This Article Full Text Full Text (PDF) Other Versions of this Article: JVI.02723-07v1 82/12/5922    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Google Scholar Articles by Lin, Y.-C. J. Articles by Evans, D. H. PubMed PubMed Citation Articles by Lin, Y.-C. J. Articles by Evans, D. H.

 Previous Article  |  Next Article 

Journal of Virology, June 2008, p. 5922-5932, Vol. 82, No. 12
0022-538X/08/$08.00+0     doi:10.1128/JVI.02723-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Vaccinia Virus DNA Ligase Recruits Cellular Topoisomerase II to Sites of Viral Replication and Assembly

Department of Medical Microbiology and Immunology,¹ Alberta Institute for Viral Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada,² Manitoba Institute of Cell Biology, 675 McDermot Ave., Room ON5027, Winnipeg, Manitoba R3E 0V9, Canada³

Received 21 December 2007/ Accepted 3 April 2008

Vaccinia virus replication is inhibited by etoposide and mitoxantrone even though poxviruses do not encode the type II topoisomerases that are the specific targets of these drugs. Furthermore, one can isolate drug-resistant virus carrying mutations in the viral DNA ligase and yet the ligase is not known to exhibit sensitivity to these drugs. A yeast two-hybrid screen was used to search for proteins binding to vaccinia ligase, and one of the nine proteins identified comprised a portion (residue 901 to end) of human topoisomerase IIβ. One can prevent the interaction by introducing a C₁₁-to-Y substitution mutation into the N terminus of the ligase bait protein, which is one of the mutations conferring etoposide and mitoxantrone resistance. Coimmunoprecipitation methods showed that the native ligase and a Flag-tagged recombinant protein form complexes with human topoisomerase II/β in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase II and IIβ antigens are recruited to cytoplasmic sites of virus replication and that less topoisomerase was recruited to these sites in cells infected with mutant virus than in cells infected with wild-type virus. Immunoelectron microscopy confirmed the presence of topoisomerases II/β in virosomes, but the enzyme could not be detected in mature virus particles. We propose that the genetics of etoposide and mitoxantrone resistance can be explained by vaccinia ligase binding to cellular topoisomerase II and recruiting this nuclear enzyme to sites of virus biogenesis. Although other nuclear DNA binding proteins have been detected in virosomes, this appears to be the first demonstration of an enzyme being selectively recruited to sites of poxvirus DNA synthesis and assembly.

Published ahead of print on 16 April 2008.