The herpes simplex virus type 1 temperature-sensitive mutant ts1222 has a single basepair deletion in the small subunit of ribonucleotide reductase

Abstract

The herpes simplex virus type 1 (HSV-1) temperature-sensitive (ts) mutant, ts1222, 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 ts1222 did not induce detectable amounts of enzyme activity at both 31 degrees and 39.5 degrees, it replicated as well as wild-type virus at 31 degrees in exponentially growing tissue culture cells under one step growth conditions. At 39.5 degrees, however, ts1222 behaved as a ts 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 ts phenotype of the mutant. In the presence of hydroxyurea, which inactivates both the cellular and virus ribonucleotide reductases, growth of the mutant at 31 degrees 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 ts1222 is dependent on the cellular ribonucleotide reductase for growth at this temperature. In serum-starved cells, growth of the mutant virus at 31 degrees was severely impaired. Thus, like thymidine kinase, the HSV-coded ribonucleotide reductase is required for virus multiplication in resting tissue culture cells.

Selectable insertion and deletion mutagenesis of the human cytomegalovirus genome using the Escherichia coli guanosine phosphoribosyl transferase (gpt) gene

“…The role of HSV-1 genes in contact-inhibited cells or in animal models has been employed to reveal phenotypes not otherwise obvious in cell culture (Bolovan et al, 1994;Preston et al, 1988;Roizman & Sears, 1993). The normal growth of a US3 mutant in both of these situations suggests that US3 does not play a role that is critical to virus growth, and leaves open the question of how this function may be important to CMV biology or pathogenesis.…”

Ribonucleotide Reductase Encoded by Herpes Simplex Virus Is a Determinant of the Pathogenicity of the Virus in Mice and a Valid Antiviral Target

“…Second, an HSV-1 insertion mutant which lacks most of RR1 (including the region sharing homology with RR1 from other viruses) induces no detectable RR activity and produces only a four-to fivefold lower yield of progeny virus in exponentially gx'owing tissue culture cells than does the parent virus (Goldstein & Weller, 1988). Furthermore, in our laboratory we have recently isolated a ts mutant, ts1222, which fails to induce detectable levels of RR activity at both permissive temperature (PT) and NPT (Preston et al, 1988). It is possible that under some conditions the cellular enzyme might substitute for the virus enzyme.…”

“…1). Cells infected with this virus at either 31 °C or the NPT exhibit no detectable virusspecific RR activity (Preston et al, 1988). The spontaneous revertant ts ÷ 1222 rev 1, which is able to grow at the NPT and has RR activity, was obtained from a plaque-purified low passage stock of ts1222.…”

RR1 and RR2 gene deletion affects the immunogenicity of a live attenuated pseudorabies virus vaccine candidate in natural pig host

“…A RR1-deficient HSV-1 mutant showed poor replication in non-dividing cells [14] . However, the virus growth in dividing cells was found to be much better given that deoxyribonucleotides for virus replication were provided by cellular nucleotide metabolism [13] .…”