Genetic characterization of the vaccinia virus DNA polymerase: cytosine arabinoside resistance requires a variable lesion conferring phosphonoacetate resistance in conjunction with an invariant mutation localized to the 3'-5' exonuclease domain. J Virol 1993 Jul;67(7):4323-36
Date
07/01/1993Pubmed ID
8389930Pubmed Central ID
PMC237803DOI
10.1128/JVI.67.7.4323-4336.1993Scopus ID
2-s2.0-0027163809 (requires institutional sign-in at Scopus site) 47 CitationsAbstract
In this report, we describe the isolation, molecular genetic mapping, and phenotypic characterization of vaccinia virus mutants resistant to cytosine arabinoside (araC) and phosphonoacetic acid (PAA). At 37 degrees C, 8 microM araC was found to prevent macroscopic plaque formation by wild-type virus and to cause a 10(4)-fold reduction in viral yield. Mutants resistant to 8 microM araC were selected by serial passage of a chemically mutagenized viral stock in the presence of drug. Because recovery of mutants required that initial passages be performed under less stringent selective conditions, and because plaque-purified isolates were found to be cross-resistant to 200 micrograms of PAA per ml, it seemed likely that resistance to araC required more than one genetic lesion. This hypothesis was confirmed by genetic and physical mapping of the responsible mutations. PAAr was accorded by the acquisition of one of three G-A transitions in the DNA polymerase gene which individually alter cysteine 356 to tyrosine, glycine 372 to aspartic acid, or glycine 380 to serine. AraCr was found to require one of these substitutions plus an additional T-C transition within codon 171 of the DNA polymerase gene, a change which replaces the wild-type phenylalanine with serine. Congenic viral stocks carrying one of the three PAAr lesions, either alone or in conjunction with the upstream araCr lesion, in an otherwise wild-type background were generated. The PAAr mutations conferred nearly complete resistance to PAA, a slight degree of resistance to araC, hypersensitivity to aphidicolin, and decreased spontaneous mutation frequency. Addition of the mutation at codon 171 significantly augmented araC resistance and aphidicolin hypersensitivity but caused no further change in mutation frequency. Several lines of evidence suggest that the PAAr mutations primarily affect the deoxynucleoside triphosphate-binding site, whereas the codon 171 mutation, lying within a conserved motif associated with 3'-5' exonuclease function, is postulated to affect the proofreading exonuclease of the DNA polymerase.
Author List
Taddie JA, Traktman PMESH terms used to index this publication - Major topics in bold
AnimalsAphidicolin
Base Sequence
Cell Line
Cytarabine
DNA-Directed DNA Polymerase
Drug Resistance
Exonucleases
In Vitro Techniques
Mice
Molecular Sequence Data
Mutation
Nucleic Acid Synthesis Inhibitors
Oligodeoxyribonucleotides
Phosphonoacetic Acid
Restriction Mapping
Sequence Alignment
Species Specificity
Structure-Activity Relationship
Vaccinia virus
Virus Replication
