Vaccinia virus DNA ligase recruits cellular topoisomerase II to sites of viral replication and assembly.
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 IIbeta. One can prevent the interaction by introducing a C(11)-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 IIalpha/beta in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase IIalpha and IIbeta 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 IIalpha/beta 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.
Lin
Y-C James
YC
Department of Medical Microbiology and Immunology, Alberta Institute for Viral Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Li
Jianhong
J
Irwin
Chad R
CR
Jenkins
Heather
H
DeLange
Luke
L
Evans
David H
DH
D000911:Antibodies, Monoclonal / Q000378:metabolism; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D011088:DNA Ligases / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism* / Q000648:ultrastructure; D018076:DNA, Complementary; D004279:DNA, Viral / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014616:Vaccinia virus / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D010948:Viral Plaque Assay; D019065:Virus Assembly / Q000187:drug effects / Q000502:physiology*; D014779:Virus Replication / Q000187:drug effects / Q000502:physiology*
D000911:Antibodies, Monoclonal; D000972:Antineoplastic Agents, Phytogenic; D018076:DNA, Complementary; D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D005047:Etoposide; D004250:DNA Topoisomerases, Type II; D011088:DNA Ligases