Genetic characterization of the vaccinia virus DNA polymerase: identification of point mutations conferring altered drug sensitivities and reduced fidelity

Abstract

We determined that 85 ,iM aphidicolin was sufficient to block macroscopic plaque formation by vaccinia virus and to cause a 104-fold reduction in viral yield from a wild-type infection. A chemically mutagenized viral stock was passaged sequentially in the presence of drug, and plaque-purified viral stocks resistant to aphidicolin were isolated and characterized. By use of a marker rescue protocol, the lesion in each mutant was found to map within the same 500-bp fragment within the DNA polymerase gene. All of the mutants were found to contain a single nucleotide change in the same codon. In nine of these mutants, the alanine residue at position 498 was changed to a threonine, whereas a 10th mutant sustained a valine substitution at this position. Congenic viral strains which carried the Aphr lesion in an unmutagenized wild-type background were isolated. The Thr and Val mutations were found to confer equivalent levels of drug resistance. In the presence of drug, viral yields were 25% of control levels, and the levels of viral DNA synthesized were 30 to 50% of those seen in control infections. The two mutations also conferred an equivalent hypersensitivity to the cytosine analog

Transient expression of the vaccinia virus DNA polymerase is an intrinsic feature of the early phase of infection and is unlinked to DNA replication and late gene expression

“…5). AraC and aphidicolin are known to effectively inhibit viral DNA replication (38), and examination of metabolically labeled proteins synthesized during infections performed with araC or aphidicolin verified that both drugs prevent any detectable late protein synthesis (data not shown). These results confirm our proposition that DNA polymerase synthesis is regulated independently of DNA replication and of intermediate and late gene expres-sion.…”

“…DMEM lacking methionine was purchased from Flow Laboratories (Irvine, United Kingdom). Stocks of aphidicolin (10 mM), araC (1 mM), PAA (10 mg/ml), and cycloheximide (10 mg/ml) were prepared and stored as previously reported (38).…”

“…In a series of time course experiments, mouse L cells were infected at 39.5°C with wt virus at an MOI of 15. AraC, aphidicolin, or PAA was added to the culture medium following adsorption of the inoculum (38). At various times postinfection, cultures were either pulselabeled with [35S]methionine in preparation for the production of protein lysates or harvested for the purpose of isolating of total mRNA.…”

“…The ordinate represents the intensity of the radiolabeled signals in arbitrary units; the abscissa represents hours postinfection. (38). ARP-12 has been shown to contain a mutation within the DNA polymerase gene which renders the virus resistant to high levels of aphidicolin; the availability of this virus allowed us to separate the inhibitory effect of aphidicolin on viral DNA replication from other effects that the drug may have.…”

“…We have chosen to investigate the regulation of the vaccinia virus DNA polymerase gene as a representative member of an important group of early genes. The 116-kDa polymerase is an essential component of the viral replication machinery; several temperature-sensitive (ts) and drug-resistant alleles of the enzyme have been characterized, and their impact on viral infection has been demonstrated (6,8,37,38,42). In this report, we describe both the temporal pattern of DNA polymerase synthesis and the steady-state levels of the polymerase mRNA throughout wild-type (wt) infections.…”