FIG. 7.

Generation of a KSHV mutant with a deletion of the vIRF gene. (A) A Kan cassette PCR product flanking sequences from outside the vIRF gene was electroporated into E. coli previously transformed with BAC36 and plasmid pGETrec. Episomal DNA isolated from bacterial colonies containing the KSHV mutant in which the vIRF gene was replaced with Kan after a second round of transformation into E. coli to eliminate the pGETrec plasmid was analyzed by PCR assay, restriction enzyme digestion, and Southern blot hybridization. The mutant virus was then reconstituted in 293 cells and used for phenotype analysis. (B) PCR assay to confirm the deletion of the vIRF gene in BAC36. BAC36 produced a band of 1.3 kb, while the mutant virus produced a band of 1.7 kb because of the size differences between the vIRF gene and Kan. (C) KpnI digestion of BAC36 and mutant virus and Southern blot hybridization with Kan as the probe.