Minute virus of mice (MVM) is an autonomous parvovirus which exhibits teratogenic and tumor-suppressive activity in vivo. We propose to define precisely the mechanism by which MVM replicates its genome and to elucidate the functions that both viral and cellular proteins perform in the replication process. The primary gene products of MVM will be enumerated, and the specific transcripts coding for them identified, by in vitro translation of infected cell RNA purified by hybridization to cloned segments of the viral genome. The coding regions which are spliced together to produce these polypeptides will be determined by cloning and sequencing cDNA copies of each vira mRNA species. The unique and shared regions of each viral gene will then be located within the entire DNA sequence of MVM, which has recently been determined. Specific mutations will be introduced into unique coding sequences in an infectious viral genome which as been cloned into a bacterial plasmid. Similarly, mutations will be introduced into the terminal regions of the genome, known to contain all of the cis-acting functions essential for viral DNA replication and progeny virion maturation. Mutant plasmids will then be transfected into appropriate host cells and the biological functions of these regions in the viral life cycle analyzed. A parallel approach, designed also to identify cellular factors essential for viral DNA synthesis, will examine the components of viral DNA replication complexes. These will be isolated by both conventional purification schemes and by affinity chromatography on avidin-Sepharose following in vivo pulse-labelling with biotinyl nucleotides. The proteins present in these complexes will be separated and characterized as to genetic origin using immunological reagents, 2-D gel electrophoresis and peptide-mapping. In addition, each component of the complex will be evaluated for specific enzymatic activities. Finally, monoclonal antibodies against each will be prepared and used to purify the protein and further investigate its function. These purified components will be used to reconstruct the viral DNA replication machinery in vitro. Since parvoviruses have a limited genetic capacity and rely extensively upon host cell functions for their growth, we anticipate that our studies will also provide some basic insights into the mechanism of eukaryotic cell DNA replication.
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