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 1 (HSV-1)-encoded ur acil 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 th e UNG, TK, or dUTPase activity were generated by disruption of the enz yme coding regions with the Escherichia coli beta-galactosidase (beta- gal) gene in strain 17syn(+). To establish a baseline RMF of strain 17 syn(+), the beta-gal gene was inserted into the UL3 locus. In all of t he viruses, the beta-gal insert served as a phenotypic marker of RMP. A mutant plaque,vas identified by the lack of beta-gal activity and, i n selected cases, positive in situ hybridization for beta-gal sequence s. Replication kinetics in NIH 3T3 cells demonstrated that all of the mutants replicated efficiently, generating stocks with equivalent tite rs. Two independently generated UL3-beta-gal viruses were examined and established a baseline RMF of similar to 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 f unction. 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 act ivity. The RMF of tito independent UNG(-) viruses showed no significan t difference from the baseline RMF in limited passage; however, follow ing successive passage, the data suggested that UNG activity serves as an antimutator. These results have implications for the natural histo ry of HSV and the development of antiviral therapies.