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

Richard Pyles, Richard L. Thompson

Research output: Contribution to journalArticle

40 Citations (Scopus)

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 1994
Externally publishedYes

Fingerprint

Human herpesvirus 1
Uracil-DNA Glycosidase
Human Herpesvirus 1
Mutation Rate
Thymidine Kinase
Cultured Cells
Viruses
mutation
mutants
Mutation
DNA
mice
thymidine kinase
viruses
cells
Galactosidases
NIH 3T3 Cells
DNA Viruses
Simplexvirus
Natural History

ASJC Scopus subject areas

  • Immunology

Cite this

@article{946c1588690840e6921cb8272a25d52c,
title = "Mutations in accessory DNA replicating functions alter the relative mutation frequency of herpes simplex virus type 1 strains in cultured murine cells",
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.",
author = "Richard Pyles and Thompson, {Richard L.}",
year = "1994",
month = "7",
language = "English (US)",
volume = "68",
pages = "4514--4524",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "7",

}

TY - JOUR

T1 - Mutations in accessory DNA replicating functions alter the relative mutation frequency of herpes simplex virus type 1 strains in cultured murine cells

AU - Pyles, Richard

AU - Thompson, Richard L.

PY - 1994/7

Y1 - 1994/7

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0028318584&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028318584&partnerID=8YFLogxK

M3 - Article

VL - 68

SP - 4514

EP - 4524

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 7

ER -