Molecular Basis of Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) Cytotoxicity in EBV Latency III B-cells: LMP1 induces type II Ligand independent Autoactivation of CD95/Fas with Caspase-8 mediated Apoptosis

Centre National de la Recherche Scientifique, UMR CNRS 6101, Faculté de Médecine de Limoges, Université de Limoges, CHU Dupuytren, Laboratoire d'Hématologie, 2 Rue du Docteur Marcland, 87025 Limoges Cedex, France; Institute of Clinical Molecular Biology and Tumor Genetics, Helmholtz Center Munich German Research Centre for Environment and Health (GmbH), D-81377 Munich, Germany; INSERM ERI-8, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq, France; CNRS UMR 8161, Institut de Biologie de Lille, Equipe cancers viro-induits, BP 447, 59021 Lille Cedex, France

^* To whom correspondence should be addressed. Email: chantal.vignoles{at}unilim.fr.

	Abstract

Epstein-Barr-Virus (EBV) oncoprotein LMP1 (Latent Membrane Protein 1) is thought to be the major transforming protein in various cell types, by rerouting the TNF receptor family signalling pathway. Despite this implication in EBV-associated transformation of cells, LMP1 toxicity is a well-known but poorly studied experimental feature, perhaps because it contradicts its role in transformation. We show that LMP1 physiological levels are very heterogeneous ant that highest levels of LMP1 correlate with Fas-overexpression and spontaneous apoptosis in LCLs. To understand the cytotoxic effect of LMP1 in LCLs, we cloned wild type LMP1 into a doxycyclin double inducible episomal vector pRT-1, with a truncated version of NGFR as a surrogate marker of inducibility. We found that LMP1 overexpression induced apoptosis in LCL B-cells, as shown by annexin-V labelling, sub-G1 peak and PARP cleavage. Knocking down Fas expression by siRNA abolished LMP1-induced apoptosis. Absence of detectable levels of FASL mRNA suggested a ligand-independent activation of Fas. LMP1 induced Fas overexpression with its relocalization in lipid raft microdomains of the membrane. Fas immunoprecipitation detected FADD and caspase-8, suggesting a Fas-dependent formation of DISC. Caspase-8, -9, -3 and-7 were activated by LMP1. Caspase-8 activation was associated with BID cleavage and tBID mitochondrial relocalization, consistent with type II apoptosis. Therefore, our results are in agreement with a model where LMP1-dependent NF-B activation induced Fas overexpression and autoactivation that could overwhelm the antiapoptotic effect of NF-B, revealing an ambivalent function of LMP1 in cell survival and programmed cell death.