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

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-deg rees-C, 8 muM araC was found to prevent macroscopic plaque formation b y wild-type virus and to cause a 10(4)-fold reduction in viral yield. Mutants resistant to 8 muM araC were selected by serial passage of a c hemically mutagenized viral stock in the presence of drug. Because rec overy of mutants required that initial passages be performed under les s stringent selective conditions, and because plaque-purified isolates were found to be cross-resistant to 200 mug 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 r esponsible mutations. PAA(r) was accorded by the acquisition of one of three G-A transitions in the DNA polymerase gene which individually a lter cysteine 356 to tyrosine, glycine 372 to aspartic acid, or glycin e 380 to serine. AraC(r) was found to require one of these substitutio ns plus an additional T-C transition within codon 171 of the DNA polym erase gene, a change which replaces the wild-type phenylalanine with s erine. Congenic viral stocks carrying one of the three PAA(r) lesions, either alone or in conjunction with the upstream araC(r) lesion, in a n otherwise wild-type background were generated. The PAA(r) mutations conferred nearly complete resistance to PAA, a slight degree of resist ance to araC, hypersensitivity to aphidicolin, and decreased spontaneo us mutation frequency. Addition of the mutation at codon 171 significa ntly augmented araC resistance and aphidicolin hypersensitivity but ca used no further change in mutation frequency. Several lines of evidenc e suggest that the PAA(r) mutations primarily affect the deoxynucleosi de triphosphate-binding site, whereas the codon 171 mutation, lying wi thin a conserved motif associated with 3'-5' exonuclease function, is postulated to affect the proofreading exonuclease of the DNA polymeras e.