the context of latent virus infection. The EBV-negative parental BL31 cells, along with BL31 cells infected with BAC-derived EBNA3A, EBNA3B or EBNA3C knockout viruses (3AKO, 3BKO and 3CKO respectively), their revertants (3Arev, 3Brev and 3Crev respectively) or wild type (WT, derived from the prototype B95.8 strain) viruses (described in [22]) were treated with ionomycin. After 48 hours, more than 80% of the parental EBV-negative BL31 cells were dead (Figure 2A) and exhibited considerable cleavage of PARP (Figure S2). As expected, infection of BL31 cells with WT EBV or any of the revertant viruses improved survival dramatically. Interestingly – as predicted by Kelly et al [25] – BL31 cells infected with the 3AKO, 3BKO or 3CKO viruses were also resistant to ionomycin-induced apoptosis, with at least 90% of cells remaining viable after 48 hours. This survival was also reflected by absence of PARP cleavage in these cell lines (Figure S2). This demonstrates that in BL31 cells the absence of EBNA3A, EBNA3B or EBNA3C individually has little or no effect on the EBV-mediated protection against ionomycin-induced apoptosis and is in marked contrast to the contribution both EBNA3A and EBNA3C make to the EBV-mediated protection of similar BL cells against apoptosis induced by genotoxic drugs such as cisplatin [22]. In order to show that these observations were not specific to BL31 cells, experiments were repeated using a panel of BL2 cells (which lack BIM, but carry WT p53) infected with recombinant 3AKO, 3BKO, 3CKO, revertant or WT EBV. The results obtained from the BL2 panel of cells are similar to those obtained with the BL31 panel (data not shown). Together these results indicate that individually deleting EBNAs 3A, 3B or 3C does not alter the protection conferred by EBV against ionomycin-induced apoptosis. The data also confirm the observations that the apoptotic pathway induced by ionomycin is independent of both p53 and BIM. While removing EBNA3A, EBNA3B or EBNA3C individually did not alter the capacity of EBV to protect against ionomycin, the possibility of functional redundancy between the EBNA3 proteins could not be ruled out. Therefore, a recombinant EBV harbouring a deletion of the entire EBNA3 locus (E3KO) was constructed and BL31 and BL2 cells were established by infection with E3KO or its revertant (E3rev) [31]. While few of the parental BL31 cells remain viable after 48 hours of exposure to ionomycin, BL31 cells infected with the E3KO, WT and revertant viruses are all resistant to ionomycin-induced apoptosis, as measured by viability and PARP cleavage (Figures 2B and S2). Experiments with a BL2 panel of cells produced essentially similar results (data not shown). All these results eliminate the possibility that the EBNA3s can functionally substitute for each other in the individual deletion viruses, and confirm that the EBNA3s are not involved in the EBV-mediated protection of BL cells from ionomycin-induced apoptosis. The BHRF1 locus provides partial resistance to ionomycin-induced apoptosis Previous studies showed that the EBV viral BCL-2 homologue, BHRF1, is only expressed early upon infection of resting B cells or during lytic reactivation [16,32]. Although ionomycin treatment does not consistently induce the viral lytic cycle in any of the panel of BL cells studied here (for examples see Figure S3), Kelly and colleagues have shown that BHRF1 can be expressed at a low level during latency in ‘Wp-restricted’ BL-derived cell lines (eg. Sal-BL and Oku-BL) and can suppress the apoptotic pathway activated by ionomycin in BL cells when it is delivered by transfection [25]. By western blotting with an anti-BHRF1 monoclonal antibody, we confirmed expression of BHRF1 in Oku-BL and Sal-BL, but detected no expression in various untreated or treated BL2, BL31 EBV Blocks NOXA Induction and BL41 converts or in Mutu III cells (Figure S3). However, one could not formally exclude the possibility that BHRF1 might be functionally active at a level that is undetectable by western blotting extracts from these latently infected cells. Therefore, using recombination in the B95-8 EBV-BAC, viruses were constructed from which the BHRF1 locus was deleted (BHLOC KO) and subsequently replaced to produce a revertant virus (BHLOC rev – Figure 3A and B). The deletion removes not only the BHRF1 coding sequence, but also three miRNAs that have been shown to inhibit apoptosis early after infection of primary B cells [33]. The recombinant viruses were then used to infect EBV-negative BL31 cells and stably infected cells were selected using hygromycin and allowed to grow out as a bulk population. Attempts to produce BL2 lines infected with the BHLOC KO virus were unsuccessful for reasons we don’t understand. Protein extracts from the BL31 cells established with these recombinant EBVs were analysed by western blotting using antibodies for each of the EBV latent proteins. It was confirmed that these cells display no consistent changes to the levels of the EBV latent proteins – EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C and LMP1; however in the two BHLOC KO lines the amount of EBNA-LP appeared slightly elevated relative to wild type and revertant infections (Figure 3C) – this was also reported in a recent study focusing on the BHRF1 miRNAs [34]. In order to determine the contribution made by the BHRF1 locus to EBV-mediated protection against ionomycin-induced apoptosis, the response to ionomycin of BL31 carrying BHLOC KO or BHLOC rev viruses were examined. Figure 4A shows that 24 hours after exposure to ionomycin, only 25% of EBV-negative BL31 cells remain viable, compared to .80% of BL31 cells infected with the WT-EBV or BHLOC rev. It was apparent from multiple experiments that BL31 cells infected with the BHLOC KO virus showed partial resistance to ionomycin-induced apoptosis – with about 50% of the cell population remaining viable after 24 hours. Western blotting for PARP echoes this result, with a substantial amount of cleaved PARP detectable in EBV-negative BL31 cells, very little or no cleaved PARP in WT- or BHLOC rev-infected BL31 cells, and an intermediate level of PARP cleavage in BL31-BHLOC KO (Figure 4B). These data indicate that the BHRF1 locus is involved in the EBV-mediated protection of BL cells against ionomycin-induced apoptosis. However, since the loss of the BHRF1 locus consistently resulted in only a partial loss of protection, it is likely that another EBV gene product(s) is also involved in the resistance to ionomycin. EBV inhibits the induction of NOXA by ionomycin To identify cellular factors that might be involved in the apoptotic pathway triggered by ionomycin in BL cells and blocked by EBV, we surveyed the changes in expression of various pro- apoptotic proteins in BL cells exposed to ionomycin. Initially BAD, BID, PUMA and NOXA were investigated by western blotting (Figures 5A, S3 and S4). Although the steady-state level of NOXA varied little between the various cell lines used here, it was the only one of these pro-apoptotic factors that consistently showed an increase in response to ionomycin (Figures 5A and S4). The gene encoding NOXA is a known transcriptional target of the tumour suppressor p53 and its expression is upregulated during p53-mediated cell death [35]. However, western blot analysis reproducibly showed an increase in NOXA protein levels in Mutu I and EBV-negative BL31 cells exposed to ionomycin and since both lines express only mutant p53 [22,36], this indicates that the induction is not dependent on p53-mediated transactivation. In contrast, NOXA protein levels were unaffected (or decreased) in Mutu III cells, BL31 cells latently infected with wild type B95.8 PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e28506