Genetic characterization of the vaccinia virus DNA polymerase: identification of point mutations conferring altered drug sensitivities and reduced fidelity. J Virol 1991 Feb;65(2):869-79
Date
02/01/1991Pubmed ID
1898973Pubmed Central ID
PMC239827DOI
10.1128/JVI.65.2.869-879.1991Scopus ID
2-s2.0-0026020782 (requires institutional sign-in at Scopus site) 48 CitationsAbstract
We determined that 85 microM aphidicolin was sufficient to block macroscopic plaque formation by vaccinia virus and to cause a 10(4)-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 1-beta-D-arabinofuranosylcytosine (araC); strains carrying the Thr mutation were moderately hypersensitive to the pyrophosphate analog phosphonoacetic acid and the adenosine analog araA, whereas the Val mutation conferred acute hypersensitivity to these inhibitors. The Val mutation also conferred a mutator phenotype, leading to a 20- to 40-fold increase in the frequency of spontaneous mutations within the viral stock.
Author List
Taddie JA, Traktman PMESH terms used to index this publication - Major topics in bold
Amino Acid SequenceAnimals
Aphidicolin
Base Sequence
Cell Line
DNA, Viral
DNA-Directed DNA Polymerase
Diterpenes
Drug Resistance, Microbial
Genes, Viral
Molecular Sequence Data
Mutagenesis, Site-Directed
Mutation
Restriction Mapping
Vaccinia virus
Viral Plaque Assay
Viral Structural Proteins
Virus Replication
