Construction and transposon mutagenesis in Escherichica 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 const ructed as a stable F plasmid in Escherichia coli. The clone, pBecker1, was colinear with PRV-Becker genomic DNA, lacking detectable rearrangements, de letions, or inversions. The transfection of pBecker1 into susceptible eukar yotic cells resulted in productive viral infection. Virus isolated followin g 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 inse rtion mutants, identification of essential viral genes, and simplified cons truction of viral revertants. The serial passage of a viral insertion mutan t demonstrated the transposon insertion to be stable. However, the F-plasmi d insertion present in the viral gG locus was found to undergo a spontaneou s deletion following transfection into eukaryotic cells. The implications o f F-plasmid insertion into the viral genome with regard to phenotype and ge nomic stability are discussed.