The best-studied member of the poxvirus family, vaccinia is a large and complex virus. The virion is 200 nm in diameter, contains perhaps 100 different polypeptides, and encloses the 180 kilobase DNA genome. Vaccinia set the precedent for virions encapsidating enzymes as well as structural proteins. Upon entry into susceptible cells, these core enzymes initiate a rapid and lytic infectious cycle. Expression of the viral genes is temporally controlled, and DNA replication is required for the activation of the late class of viral genes. Infection is unusually automonous of host cell functions. Replication proceeds exclusively in the cytoplasm of infected cells, and vaccinia is thought to encode virtually all of the enzymes involved in transcription and DNA replication. For these reasons, the development of vaccinia as a model system for the study of eucaryotic DNA replication is proposed. The recent identification and characterization of the gene for the viral DNA polymerase (by the P.l.) was accomplished by combining genetics, enzymology and molecular biology. This approach will be expanded to identify many of the genes and proteins involved in DNA replication. (1) Existing knowledge about vaccinia enzymes will be used to identify homologous polypeptides among the translation products of viral mRNA hybrid-selected to fragments of the vaccinia genome. Genes so identified will be transcriptionally mapped. (2) The genomic lesions of DNA replication mutants will be mapped by marker rescue with progressively smaller fragments of the vaccinia genome. The transcripts and polypeptides encoded by the affected genes will be defined. (3) Genetic techniques will be used to select for temperature-sensitive and drug-resistant mutants with lesions in replication enzymes. (4) The DNA polymerase gene will be used as a model system for random and targeted in vitro mutagenesis. Altered copies of the gene will be reintroduced into the viral genome via marker rescue, and polymerase mutants will be isolated. This approach will be refined for future application to genes which have eluded classical genetic analysis. (5) The cytoplasmic localization of vaccinia infection, free from structural or functional interference from nuclear components, will be exploited in the refinement of an in vitro DNA replication system. Replication in fractionated cytoplasmic extracts will be used to analyze the lesions of replication mutants and to identify individual components of the replication machinery. In vitro complementation will provide a unification of genetic and biochemical analyses.
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