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

Abstract

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 drugresistant 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 II␤. 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 II␣/␤ 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 II␤ 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␣/␤ 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.Topoisomerases play a critical role in modulating DNA superhelical density and in unknotting and decatenating the structures formed during replication, recombination, and repair (reviewed in references 2 and 5). Mammalian cells carry several different kinds of topoisomerases, classified as being either type I or type II enzymes. Both classes of enzyme catalyze DNA strand breakage and rejoining, but type I enzymes catalyze reactions involving single-strand breaks, while the type II topoisomerases catalyze double-strand cleavage reactions. The mammalian type IB enzyme (Topo I) is well adapted for removing the superhelical stress created by the replication and transcription machinery. The two closely related type IIA enzymes (Topo II␣ and Topo II␤) may also catalyze these same reactions but probably play a more critical role in processes like chromatin reorganization and chromosome segregation. A third class of type IA enzymes (Topo III␣ and Topo III␤)

Human genome-wide RNAi screen reveals a role for nuclear pore proteins in poxvirus morphogenesis

“…We found that knockdown of Nup62 inhibited the production of infectious VACV in a one-step growth experiment, but had no effect on entry and only modest effects on DNA replication and early and late viral gene expression. In addition, Nup62 knockdown did not prevent the previously reported association of cellular topoisomerase II with viral factories (34). However, TEM revealed a severe block in VACV morphogenesis beyond the IV stage.…”

“…Cellular topoisomerase II, which is present predominantly in the nucleus, associates with the VACV DNA ligase in cytoplasmic viral factories (34). Localization of topisomerase II with viral factories was not prevented by knockdown of Nup62 (Fig.…”

Triad of human cellular proteins, IRF2, FAM111A, and RFC3, restrict replication of orthopoxvirus SPI-1 host-range mutants

“…Nevertheless, that result relates to our current finding that the cellular DNA replication proteins FAM111A and RFC are host factors that restrict replication of the SPI-1 mutant. Thus far, investigations of poxvirus DNA replication have focused on viral proteins, except to show that cellular topoisomerase II is recruited by the viral DNA ligase to sites of viral DNA replication (30) and that cellular DNA ligase 1 can substitute for the viral ligase (29). At this time we can only suggest a speculative model for the roles of SPI-1, IRF2, FAM111A, and RFC3 in host restriction of poxviruses that may be useful for designing further studies (Fig.…”

“…Barrier-to-autointegration factor is an example of a host DNA-binding protein that inhibits VACV replication unless inactivated by the poxvirus B1 kinase (28). No cellular proteins essential for poxvirus DNA replication have been identified, although DNA ligase 1 can substitute for the VACV DNA ligase (29) and topoisomerase II is recruited to sites of viral DNA replication (30).…”

Poxvirus Proteomics and Virus-Host Protein Interactions

“…Additional experiments suggest that this virus-host PPI plays a positive role in promoting viral growth and perhaps contributes to the virulence of VACV in neural cells (109). Finally, a Y2H screen was used to investigate protein binding partners of VACV ligase (the A50R gene), and one of the nine host proteins identified was human topoisomerase II (95). This particular interaction was further confirmed by coimmunoprecipitation methods that showed that native ligase and a Flagtagged recombinant protein form complexes with human topoisomerase II in virus-infected cells (95).…”