Vaccinia virus encodes a type I DNA topoisomerase that is highly conserved in all known poxviruses. Although the structure and catalytic activity of the enzyme were well studied, little was known about its biological function. The viral topoisomerase was thought to be essential, and roles in DNA replication, recombination, concatemer resolution, and transcription were suggested. Here, we demonstrated that the topoisomerase is not essential for replication of vaccinia virus in cultured cells, although deletion mutants formed fewer and smaller plaques on cell monolayers than wild-type virus. Purified mutant virus particles were able to bind and enter cells but exhibited reduced viral early transcription and a delay in DNA replication. Infecting with a high number of virus particles increased early mRNA and accelerated viral DNA synthesis. Processing of viral DNA concatemers into unit-length genomes was unimpaired at either a low or high multiplicity of infection. The data suggest that the primary, perhaps only, role of the poxvirus topoisomerase is to increase early transcription, which takes place within virus cores in the cytoplasm of infected cells. Because the topoisomerase functions early in infection, drugs capable of penetrating the virus core and irreversibly damaging DNA by trapping nicked DNA–topoisomerase intermediates could make potent antiviral agents
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