Poxviruses encode enzymes and factors needed for replication of their genomes within the cytoplasm of infected cells. Vaccinia virus, the prototypic member of the poxvirus family, provides a unique system for combining biochemical and genetic approaches for investigating mechanisms of DNA replication. The DNA is synthesized as concatemers that are resolved into unit length genomes and packaged during virus assembly. Studies with conditional lethal mutants indicate that five VACV early proteins are required for DNA replication: namely E9 DNA polymerase, D4 uracil DNA glycosylase, A20 processivity factor, B1 protein kinase and D5 nucleoside triphosphatase (NTPase). The DNA polymerase catalyzes primer- and template-dependent synthesis and possesses 3 to 5 exonucleolytic activity. The essential role of D4 in DNA replication is independent of its uracil DNA glycosylase activity, which presumably has a facultative repair function. The A20 and D4 proteins interact and together provide processivity for the DNA polymerase. The B1 kinase was recently shown to phosphorylate a cellular DNA-binding protein called BAF and prevent the latter from blocking VACV DNA replication. The fast stop DNA replication phenotype of conditional lethal D5 mutants suggested a function at the replication fork. D5 also interacts with A20 and forms multimers. Potential roles for D5 have come from extensive protein sequence analyses, which indicate that the 90-kDa D5 protein is a member of the helicase superfamily III within the AAA+ class of NTPases, which includes the replicative helicases of some other DNA and RNA viruses. Recently, the D5 protein was shown to have a second function as a DNA primase.

Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2010
Total Cost
$931,112
Indirect Cost
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State
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Senkevich, Tatiana G; Bruno, Daniel; Martens, Craig et al. (2015) Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing. Proc Natl Acad Sci U S A :
Moss, Bernard (2013) Poxvirus DNA replication. Cold Spring Harb Perspect Biol 5:
Bengali, Zain; Satheshkumar, P S; Yang, Zhilong et al. (2011) Drosophila S2 cells are non-permissive for vaccinia virus DNA replication following entry via low pH-dependent endocytosis and early transcription. PLoS One 6:e17248
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Senkevich, Tatiana G; Koonin, Eugene V; Moss, Bernard (2009) Predicted poxvirus FEN1-like nuclease required for homologous recombination, double-strand break repair and full-size genome formation. Proc Natl Acad Sci U S A 106:17921-6
Satheshkumar, P S; Anton, Luis C; Sanz, Patrick et al. (2009) Inhibition of the ubiquitin-proteasome system prevents vaccinia virus DNA replication and expression of intermediate and late genes. J Virol 83:2469-79
De Silva, Frank S; Paran, Nir; Moss, Bernard (2009) Products and substrate/template usage of vaccinia virus DNA primase. Virology 383:136-41
De Silva, Frank S; Moss, Bernard (2008) Effects of vaccinia virus uracil DNA glycosylase catalytic site and deoxyuridine triphosphatase deletion mutations individually and together on replication in active and quiescent cells and pathogenesis in mice. Virol J 5:145