Vaccinia, the prototypic poxvirus, is a complex DNA virus which replicates entirely within the cytoplasm of infected cells. Physical autonomy from the host nucleus is accompanied by genetic autonomy: the 192 kb genome encodes virtually all of the functions required for viral replication. Vaccinia replicates to high titers after inducing a rapid and synchronous infectious cycle in which temporally distinct phases of gene expression, DNA replication and morphogenesis are easily studied. Viral genes, which lack introns, are regulated by short motifs which determine their temporal expression. Genetic analysis is facilitated by the haploid nature of the genome and the high levels of recombination which permit exchange of endogenous sequences with those introduced by transfection. Despite the fact that the cis and trans components of transcription and replication are unique to the virus, many viral proteins are both structurally and functionally analogous to their eucaryotic counterparts. This observation ensures that an understanding of vaccinia virus will yield information of general relevance. This proposal represents our ongoing interest in identifying and characterizing the repertoire of viral proteins which mediate viral DNA replication. To date these proteins include the essential DNA polymerase, DS protein and 34 kDa protein kinase, as well as three enzymes involved in nucleotide metabolism; the virus also encodes a DNA ligase and topoisomerase 1 whose roles in DNA replication are speculative. Using a variety of genetic and biochemical approaches, we will: (1) characterize the B1 kinase in depth by performing structure/function analysis, define its consensus recognition motif, identify in vivo substrates and accessory proteins, and dissect its role in regulating virus replication; (2) purify the D5 protein and determine its biochemical and enzymatic properties, identify other components of the replication apparatus with which it interacts, and pursue its possible involvement in homologous recombination; (3) seek to identify other elements of the replication apparatus, such as an initiating nuclease, single-strand DNA binding protein, and DNA helicase; the phosphoprotein encoded by the I3 gene will also be studied as a candidate DNA binding protein, and genetic strategies for isolating additional DNA mutants will be implemented; (4) characterize the kinetic parameters, pharmacological sensitivities, and fidelity of the wt DNA polymerase and eight mutant enzymes which have altered drug sensitivities and/or fidelity. Purified polymerase will be used in an in vitro complementation assay to identify viral components which enable the polymerase to move processively through barriers of template secondary structure. These studies should enrich our understanding of vaccinia virus replication and provide comparative information for investigators probing similar questions in other systems. The importance of understanding replication enzymes in detail is underscored by the extent to which anti-viral and anti-neoplastic therapies are aimed at inhibiting these enzymes. Vaccinia's historical role as the virus used in the successful vaccination campaign to eradicate smallpox, and its current development as a recombinant vaccine, confer biomedical significance on what is intrinsically an interesting and accessible experimental system.
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