Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alphaherpesvirus.
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.
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
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D000483:Alleles; D000818:Animals; D002460:Cell Line; D004251:DNA Transposable Elements; D004926:Escherichia coli; D005818:Genetic Engineering; D011558:Herpesvirus 1, Suid / Q000235:genetics* / Q000472:pathogenicity*; D008297:Male; D016254:Mutagenesis, Insertional; D010957:Plasmids; D011557:Pseudorabies / Q000473:pathology / Q000821:virology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013552:Swine
D004251:DNA Transposable Elements