The herpes simplex virus type 1 temperature-sensitive mutantts1222 has a single base pair deletion in the small subunit of ribonucleotide reductase

Abstract The herpes simplex virus type 1 (HSV-1) temperature-sensitive ( ts ) mutant, ts 1222, has a defect within the gene specifying the small subunit of ribonucleotide reductase. Sequence determination of the lesion revealed that the mutant DNA had a single base pair deletion at the 3′ end of the gene. The mutation altered the translational reading frame such that the codons of all but one of the last 15 amino acids of the protein were changed and the termination codon removed. Although ts 1222 did not induce detectable amounts of enzyme activity at both 31° and 39.5°, it replicated as well as wild-type virus at 31° in exponentially growing tissue culture cells under one step growth conditions. At 39.5°, however, ts 1222 behaved as a is mutant. These findings suggest that at low temperatures the virus-coded enzyme is dispensable for virus growth in actively dividing tissue culture cells but at high temperatures the enzyme is essential for virus replication. Under these conditions altered properties of the host cell contribute to the is phenotype of the mutant. In the presence of hydroxyurea, which inactivates both the cellular and virus ribonucleotide reductases, growth of the mutant at 31° was inhibited more than wild-type virus replication. Growth of the mutant at the permissive temperature was also sensitive to high concentrations of thymidine whereas wild-type virus multiplication was resistant to the nucleoside. It is therefore likely that ts 1222 is dependent on the cellular ribonucleotide reductase for growth at this termperature. In serum-starved cells, growth of the mutant virus at 31° was severely impaired. Thus, like thymidine kinase, the HSV-coded ribonucleotide reductase is required for virus multiplication in resting tissue culture cells.