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 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 beta-galactosidase (beta-gal) gene in strain 17syn+. To establish a baseline RMF of strain 17syn+, the beta-gal gene was inserted into the UL3 locus. In all of the viruses, the beta-gal insert served as a phenotypic marker of RMF. A mutant plaque was identified by the lack of beta-gal activity and, in selected cases, positive in situ hybridization for beta-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-beta-gal viruses were examined and established a baseline RMF of approximately 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.
Pyles
R B
RB
Department of Molecular Genetics, Biochemistry and Microbiology, College of Medicine, University of Cincinnati, Ohio 45267-0524.
Thompson
R L
RL
D016475:3T3 Cells; D000818:Animals; D002478:Cells, Cultured; D045647:DNA Glycosylases; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D007700:Kinetics; D051379:Mice; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism; D011817:Rabbits; D013937:Thymidine Kinase / Q000378:metabolism; D051981:Uracil-DNA Glycosidase; D014779:Virus Replication; D001616:beta-Galactosidase / Q000235:genetics
D004279:DNA, Viral; D013937:Thymidine Kinase; D001616:beta-Galactosidase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase