A double-strand break in a herpesvirus genome stimulates targeted homologous recombination with exogenous, cloned viral sequences.
A method is described for the highly efficient recovery of recombinant pseudorabies virions; the approach should be applicable to other herpesviruses. Pseudorabies virus (PRV) strain PRV509 contains a unique EcoRI site in its genome, largely replacing the glycoprotein gC gene. By digesting PRV509 DNA with EcoRI prior to cotransfection with plasmid DNA that harbored a cloned copy of gC, we isolated recombinant viruses containing the cloned gC allele at a frequency exceeding 75%. This represented u to a 37-fold increase over the use of intact viral DNA in cotransfection experiments, and may eliminate the need for phenotypic screening of recombinants. Closer analysis of the recombinant viruses revealed that genetic markers up to 1 kilobase pair apart could be recombined into the genome using the EcoRI-digested DNA. Overall, the increased frequency of recombinant viruses can be explained if homologous recombination at sites of double-strand breakage is a more efficient repair mechanism than the re-annealing and ligation of the break itself.
Ryan
P
P
Department of Microbiology and Immunology, University of Tennessee at Memphis 38163, USA.
Shankly
F L
FL
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D003434:Crossing Over, Genetic; D004249:DNA Damage; D004279:DNA, Viral / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D016679:Genome, Viral; D011558:Herpesvirus 1, Suid / Q000235:genetics*; D011995:Recombination, Genetic; D013552:Swine; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics*
D004279:DNA, Viral; D014759:Viral Envelope Proteins; C042966:pseudorabies virus glycoproteins; D015246:Deoxyribonuclease EcoRI