EBV Blocks NOXA Induction Figure 2. Deleting EBNA3A, 3B or 3C or the complete EBNA3 locus has little or no effect on ionomycin-induced apoptosis. (A) EBV- negative BL31 cells and their converts infected with recombinant B95.8 EBV (WT), EBNA3A, -3B and -3C-knockout EBVs, or their respective revertants were treated with 1 mg/ml ionomycin (IM) for 48 hours. (B) EBV-negative BL31 cells and their converts infected with recombinant B95.8 EBV (WT), EBNA3 locus knockout (E3KO) or revertant (E3rev) EBVs were treated with 1 mg/ml ionomycin for 48 hours. In both (A) and (B) viable cells that exclude trypan blue were counted after 48 hours and expressed as a percentage relative to the starting population. The mean and standard deviation from three independent experiments are shown. doi:10.1371/journal.pone.0028506.g002 recombinant virus or infected with the P3HR1-like, EBNA2 knockout virus (E2KO). This indicates that only a limited pattern of EBV gene expression is necessary to block NOXA accumula- tion. In a similar BL2 series of cells and Oku- and Sal-BL cells it was only in the EBV-negative BL2 cells that NOXA accumulated and apoptosis was induced in response to ionomycin. These cells all carry wild type, transactivation competent p53 [22], but ionomycin did not induce its stabilization (data not shown), again consistent with NOXA induction being p53-independent. Quan- titative reverse transcriptase PCR (qRT-PCR) analysis of RNA from BL31 and BL2 cells established that this induction of NOXA expression involves the modulation of NOXA mRNA and may therefore be regulated at the level of transcription; in both the EBV-infected converts this induction is inhibited (Figure 5B). Together these data indicate that NOXA is consistently up- regulated in EBV-negative and latency I BL-derived cells that PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e28506