The studies proposed will examine, in molecular detail, the mechanism by which the single-stranded DNA genome of the autonomous parvovirus MVM is replicated. The roles that both viral and cellular proteins play in the replication process will be investigated, and the target sequences within the viral DNA upon which they act will be analysed. Both trans- and cis-acting functions relevant to virus replication encoded in viral DNA will be analysed by developing further the genetics of the virus, concentrating upon the replicative consequences of mutations introduced into the non-structural (NS) protein genes and the palindromic termini of the genome. these effects will be assayed by the ability of cloned viral mutants to undergo detectable replication events after transfection into permissive host cells, and by the analysis of infection by virus stocks produced in cell lines expressing viral gene products capable of complementing such mutants. The various forms of the two non-structural proteins will be isolated and purified using conventional chromatographic procedures combined with immunoaffinity, and where appropriate, by recognition site affinity chromatography. Specific assays of target DNA sequence binding, nicking, terminal protein, single- and double-stranded DNA binding, topoisomerase and helicase activities will be conducted with purified NS proteins, or through immunoprecipitation-linked procedures on unfractionaed extracts. Terminal structures important for packaging and processing of the genome during replication will be explored by the construction of """"""""minimal replicon"""""""" genomes, carrying selectable marker genes, capable of replicating in complementing cell lines. Viral and host components important to the mechanics of viral DNA replication will be characterized by using immunological reagents, 2-D gel electrophoresis, peptide mapping, specific polymerase and accessory enzyme assays, and inhibitor studies of in vitro DNA synthesis, to analyze the proteins present in affinity-purified viral replication complexes isolated from infected cells.
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