EBV Blocks NOXA Induction Figure 3. Construction of an EBV-BAC recombinant virus from which BHRF1 locus has been deleted. (A) Schematic representation of the recombinant EBVs generated with a deletion in the BHRF1 locus (BHLOC KO). Recombinant EBV-BACs harbouring a deletion of the complete BHRF1 locus were constructed. The BAC region is inserted at the site of the 10 kb deletion of B95-8 (D). Vertical arrows indicate the restriction enzyme sites and positions (within the EBV genome sequence, Accession No V01555) between which the BHRF1 locus was deleted. This region was reinserted to generate the revertant virus (BHLOC rev). The schematic is not drawn precisely to scale. (B) Validating BHRF1 knockout and revertant EBV-BACs. DNA from the BHRF1 locus (BHLOC)-knockout (KO – BAC JY28+2.4) and its revertant (rev – BAC JY32+2.3) EBV-BACs as well as the wild-type EBV-BAC (WT) were analysed by restriction digestion with AgeI (top) or EcoRI (bottom) followed by pulsed field gel electrophoresis. A mixture of l-DNA BstEII and l- DNA mono cut mix ladders (NEB) were used as a size marker (Ladder). Bands whose sizes are changed by the deletion of the BHRF1 locus (13 kb AgeI fragment, and the Bam W-containing EcoRI fragment lacking BHRF1 - W-DBHRF1) are indicated. The EcoRI restriction fragments that include the Bam W repeats and the terminal repeats (TR) are also indicated. (C). Protein extracts from BL31, BL31 BHLOC KO, revertants and an LCL were separated by SDS-PAGE and western immunoblotted with antibodies specific to the EBV proteins indicated (and the cellular protein c-tubulin as a loading control). doi:10.1371/journal.pone.0028506.g003 undergo apoptosis in response to ionomycin. The accumulation of NOXA is independent of p53-status and can be inhibited by a factor(s) encoded by EBV in latency state III, but does not seem to require EBNA2, (or the LMPs) that are absent from the E2KO- infected cells, Oku- BL and Sal-BL. NOXA contributes to ionomycin-induced apoptosis NOXA and its mRNA are consistently up-regulated in EBV- negative BL cells that undergo apoptosis in response to ionomycin. However, NOXA expression appears to be largely unaffected in EBV-positive BL cells that are resistant to ionomycin. It was PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e28506