Rapid identification of essential and nonessential herpesvirus genes by direct transposon mutagenesis.
Herpesviruses are important pathogens in animals and humans. The large DNA genomes of several herpesviruses have been sequenced, but the function of the majority of putative genes is elusive. Determining which genes are essential for their replication is important for identifying potential chemotherapy targets, designing herpesvirus vectors, and generating attenuated vaccines. For this purpose, we recently reported that herpesvirus genomes can be maintained as infectious bacterial artificial chromosomes (BAC) in Escherichia coli. Here we describe a one-step procedure for random-insertion mutagenesis of a herpesvirus BAC using a Tn1721-based transposon system. Transposon insertion sites were determined by direct sequencing, and infectious virus was recovered by transfecting cultured cells with the mutant genomes. Lethal mutations were rescued by cotransfecting cells containing noninfectious genomes with the corresponding wild-type subgenomic fragments. We also constructed revertant genomes by allelic exchange in bacteria. These methods, which are generally applicable to any cloned herpesvirus genome, will facilitate analysis of gene function for this virus family.
Brune
W
W
Department of Virology, Max von Pettenkofer-Institut, Ludwig-Maximilians-Universität München, Germany.
Ménard
C
C
Hobom
U
U
Odenbreit
S
S
Messerle
M
M
Koszinowski
U H
UH
D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics; D004251:DNA Transposable Elements; D004279:DNA, Viral / Q000235:genetics; D020043:Genes, Essential; D005814:Genes, Viral; D008969:Molecular Sequence Data; D018146:Muromegalovirus / Q000235:genetics* / Q000254:growth & development; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA
D004251:DNA Transposable Elements; D004279:DNA, Viral