BL2 cells challenged with ionomycin, confirming that ionomycin- induced apoptosis is at least partly mediated by NOXA and that inhibiting its expression can promote B cell survival. The presence of shRNA against NOXA did not prevent its induction by ionomycin, but since NOXA was induced from a lower baseline level, the improved survival manifested as delayed apoptosis (apparent at 24 hours post treatment) rather than as complete resistance – if the cells were left long enough they died. We assume the rate at which new RNA was synthesised outstripped the capacity of the shRNA to inactivate NOXA message. Despite these technical drawbacks inherent in shRNA experiments, the results were highly reproducible and consistent in two different cell backgrounds. It was more surprising to see that NOXA levels did not increase in EBV-positive BL cells lacking the BHRF1 locus, since they are partially sensitive to ionomycin and staurosporine treatment. This suggests that these drugs trigger an additional pro-apoptotic mechanism in addition to NOXA induction, which is blocked by the active component of the BHRF1 locus – this could be BHRF1, expressed at a low level as hypothesised by Kelly et al, mimicking BCL-2 and neutralizing multiple pro-apoptotic factors. The increase in NOXA protein after drug-treatment was mirrored by an increase in NOXA mRNA, suggesting that the regulation is at the level of RNA turnover or transcription. Since wild type p53 is absent from BL41, BL31 and Mutu I cells the induction of NOXA expression by ionomycin does not involve p53-mediated activation of NOXA transcription. Consistent with this, ionomycin does not induce the stabilization and accumulation of wild type p53 in BL2 cells (data not shown). However, it is currently unclear by what signalling pathway ionomycin (or staurosporine) induces the accumulation of NOXA. Furthermore, using the various mutants of EBV described above, we were unable to identify the viral gene or gene product(s) that is responsible for the block in NOXA accumulation. None of the latency-associated proteins, the EBERs, BHRF1, the BHRF1 miRNAs or most of the BART miRNAs, appear to be necessary to block the ionomycin-induced increase in NOXA. Of the currently recognised latency-associated gene products only the BART miRNAs that are not missing from the B95.8 genome (miR- BARTs 1, 2, 3, 4 and 15 [40], or the W1W2 repeat region of EBNA-LP [15] remain as candidates for this regulation. However, at this stage we cannot formally exclude a contribution by products from other regions of the EBV genome not presently associated with latency. Finally, etoposide – a DNA topoisomerase II inhibitor and therefore another drug that can cause damage to DNA – also triggered apoptosis that was blocked by latency III EBV gene expression in Mutu and BL31 cells. Apoptosis did not involve the induction of NOXA, but resistance required expression of EBNA3A and EBNA3C, in this respect etoposide has a similar mode of action to cisplatin and nocodazole. These data are consistent with the hypothesis of two distinct pathways and suggest that EBNAs 3A and 3C might play a specific role in the DNA damage response, in addition to the regulation of apoptosis per se. Materials and Methods Cell culture Burkitt’s lymphoma-derived cell lines: BL41, BL41/B95.8, BL41/P3HR1, Oku-BL, Sal-BL, BL2, BL31, Mutu I-clone 179 and Mutu III [described in [21,22]; lymphoblastoid cell lines: LCL-BF and LCL-CH (described in [24,30]) and HEK293 cells [31] were all cultured in RPMI 1640 medium containing L- glutamine (Invitrogen, UK) supplemented with 10% fetal calf EBV Blocks NOXA Induction serum and penicillin-streptomycin at 37uC in a humidified incubator with 10% CO2. BL2 and BL31 cell lines were additionally supplemented with 1 mM sodium pyruvate (Sigma, UK) and 50 mM a-thioglycerol (Sigma). Hygromycin B (Roche, UK) was added at a concentration of 100 mg/ml to all BL cell lines containing recombinant hygromycin-resistant EBV. BL2 cells infected with a recombinant EBNA2-knockout virus (BL2+E2KO) [20]. For routine passage, cells were split 1:3 every 2 or 3 days. For experiments, all cells were re-suspended in fresh medium at a density of 36105 cells/ml 24 hours prior to manipulation. Generation and use of recombinant viruses BL31 and BL2 cell lines carrying recombinant EBNA3 mutants and their revertants were described previously [22,31]. EBV containing a deletion of the BHRF1 locus was generated using recA-mediated recombination. Briefly, the region of the EBV genome containing the BHRF1 locus was sub-cloned, and digested with SmaI/PstI, treated with Klenow and re-ligated, to remove the fragment corresponding to positions 41399–43125 of the EBV genome (Accession number V01555). Approximately 500 bp on each flank of this deletion enabled homologous recombination with the B95-8 EBV-BAC genome [41], using a markerless insertion technique [42], to generate BHLOC KO EBV. Revertants of this (BHLOC rev) were generated using the same region of EBV lacking the deletion, which was reintroduced into the EBV-BAC genome to revert the mutant to the wild-type sequence. Mutant and revertant viruses were tested for the presence/absence of the desired mutation, and screened for undesired deletions/alterations by restriction digestion and pulsed- field gel electrophoresis. Recombinant EBV-BAC was transfected into HEK-293 cells that were selected with hygromycin, cloned, screened and used to generate virus as described elsewhere [43]. These recombinant viruses were used to infect BL31 and BL2 cells as described previously [22,31]. Induction of cell death and measurement of cell viability Ionomycin (Sigma), staurosporine (Sigma) and etoposide (Calbiochem) were reconstituted in DMSO to form stock solutions. Ionomycin was added to the cell medium to give a final concentration of 1 mg/ml unless otherwise stated. Staurosporine was used at a final concentration of 0.25 mM and etoposide was used at a final concentration of either 500 or 800 ng/ml. Viable cell counts were performed using a hemocytometer and based on the exclusion of trypan blue dye, or in some experiments, cell viability was measured using the CellTiter-GloTM Luminescent Cell Viability Assay (Promega) according to the manufacturer’s instructions. In this assay ATP activity is used as a measure of viable cells. Western blot analysis This was performed essentially as previously described [44]. Briefly, protein extracted using RIPA buffer were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) and transferred to Protran nitrocellulose membranes (Schleicher & Schuell Bioscience). Membranes were blocked with 5% skimmed-milk in phosphate buffered saline (PBS) containing 0.05% Tween 20, probed with appropriate primary and HRP- conjugated secondary or tertiary antibodies. An ECL kit (Amersham Biosciences) was used for visualization. Primary antibodies were used according to the manufacturer’s instructions: rabbit polyclonal anti-poly(ADP-ribose) polymerase or PARP (Roche), mouse monoclonal anti-c-tubulin (GTU-88; Sigma), mouse monoclonal anti-NOXA (114C307; Calbiochem), rabbit polyclonal anti-BIM (Stressgen), rabbit polyclonal anti-PUMA PLoS ONE | www.plosone.org 13 December 2011 | Volume 6 | Issue 12 | e28506