cells that are not positively selected by high-affinity binding of antigen are removed. During B cell development, therefore, multiple regulators of apoptosis act to cull B cells at different stages in order to maintain homeostasis of the B cell compartment [17]. Inhibition of apoptosis by EBV is also generally considered to make a major contribution to the malignant transformation of infected cells, and hence the development of EBV-associated cancers – reviewed in [18,19]. Numerous studies of EBV and apoptosis have identified most of the viral latency-associated gene products as having some anti- apoptotic activity (reviewed in [18,19]), and a great deal of effort has gone into showing that the presence of latent EBV in BL- derived cells rescues them from apoptosis triggered by a wide range of exogenous stimuli – including cytotoxic drugs, cytokine deprivation, anti-IgM andc-irradiation [13,20,21,22]. This means comparison between studies is often difficult. For instance, we recently showed that the EBV-mediated protection against apoptosis specifically induced by either the DNA cross-linking agent cisplatin, the cyclin-dependent kinase inhibitor roscovitine or the mitotic spindle poison nocodazole is dependent on the co- expression of EBV latency-associated proteins EBNA3A and EBNA3C [21,22]. This correlates with the down-regulation of the pro-apoptotic BH3-only protein BIM, which depends on expres- sion of both EBNA3A and EBNA3C. This protection is independent of the tumour suppressor p53, which is often mutated in BL cells, including BL31 used in the study. However, we noted that when similar experiments were repeated using BL2 cells and their EBV-infected counterparts, both of which carry wild type p53 but fail to express BIM, latent EBV was unable to protect against apoptosis induced by the same agents [22]. This is because these genotoxic agents induce the stabilization and activation of the wild type p53 present in BL2 and the activation of an apoptotic pathway that is independent of BIM. Consistent with this, EBV does not block p53-induced apoptosis after DNA-damage to LCLs [23,24]. In contrast with these observations, Kelly and colleagues found that latent EBV protects both BL31 cells (BIM-positive and carrying a mutant p53) and BL2 cells (BIM-negative and carrying wild type p53) equally well against apoptosis induced by the calcium-ionophore ionomycin and that the EBNA3 proteins appeared to play no role in this [20,25]. We hypothesised that this must depend on a second pathway by which BL cells can be triggered to undergo apoptosis, that this is independent of EBNA3- , BIM- and p53-status, but can also be blocked very effectively by EBV. Here we have explored this alternative pathway and established that it involves the p53-independent accumulation of another pro-apoptotic, BH3-only protein, NOXA. The mecha- nism by which EBV protects the cells from this agent does not involve any of the well-characterised latency factors (including EBNA3A and EBNA3C), but is partly dependent on products of the EBV BHRF1 locus – apparently acting alongside, but independently of NOXA. Results Confirmation that EBV latency-associated gene expression confers resistance against ionomycin-induced apoptosis EBV-negative BL41 and two EBV-positive BL41 cell lines converted by infection using different virus strains, B95.8 and P3HR1, were treated with ionomycin at a concentration of 1 mg/ ml for 48 hours (Figure 1A). BL41 is for (WT) p53 because it carries only a single, mutant allele [26]. After treatment for 48 hours, fewer than 20% of BL41 cells remain viable. In contrast, EBV Blocks NOXA Induction BL41/B95.8 cells, which display the latency III phenotype, appear to be completely resistant to ionomycin-induced apoptosis and continue to proliferate. A similar response was observed in BL41/ P3HR1 cells, which express a restricted set of latent proteins, as a result of a deletion in the region encoding EBNA2 and the two unique C-terminal exons of EBNA-LP (reviewed in [27]). As a result BL41/P3HR1 cells only express the latent proteins EBNA1, EBNA3A, EBNA3B, EBNA3C, a truncated form of EBNA-LP, and the EBERs and BARTs; the LMPs are not expressed because generally EBNA2 is required to transactivate their expression. Similarly, the responses of more recently described BL lines, Oku-BL and Sal-BL, were also analysed (Figure 1A). These early passage cell lines were established from BL biopsy material [28] and found to carry EBV genomes with EBNA2 deletions similar to that of the P3HR1 strain. These cell lines are termed ‘Wp- restricted’ because the EBNA2 deletion causes the Wp promoter, rather than Cp, to remain the main originator of EBNA transcripts. Oku-BL and Sal-BL therefore express the same subset of EBV genes as BL41/P3HR1. However unlike BL41, both Oku- BL and Sal-BL carry wild-type (WT) p53 [22]. The data confirm that the ionomycin-resistant phenotype is not specific to BL41 and suggest that the restricted set of EBV proteins associated with the P3HR1 strain is sufficient to protect BL cells from ionomycin- induced apoptosis irrespective of the p53-status of cells. BL2 cells – which have WT p53, but fail to express the pro- apoptotic factor BIM [22] – were infected with a recombinant EBV-BAC from which the EBNA2 open reading frame (ORF) had been deleted to produce a P3HR1-like virus (E2KO), but carrying a full length EBNA-LP [20]. As seen with EBV-negative BL41, BL2 cells were susceptible to ionomycin-induced apoptosis, as measured by viability. In contrast, BL2 cells latently infected with recombinant E2KO virus were protected from ionomycin-induced apoptosis (Figure 1B). These results confirm that the limited pattern of EBV gene expression associated with P3HR1, Oku-BL, Sal-BL and an E2KO virus is sufficient to enhance survival of ionomycin treated cells, that BIM is not involved in this pathway to apoptosis and that the p53 context is unimportant. Throughout, in order to confirm that death was a result of cells undergoing apoptosis, protein extracts prepared from treated and untreated cells were separated by SDS-PAGE and analysed by western blotting using an antibody which detects both full length poly(ADP-ribose) polymerase (PARP) and its cleaved fragments. PARP is proteolytically cleaved from a full-length 113 kDa protein to 89 kDa and 24 kDa fragments by caspases activated during apoptosis. PARP cleavage is widely accepted to be a hallmark of programmed cell death [29]. All these data are shown in supplementary Figure S1. Having confirmed that the EBV-mediated protection of BL cells from ionomycin-induced apoptosis only requires the expression of a limited set of viral genes, the number of candidate genes was reduced further by assessing the response of cells that exhibit type I latency. This is a restricted form of EBV latency in which only EBNA1, the EBERs and BARTs are expressed, and is the EBV gene expression pattern found in the majority of primary EBV- positive BL. The Latency I BL-derived line, Mutu I, was used for investigation. Mutu cells that had drifted in culture to produce the full latency III pattern of EBV gene expression (Mutu III) were analysed for comparison. Figures 1C and S1 show that Mutu III cells were resistant to ionomycin-induced apoptosis and continued to proliferate. In contrast, fewer than 20% of Mutu I cells survived after 48 hours of exposure to ionomycin and complete cleavage of full-length PARP was observed, consistent with death by apoptosis in response to ionomycin. These data demonstrate that type I PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28506