EBV Blocks NOXA Induction Figure 9. The BHRF1 locus makes a significant contribution to resistance against both staurosporine and etoposide, but the EBNA3s only protect against genotoxin-induced damage. (A) EBV-negative BL31, its recombinant B95.8 (WT) EBV convert and BL31 cell lines established with recombinant BHRF1 locus-knockout (BHLOC KO) and revertant (BHLOC rev) EBVs were treated with 0.25 mM staurosporine (STS, left panel) or 800 ng/ml etoposide (ETP, right panel) for up to 24 hours. Cell viability was determined after 24 hours using the CellTiter-Glo assay and expressed as a percentage of DMSO vehicle-treated cells. (B) Experiments and analysis described in (A) performed on BL31 converts made with EBNA3 locus knockouts (E3KO) and revertant virus. doi:10.1371/journal.pone.0028506.g009 Deletion of the BHRF1 locus partially sensitized latently infected BL31 cells to these drug treatments, indicating that the locus must be involved in mediating resistance. However at this stage we cannot say whether this is due to the action of BHRF1 protein (a BCL-2 homologue) or the BHRF1 miRNAs or both. Furthermore, since the reduction in survival is only partial, this implies that another EBV factor(s) contributes to the resistance phenotype by an as yet unidentified mechanism. BH3-only proteins are critical mediators of the intrinsic apoptotic pathway (reviewed in [39]). Therefore, in order to identify cellular factors that might be associated with the apoptotic pathway activated by ionomycin (and staurosporine), the expres- sion of pro-apoptotic BH3-only members of the BCL-2 family – BAD, BID, NOXA and PUMA – was investigated. Of these NOXA was the only protein consistently induced in EBV-negative and latency I BL cells that were sensitive to treatment with both ionomycin and staurosporine. In contrast, up-regulation of NOXA did not occur in any EBV-positive BL cells exhibiting resistance to the drugs. These data suggested that apoptosis triggered by ionomycin and staurosporine involves the induction of NOXA expression and that EBV rescues BL cells from apoptosis, at least in part, by inhibiting the accumulation of NOXA. ShRNA- mediated knock-down of NOXA expression was able to substantially enhance the survival of EBV-negative BL31 and PLoS ONE | www.plosone.org 12 December 2011 | Volume 6 | Issue 12 | e28506