Hydroxyurea-resistant vaccinia virus: overproduction of ribonucleotide reductase.
Repeated passages of vaccinia virus in increasing concentrations of hydroxyurea followed by plaque purification resulted in the isolation of variants capable of growth in 5 mM hydroxyurea, a drug concentration which inhibited the reproduction of wild-type vaccinia virus 1,000-fold. Analyses of viral protein synthesis by using [35S]methionine pulse-labeling at intervals throughout the infection cycle revealed that all isolates overproduced a 34,000-molecular-weight (MW) early polypeptide. Measurement of ribonucleoside-diphosphate reductase (EC 1.17.4.1) activity after infection indicated that 4- to 10-fold more activity was induced by hydroxyurea-resistant viruses than by the wild-type virus. A two-step partial purification which yielded greater than 90% of the induced ribonucleotide reductase activity in the fraction obtained by 35% saturation with ammonium sulfate resulted in a substantial enrichment for the 34,000-MW protein from extracts of wild-type and hydroxyurea-resistant-virus-infected, but not mock-infected, cells. In the presence of the drug, the isolates incorporated [3H]thymidine into DNA earlier and at a rate substantially greater than that of the wild type, although the onset of DNA synthesis was delayed in both cases. In the absence of the drug, the attainment of a maximum viral DNA synthesis rate was accelerated after infection by drug-resistant isolates. The drug resistance trait was markedly unstable in all isolates. In the absence of selective pressure, plaque-purified isolates readily segregated progeny that displayed a wide range of resistance phenotypes. The results of this study indicate that vaccinia virus encodes a subunit of ribonucleotide reductase which is a 34,000-MW early protein whose overproduction confers hydroxyurea resistance on reproducing viruses.
D004279:DNA, Viral / Q000096:biosynthesis; D004352:Drug Resistance, Microbial; D006918:Hydroxyurea / Q000494:pharmacology*; D008970:Molecular Weight; D010641:Phenotype; D012262:Ribonucleoside Diphosphate Reductase / Q000096:biosynthesis* / Q000302:isolation & purification; D012264:Ribonucleotide Reductases / Q000096:biosynthesis*; D014616:Vaccinia virus / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis
D004279:DNA, Viral; D014764:Viral Proteins; D012264:Ribonucleotide Reductases; D012262:Ribonucleoside Diphosphate Reductase; D006918:Hydroxyurea