Mutations in accessory DNA replicating functions alter the relative mutation frequency of herpes simplex virus type 1 strains in cultured murine cells

The contribution of the herpes simplex virus type I (HSV-1)-encoded uracil DNA glycosylase (UNG), thymidine kinase (TK), and dUTPase to the relative mutant frequency (RMF) of the virus in cultured murine cells was examined. A panel of HSV-1 mutants that lacked singly or doubly the UNG, TK, or dUTPase activity were generated by disruption of the enzyme coding regions with the Escherichia coli β-galactosidase (β-gal) gene in strain 17syn⁺. To establish a baseline RMF of strain 17syn⁺, the β-gal gene was inserted into the UL3 locus. In all of the viruses, the β-gal insert served as a phenotypic marker of RMF. A mutant plaque was identified by the lack of β- gal activity and, in selected cases, positive in situ hybridization for β- gal sequences. Replication kinetics in NIH 3T3 cells demonstrated that all of the mutants replicated efficiently, generating stocks with equivalent titers. Two independently generated UL3-β-gal viruses were examined and established a baseline RMF of ~0.5% in both NIH 3T3 and LM TK^- cells. Loss of dUTPase activity resulted in viruses with fivefold-increased RMFs, indicating that the HSV-1 dUTPase has an antimutator function. The RMF observed for the tk^- viruses was reduced as much as 40-fold (RMF of 0.02%), suggesting that the viral TK is a mutator activity. The RMF of two independent UNG^- viruses showed no significant difference from the baseline RMF in limited passage; however, following successive passage, the data suggested that UNG activity serves as an antimutator. These results have implications for the natural history of HSV and the development of antiviral therapies.