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. 2010 Jul 29;6(7):e1001001. doi: 10.1371/journal.ppat.1001001

Figure 6. Generation and characterization of γHV68 BAC carrying the TTS/A mutation.

Figure 6

(A) Diagram of the strategy to generate recombinant γHV68. Briefly, wild-type RTA or the STS/A and TTS/A alleles were PCR amplified with overlapping PCR primers. Purified PCR products were transfected into NIH3T3 cells, together with the BAC clone containing a transposon within the transactivation domain of RTA. Recombinant viruses in the supernatant were used to infect NIH3T3 cells. Circular BAC DNA was purified and electroporated into DH10B cells. Chl, chloramphenicol; Kan, kanamycin. (B and C) BACs containing γHV68 genome were analyzed by KpnI (B) or EcoRI (C) digestion, and resolved on 0.8% agarose gels stained with ethidium bromide. The white arrows indicate the specific fragment shift caused by homologous recombination within the RTA locus. NR, RTA-null rescued. (D) 293T cells were transfected with γHV68 M3 reporter plasmid and BAC.NR or BAC.TTS/A. At 28 h post-transfection, luciferase activity and β-galactosidase activity were determined and M3 transcriptional activation by RTA was shown. (E) Transfection of 293T cells with the BAC.TTS/A DNA and a plasmid containing TRAF6, and qRT-PCR were carried out as in Figure 4F.