DNA replication in baculovirus may be prone to generation of defective genomes.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Queen's University, Kingston, Ontario K7L 3Y6, Canada. Phone: (613) 533-2463. Fax: (613) 533-6796. E-mail: carstens{at}post.queensu.ca.

dagger Present address: Canadian Food Inspection Agency, Animal Disease Research Institute, Nepean, Ontario K2H 8P9, Canada.

Dagger Present address: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892.

Journal of Virology, July 1999, p. 5473-5480, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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