EBV Blocks NOXA Induction Figure 8. Comparison of staurosporine- and etoposide-induced apoptosis in Mutu BL cells. Mutu I and Mutu III cells were exposed to 0.25 mM staurosporine (STS) or 800 ng/ml etoposide (ETP) for 24 hours or 72 hours respectively. (A) Cell viability was determined using the CellTiter- Glo assay and expressed as a percentage of DMSO vehicle-treated cells. The mean and standard deviation of three independent experiments is shown. Protein was extracted from cells harvested at the start of treatment and the times indicated. Western immunoblotting was performed using antibodies that detect PARP (B) or NOXA (C). U indicates vehicle-treated control cells after 24 or 72 hours, and c-tubulin was used as a loading control. doi:10.1371/journal.pone.0028506.g008 truncated EBNA-LP, the EBER and the BART RNAs that remain in B95.8 being sufficient for protection from ionomycin. We obtained similar results with Oku-BL and Sal-BL that carry a virus with a similar genomic deletion to P3HR1. If EBV gene expression was restricted further, as in Mutu I cells (EBNA1, EBERs and BARTs only) there was no protection. Analyses utilizing recombinant EBVs carrying specific gene deletions of EBNA3A, EBNA3B, EBNA3C or the whole EBNA3 locus showed that none of these genes is necessary for the survival phenotype and that there is no functional redundancy – that is, one EBNA3 cannot substitute for either of the others. It was recently shown that BHRF1 might not be a strictly ‘lytic’ product and that the genomic locus from which it derives is also the source of three potentially anti-apoptotic miRNAs [16,25,33]. We therefore constructed a recombinant EBV from which the complete BHRF1 locus was deleted to formally test whether these potentially anti-apoptotic factors are required for protection of latently infected cells against ionomycin (and staurosporine). PLoS ONE | www.plosone.org 11 December 2011 | Volume 6 | Issue 12 | e28506