Downloaded from www.microbiologyresearch.org by IP: 52.90.170.79 On: Thu, 03 Aug 2017 06:42:54 Dengue virus M protein contains a proapoptotic sequence referred to as ApoptoM Adeline Catteau, 1 Olga Kalinina, 1 3 Marie-Christine Wagner, 2 Vincent Deubel, 3 Marie-Pierre Courageot 1 4 and Philippe Despre `s 1 Correspondence Philippe Despre `s pdespres@pasteur.fr 1,2 Unite ´ Postulante des Interactions Mole ´ culaires Flavivirus-Ho ˆ tes 1 and Plate-Forme de Cytome ´ trie 2 , Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France 3 Unite ´ de Biologie des Infections Virales Emergentes, Institut Pasteur, Lyon, France Received 13 February 2003 Accepted 21 May 2003 The induction of apoptotic cell death is a prominent cytopathic effect of dengue (DEN) viruses. One of the key questions to be addressed is which viral components induce apoptosis in DEN virus-infected cells. This study investigated whether the small membrane (M) protein was involved in the induction of apoptosis by DEN virus. This was addressed by using a series of enhanced green fluorescent protein-fused DEN proteins. Evidence is provided that intracellular production of the M ectodomains (residues M-1 to M-40) of all four DEN serotypes triggered apoptosis in host cells such as mouse neuroblastoma Neuro 2a and human hepatoma HepG2 cells. The M ectodomains of the wild-type strains of Japanese encephalitis, West Nile and yellow fever viruses also had proapoptotic properties. The export of the M ectodomain from the Golgi apparatus to the plasma membrane appeared to be essential for the initiation of apoptosis. The study found that anti-apoptosis protein Bcl-2 protected HepG2 cells against the death-promoting activity of the DEN M ectodomain. This suggests that the M ectodomain exerts its cytotoxic effects by activating a mitochondrial apoptotic pathway. The cytotoxicity of the DEN M ectodomain reflected the intrinsic proapoptotic properties of the nine carboxy-terminal amino acids (residues M-32 to M-40) designated ApoptoM. Residue M-36 was unique in that it modulated the death-promoting activity of the M ectodomain. Defining the ApoptoM-activated signalling pathways leading to apoptosis will provide the basis for studying how the M protein might play a key role in the fate of the flavivirus-infected cells. INTRODUCTION Mosquito-borne flaviviruses such as dengue (DEN), Japanese encephalitis (JE), Saint Louis encephalitis (SLE), West Nile (WN) and yellow fever (YF) viruses may cause epidemic disease outbreaks in humans. Infected patients may exhibit a wide range of acute diseases, from nonspecific febrile illness to severe haemorrhagic manifestations (DEN and YF) or encephalitic syndromes (JE, SLE and WN). Flaviviruses (family Flaviviridae) are single-stranded, enveloped RNA viruses (Chambers et al., 1990; Rice, 1996). The virion consists of three structural proteins: C (core protein), M (membrane protein) and E (envelope protein) (Chambers et al., 1990; Rice, 1996). The virion is first assembled as an immature particle, in which prM (the intracellular precursor of M) is noncovalently associated with E in a heterodimeric complex. Late in virus morphogenesis, prM is processed by subtilisin-like proteases to generate the mature M protein (Chambers et al., 1990; Rice, 1996). The M protein (7–9 kDa) consists of a 40 amino acid long ectodomain followed by the transmembrane-anchoring region including two transmembrane domains (TMDs) (Chambers et al., 1990; Rice, 1996). Three-dimensional imaging of the struc- ture of the DEN virion, showing the location of the M protein with respect to the E homodimer, was recently carried out (Kuhn et al., 2002). Several studies have shown that the M ectodomain induces a neutralizing antibody response (Bray & Lai, 1991; Vazquez et al., 2002). All four serotypes of DEN virus (DEN-1, DEN-2, DEN-3 and DEN-4) and JE, Langat, SLE, WN and YF viruses have been reported to trigger apoptosis in host cells (Despre `s et al., 1996, 1998; Duarte dos Santos et al., 2000; Jan et al., 2000; Liao et al., 1997; Marianneau et al., 1997, 1998; Parquet et al., 2001, 2002; Prikhod’ko et al., 2001; Su et al., 2002; Xiao et al., 2001; Courageot et al., 2003; Catteau et al., 2003). Apoptosis is an active process of cell death involving a number of distinct morphological changes (Hengartner, 2000; Kimura et al., 2000). Apoptosis is induced via the activation of intracellular signalling systems, a number of which converge on mitochondrial membranes to induce 3Present address: Pasteur Institute of Saint Petersburg, U1 Mira 14, 197101 Saint Petersburg, Russia. 4Present address: Laboratoire des Biomembranes et Messagers Cellulaires, CNRS-UMR 8619, Universite ´ Paris XI, Orsay, France. 0001-9163 G 2003 SGM Printed in Great Britain 2781 Journal of General Virology (2003), 84, 2781–2793 DOI 10.1099/vir.0.19163-0