Figure 7. Neither the EBNA3 nor the BHRF1 loci are necessary to block the induction of NOXA. (A) A comparison of NOXA induction in EBNA3KO and EBNA3 revertant BL31 cells after treatment with ionomycin (IM) and analysis by western immunoblotting. (B) Similar analysis of BHLOC KO BL31 lines and a revertant line. Induction of NOXA by ionomycin in BL31 is shown for comparison. Throughout c-tubulin was used as a loading control. doi:10.1371/journal.pone.0028506.g007 Neither the EBNA3s nor the BHRF1 locus contribute to the inhibition of NOXA induction Since BL31 cells latently infected with an EBNA3 knockout virus (E3KO) are resistant to ionomycin, it was predictable that EBV carrying this deletion would still block NOXA accumulation. This is formally shown in Figure 7A. However, more surprising was the observation that virus with the BHRF1 locus deletion – despite providing reduced resistance to ionomycin treatment – also suppressed NOXA accumulation as effectively as revertant and WT B95.8 EBVs (Figure 7B). EBV Blocks NOXA Induction Responses of BL-derived cells to staurosporine and etoposide confirm two distinct pathways Staurosporine is a broad-spectrum protein kinase inhibitor, is widely used as an inducer of apoptosis in a variety of cell types, but is chemically unrelated to ionomycin [37]. Etoposide is a DNA topoisomerase II inhibitor that can induce apoptosis through DNA damage-induced p53 activation [38]. Similar experiments to those described above were performed to assess the responses of BL-derived cells to staurosporine and etoposide in order to establish whether they could be categorised with either ionomycin or the genotoxins. Initially Mutu I and Mutu III were compared. Although both staurosporine and etoposide induced apoptosis in Mutu I, latency-III EBV gene expression (in Mutu III) substantially enhanced survival after both treatments (Figure 8A & B). Staurosporine induced NOXA in sensitive Mutu I cells, but in contrast, etoposide had no effect on NOXA levels and must therefore activate a separate pathway (Figure 8C). When the BHLOCKO infected cells were tested, they exhibited partial resistance to both staurosporine and etoposide, but as expected, the BHLOC KO virus still blocked NOXA accumulation (Figures 9A and S6). It seems that the BHRF1 locus enhances survival irrespective of the type of trigger, but acts independently of NOXA. When BL31 cells infected with the EBNA3 locus knockout (E3KO) series were analysed in similar assays, the deletion viruses showed similar levels of protection to B95.8-BAC (WT) EBV in response to staurosporine. In contrast when the cells were treated with etoposide, only 10–20% of the E3KO-infected cells survived, but NOXA was not induced (Figure 9B and S7). These observations suggest that staurosporine induces apoptosis in BL cells by activating the same or a similar pathway to that activated by ionomycin whereas etoposide works, like cisplatin, via a second distinct pathway associated with DNA damage. Discussion Previous work in our laboratory showed that the resistance conferred by EBV against cell death induced by agents that cause damage to DNA, including the mitotic spindle poison nocodazole and the DNA cross-linking agent cisplatin, is dependent on the co- expression of EBNA3A and EBNA3C [22]. Here we have expanded this group to include etoposide (see below). Protection correlates with the down-regulation of the BH3-only pro-apoptotic protein BIM by the combined action of EBNA3A and EBNA3C, but is only apparent in the absence of a functional p53. When BIM is not expressed, but p53 is functional (as for instance in BL2 cells), latent EBV does not appear to provide any survival advantage against apoptosis induced by these genotoxic drugs. These findings appeared to be inconsistent with the observations made by Kelly and colleagues that EBV protects both BL2 cells (that carry WT p53, but are BIM-negative) and BL31 cells (that carry mutant p53, but are BIM-positive) to a similar extent when apoptosis was induced by the calcium ionophore, ionomycin [20,25]. We hypothesised that ionomycin must activate a distinct apoptotic pathway in BL cells that is both independent of p53 status and not associated with BIM. We confirm here that latent EBV protects cells from ionomycin and also the apparently unrelated drug staurosporine via a similar pathway and show that EBV blocks the accumulation of the pro-apoptotic protein NOXA that is induced by both of these agents. The data from BL41 cells infected with P3HR1 virus and BL2 cells infected with a recombinant virus that also fail to express EBNA2 (E2KO) were consistent with a restricted pattern of viral gene expression – EBNA1, EBNA3A, EBNA3B, EBNA3C, PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e28506