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Journal of Virology, July 2008, p. 6721-6733, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.02250-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Molecular Basis of Cytotoxicity of Epstein-Barr Virus (EBV) Latent Membrane Protein 1 (LMP1) in EBV Latency III B Cells: LMP1 Induces Type II Ligand-Independent Autoactivation of CD95/Fas with Caspase 8-Mediated Apoptosis ^,||

Christophe Le Clorennec,^1,, Tan-Sothéa Ouk,^1, Ibtissam Youlyouz-Marfak,¹ Stéphanie Panteix,¹ Catherine-Claude Martin,¹ Julia Rastelli,^2, Eric Adriaenssens,³ Ursula Zimber-Strobl,² Jean Coll,^4,¶ Jean Feuillard,^1,5 and Chantal Jayat-Vignoles^1*

UMR CNRS 6101, Faculté de Médecine de Limoges, Université de Limoges, 2 Rue du Docteur Marcland, 87025 Limoges Cedex, France,¹ Institute of Clinical Molecular Biology and Tumor Genetics, Helmholtz Center Munich German Research Centre for Environment and Health (GmbH), D-81377 Munich, Germany,² INSERM ERI-8, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq, France,³ CNRS UMR 8161, Institut de Biologie de Lille, Equipe cancers viro-induits, BP 447, 59021 Lille Cedex, France,⁴ CHU Dupuytren, Laboratoire d'hématologie, 2 Avenue Martin Luther King, 87042 Limoges Cedex, France⁵

Received 17 October 2007/ Accepted 10 April 2008

The Epstein-Barr virus (EBV) oncoprotein latent membrane protein 1 (LMP1) is thought to act as the major transforming protein in various cell types, by rerouting the tumor necrosis factor receptor family signaling pathway. Despite this implication in EBV-associated transformation of cells, LMP1 toxicity is a well-known but poorly studied feature, perhaps because it contradicts its role in transformation. We show that LMP1 physiological levels are very heterogeneous and that the highest levels of LMP1 correlate with Fas overexpression and spontaneous apoptosis in lymphoblastoid cell lines (LCLs). To understand the cytotoxic effect of LMP1 in LCLs, we cloned wild-type LMP1 into a doxycycline 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 labeling, sub-G₁ peak, and poly(ADP ribose) polymerase cleavage. Knocking down Fas expression by small interfering RNA abolished LMP1-induced apoptosis. The absence of detectable levels of Fas ligand 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 (Fas-associated death domain protein) and caspase 8, suggesting a Fas-dependent formation of the death-inducing signaling complex. Caspases 8, 9, 3, and 7 were activated by LMP1. Caspase 8 activation was associated with BID cleavage and truncated-BID mitochondrial relocalization, consistent with type II apoptosis. Therefore, our results are in agreement with a model where LMP1-dependent NF-B activation induces 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.

Published ahead of print on 30 April 2008.

^|| Supplemental material for this article may be found at http://jvi.asm.org/.

These authors contributed equally to this study.

Present address: Drug Resistance Group, Centre for Cancer Research and Cell biology, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7AB, Northern Ireland.

Present address: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142.

^¶ Deceased.