Robert C. Bates

Effect of bovine parvovirus replication on DNA, RNA, and protein synthesis in S phase cells

Abstract The kinetics of replication of bovine parvovirus (BPV) and the effect of viral replication on cellular macromolecular synthesis were examined in hydroxyurea (HU)-synchronized fetal bovine spleen cells. Immediately after release of cells from HU block, 80–85% of the cells began to synthesize DNA. The production of infectious progeny BPV proceeded at a faster rate in synchronized cells infected at the beginning of S phase than in asynchronous cultures. In synchronized cells, titers of infectious virus increased at 8 hr p.i. and the maximum titer was achieved by 20 hr. BPV DNA synthesis preceded the production of progeny virus by 2 hr. Although the rates of RNA and protein synthesis in infected cells were severely reduced after 8 hr p.i., BPV replication did not affect the rate of progression of cells through S phase.

Reversible Inhibition of Bovine Parvovirus DNA Replication by Aphidicolin and L-Canavanine

The replication of the autonomous parvovirus, bovine parvovirus (BPV), has been studied in virus-infected cells. Gel electrophoresis was used to determine the effect of aphidicolin, a specific inhibitor of DNA polymerase alpha, and L-canavanine, an inhibitor of protein synthesis, on viral DNA replication. Synchronized cell cultures were infected with 32P-labelled or unlabelled BPV in the presence or absence of aphidicolin and L-canavanine. Cells were harvested at various times post-infection, and DNA was electrophoresed and blotted. When aphidicolin was added to cells at the time of infection, then removed 8 h later, BPV replicative form DNA (RF) synthesis began within 2 h after its removal. This preceded the peak of cellular DNA synthesis by 2 h, unlike an uninhibited infection, when viral RF synthesis follows the peak of S phase by 2 to 4 h. Furthermore, if aphidicolin was added at any point during the replication cycle, BPV DNA synthesis stopped. This effect was shown to be completely reversible and indicated that aphidicolin did not disrupt the replication apparatus required for viral DNA synthesis. L-Canavanine inhibited synthesis of the virus-specific proteins NP-1 and VP3 and synthesis of BPV DNA. Upon removal of L-canavanine, viral protein synthesis was detected by 30 min followed by viral DNA synthesis. These results indicate that a specific S phase function other than cellular DNA synthesis is required for initiation of BPV DNA synthesis, that DNA polymerase alpha plays a major role in BPV DNA replication in vivo, and that these inhibitors can be used to inhibit reversibly various stages of BPV DNA replication.

Hydroxyurea synchronization of bovine fetal spleen cells

Abstract Hydroxyurea (HU) was shown to be an effective synchronization agent for bovine fetal spleen (BFS) cells. Following exposure of cells to 2 mM HU for 32 h, DNA synthesis above background levels was not observed. BFS cells released from the HU block by washing began to synthesize DNA immediately. Within 2 h, 80–85% of the cells were in S phase, as determined by autoradiography, and the maximum rate of DNA synthesis occurred 2–4 h following removal of HU. The rapid induction of DNA synthesis in BFS cells and the high percentage of cells synthesizing DNA immediately after removal of HU demonstrate that HU produces a highly synchronized population of S phase BFS cells. Although RNA and protein synthesis were maintained at near normal rates early after cells were exposed to HU, the rates decreased to 40–50% of those observed in cells seeded in medium without HU by the time of release. These reduced rates of synthesis of RNA and protein in the absence of DNA synthesis may account for the low toxicity of HU for BFS cells.

Possible sequences for nuclear accumulation of parvoviral proteins

Parvoviral genomes have been searched for sequences which may code for the nuclear transport of viral proteins. Sequences similar to those which regulate the nuclear transport of T antigen and yeast mating type protein were detected within the sequences coding for capsid and non-capsid proteins.

Purified DNA polymerase γ replicates bovine parvovirus DNA to a unit-length product

Abstract DNA polymerase gamma, purified from fetal bovine liver, replicated virion single-stranded DNA from bovine parvovirus to a unit-length double-stranded DNA molecule. This product was not nicked and was covalently linked to the 3′ hairpin primer. The reaction was inhibited by dideoxythymidine 5′-triphosphate, but was unaffected by ATP or aphidicolin. Double-stranded viral DNA was not a functional template for purified DNA polymerase gamma.

IN VITRO SYNTHESIS OF PARVOVIRAL DNA

ABSTRACT We have developed a nuclear lysate system capable of synthesizing unit length parvoviral DNA in vitro . In this system RF, RI, and single stranded DNA was synthesized. It was necessary to supplement the nuclear lysates with polyamines to prevent degradation of template and product DNA. The results show that viral RF DNA pulse-labeled in vivo and in vitro was chased into single stranded viral DNA in nuclear lysates. The single stranded DNA was synthesized by a continuous mechanism through replicative intermediates. The results of density labeling experiments suggest that the single stranded DNA was of one polarity. The RF DNA synthesized in nuclear lysates had identical restriction enzyme patterns to virion DNA replicated in vitro with E. Coli DNA polymerase 1.