Analysis of DNA polymerase activities involved in bovine parvoviral DNA replication

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Virginia Polytechnic Institute and State University


The polymerase activities involved in bovine parvoviral (BPV) DNA replication in vivo and in vitro have been described. In the in vitro system, purified and partially purified enzymes were used and the replication products were analyzed by gel electrophoresis and restriction enzyme digestion. DNA pol ૪ purified from fetal bovine liver, replicated BPV ssDNA to a unit-length covalently-linked dsDNA hairpin molecule but was unable to utilize purified BPV dsDNA as a template. Partially purified calf thymus DNA pol α replicated BPV DNA to a product 1 kbp smaller than unit-length dsDNA (11 kbp). Mapping of this product showed that the middle of the genome was under-represented. Purified pol a from bovine fetal lung (BFL) cells was capable of only end-labeling BPV ssDNA. If HeLa cell DNA pol α, which consisted of the core enzyme plus cofactors C₁ C₂, was used, the products consisted of both noncovalently- and covalently-linked unit-length dsDNA hairpin molecules. Hence, purification of pol a removed factor(s) necessary for the activity of the enzyme on B PV DNA. The polymerase activities involved in vivo in BPV DNA replication were analyzed using aphidicolin, a specific inhibitor of DNA pol α, and/or L-canavanine, an inhibitor of protein synthesis. DNA present in infected cells was visualized by autoradiography of Southern blots after probing with nick-translated BPV DNA. Aphidicolin, added at any time after-infection, reversibly inhibited each step of BPV DNA synthesis. Conversely, L-canavanine slowed the replication process, inhibited the synthesis of the viral-coded proteins NP-1 and VP3, and inhibited the production of replicative intermediates (RI) and progeny ssDNA. After removal of L-canavanine, both protein and DNA synthesis resumed. These results demonstrate that 1) pol α is involved in every stage of the replication process including the production of parental replicative form (RF), daughter RF, RI, and progeny genomes, 2) taken in conjunction with the in vitro data, that a pol α holoenzyme complex is required for BPV DNA replication, and 3) viral proteins are required for RI and progeny DNA synthesis,