Isolation and characterisation of herpes simplex virus type 1 mutants which fail to induce dUTPase activity

Abstract

The herpes simplex virus (HSV) type 1 dUTPase gene was inactivated by insertion of HindIII oligonucleotide linker sequences into the KpnI site within the coding region of the cloned gene. The mutated gene was introduced into wild type herpes simplex virus by marker rescue and the recombinants were identified by the acquisition of a HindIII site within genome map coordinates 0.69 to 0.70 and the failure to induce virus-specific dUTPase activity. A spontaneous dUTPase deficient mutant, which had an identical restriction endonuclease DNA pattern to wild type virus, was also isolated from this transfection experiment. Both types of dUTPase-negative mutants failed to induce a virus-specific 39,000 mol wt polypeptide. Cells infected with the insertional mutant contained instead a novel polypeptide about 40,000 mol wt. No abnormal virus specific polypeptide was detected in cells infected with the spontaneous mutant. We conclude that the 39,000 mol wt polypeptide induced by wild type HSV-1 is the virus-coded dUTPase. Since both types of mutants grew well in exponentially growing and serum-starved tissue culture cells in the absence of wild type helper virus, the dUTPase is not required for virus replication under these conditions. Thymidine kinase deficient, dUTPase deficient double mutants were constructed by recombination of a thymidine kinase insertional mutation into dUTPase deficient virus. These mutants also grew as well as wild type virus both in normal tissue culture cells and cells lacking the cellular thymidine kinase.