Construction and transposon mutagenesis in Escherichia coli of a full- length infectious clone of pseudorabies virus, an alphaherpesvirus

Gregory A. Smith, Lynn W. Enquist*

*Corresponding author for this work

Research output: Contribution to journalArticle

207 Scopus citations

Abstract

A full-length clone of the 142-kb pseudorabies virus (PRV) genome was constructed as a stable F plasmid in Escherichia coli. The clone, pBecker1, was colinear with PRV-Becker genomic DNA, lacking detectable rearrangements, deletions, or inversions. The transfection of pBecker1 into susceptible eukaryotic cells resulted in productive viral infection. Virus isolated following transfection was indistinguishable from wild-type virus in a rodent model of infection and spread to retinorecipient regions of the brain following inoculation in the vitreous body of the eye. Mutagenesis of pBecker1 in E. coli with a mini-Tn5-derived transposon enabled the rapid isolation of insertion mutants, identification of essential viral genes, and simplified construction of viral revertants. The serial passage of a viral insertion mutant demonstrated the transposon insertion to be stable. However, the F-plasmid insertion present in the viral gG locus was found to undergo a spontaneous deletion following transfection into eukaryotic cells. The implications of F-plasmid insertion into the viral genome with regard to phenotype and genomic stability are discussed.

Original languageEnglish (US)
Pages (from-to)6405-6414
Number of pages10
JournalJournal of virology
Volume73
Issue number8
DOIs
StatePublished - Jan 1 1999

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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