Latent Epstein-Barr Virus Can Inhibit Apoptosis in B Cells by Blocking the Induction of NOXA Expression ¤ Jade Yee, Robert E. White, Emma Anderton , Martin J. Allday* Section of Virology, Division of Infectious Diseases, Faculty of Medicine, Imperial College London, London, United Kingdom Abstract Latent Epstein-Barr virus (EBV) has been shown to protect Burkitt’s lymphoma-derived B cells from apoptosis induced by agents that cause damage to DNA, in the context of mutant p53. This protection requires expression of the latency- associated nuclear proteins EBNA3A and EBNA3C and correlates with their ability to cooperate in the repression of the gene encoding the pro-apoptotic, BH3-only protein BIM. Here we confirm that latent EBV in B cells also inhibits apoptosis induced by two other agents – ionomycin and staurosporine – and show that these act by a distinct pathway that involves a p53- independent increase in expression of another pro-apoptotic, BH3-only protein, NOXA. Analyses employing a variety of B cells infected with naturally occurring EBV or B95.8 EBV-BAC recombinant mutants indicated that the block to NOXA induction does not depend on the well-characterized viral latency-associated genes (EBNAs 1, 2, 3A, 3B, 3C, the LMPs or the EBERs) or expression of BIM. Regulation of NOXA was shown to be at least partly at the level of mRNA and the requirement for NOXA to induce cell death in this context was demonstrated by NOXA-specific shRNA-mediated depletion experiments. Although recombinant EBV with a deletion removing the BHRF1 locus – that encodes the BCL2-homologue BHRF1 and three microRNAs – partially abrogates protection against ionomycin and staurosporine, the deletion has no effect on the EBV- mediated block to NOXA accumulation. Citation: Yee J, White RE, Anderton E, Allday MJ (2011) Latent Epstein-Barr Virus Can Inhibit Apoptosis in B Cells by Blocking the Induction of NOXA Expression. PLoS ONE 6(12): e28506. doi:10.1371/journal.pone.0028506 Editor: Maria G. Masucci, Karolinska Institutet, Sweden Received July 7, 2011; Accepted October 25, 2011; Published December 9, 2011 Copyright: 2011 Yee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by a Wellcome Trust programme grant (077489). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: m.allday@imperial.ac.uk ¤ Current address: Molecular Oncology Laboratory, Cancer Research UK London Research Institute, London, United Kingdom Introduction In vitro, EBV can very efficiently induce the activation and continuous proliferation of primary human B cells. The resulting ‘immortalized’ lymphoblastoid cell lines (LCLs) carry the viral genome as extra-chromosomal episomes and express only nine latency-associated EBV proteins. There are six nuclear antigens (EBNAs 1, 2, 3A, 3B, 3C & -LP), three membrane associated proteins (LMP1, LMP2A & 2B) and several untranslated RNA species (EBERs and BARTs). This programme of viral gene expression is known as latency III or the ‘growth programme’ [1,2,3]. However, data on the persistence of EBV in humans are consistent with the viral genome residing long-term in a resting memory B cell population in which no EBV proteins are expressed. It is now considered probable that to establish persistence, EBV initially infects resting (na¨ıve) B cells and drives these to proliferate as activated B-blasts. This expanding infected B-blast population then migrates into germinal centres, where the infected cells differentiate to become centroblasts, centrocytes and finally resting memory B cells. While the precise series of events that the EBV-positive B cells undergo to reach the memory compartment is unknown, it appears to involve regulated shut- down of latency-associated viral gene expression from an initial state (latency III, as found in LCLs) in B-blasts, via latency II (EBNA1, the LMPs, the EBERs and BARTs) in centroblasts and centroctyes until in quiescent memory B cells no EBV protein transcripts can be detected in a state called latency 0. When resting memory cells re-enter the cell cycle and divide, EBNA1 alone becomes detectable (termed latency I) [3,4,5]. Although persistent infection is normally asymptomatic, EBV can be the causative agent in the benign lymphoproliferation known as infectious mononucleosis, and the virus is also etiologically linked to a number of human cancers including Burkitt’s lymphoma (BL), Hodgkin’s lymphoma, diffuse large B cell lymphoma, and epithelial-derived nasopharyngeal carcinoma [1,6]. In normal individuals the accumulation of proliferating B cells is counterbalanced by differentiation into memory and also the action of EBV-specific cytotoxic T lymphocytes that recognise and destroy the proliferating B-blasts [7]. There is a great deal of evidence that EBV gene expression can very effectively suppress the process of programmed cell suicide known as apoptosis [8,9,10,11,12,13,14,15,16]. This ability of a virus to regulate apoptosis is generally considered to be important because apoptosis is a major antiviral response used by multicellular organisms for the removal of infected cells. EBV has therefore evolved multiple mechanisms to ensure the survival of infected cells long enough for the virus to establish persistent latent infection and subsequently undergo ‘lytic’ replication to produce new infectious virus. Furthermore, B cells – the primary target for EBV infection – are particularly prone to apoptosis when they pass through germinal centres because this is where PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28506