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

Richard B. Pyles, Richard L. Thompson

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)4514-4524
Number of pages11
JournalJournal of virology
Volume68
Issue number7
StatePublished - Jul 1 1994
Externally publishedYes

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ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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