Betanodavirus B2 Causes ATP Depletion-induced Cell Death via Mitochondrial Targeting and Complex II Inhibition in Vitro and in Vivo

Abstract

The betanodavirus non-structural protein B2 is a newly discovered necrotic death factor with a still unknown role in regulation of mitochondrial function. In the present study, we examined protein B2-mediated inhibition of mitochondrial complex II activity, which results in ATP depletion and thereby in a bioenergetic crisis in vitro and in vivo. Expression of protein B2 was detected early at 24 h postinfection with red-spotted grouper nervous necrosis virus in the cytoplasm. Later B2 was found in mitochondria using enhanced yellow fluorescent protein (EYFP) and immuno-EM analysis. Furthermore, the B2 mitochondrial targeting signal peptide was analyzed by serial deletion and specific point mutation. The sequence of the B2 targeting signal peptide (41RTFVISAHAA50) was identified and its presence correlated with loss of mitochondrial membrane potential in fish cells. Protein B2 also was found to dramatically inhibit complex II (succinate dehydrogenase) activity, which impairs ATP synthesis in fish GF-1 cells as well as human embryonic kidney 293T cells. Furthermore, when B2 was injected into zebrafish embryos at the one-cell stage to determine its cytotoxicity and ability to inhibit ATP synthesis, we found that B2 caused massive embryonic cell death and depleted ATP resulting in further embryonic death at 10 and 24 h post-fertilization. Taken together, our results indicate that betanodavirus protein B2-induced cell death is due to direct targeting of the mitochondrial matrix by a specific signal peptide that targets mitochondria and inhibits mitochondrial complex II activity thereby reducing ATP synthesis. The betanodavirus non-structural protein B2 is a newly discovered necrotic death factor with a still unknown role in regulation of mitochondrial function. In the present study, we examined protein B2-mediated inhibition of mitochondrial complex II activity, which results in ATP depletion and thereby in a bioenergetic crisis in vitro and in vivo. Expression of protein B2 was detected early at 24 h postinfection with red-spotted grouper nervous necrosis virus in the cytoplasm. Later B2 was found in mitochondria using enhanced yellow fluorescent protein (EYFP) and immuno-EM analysis. Furthermore, the B2 mitochondrial targeting signal peptide was analyzed by serial deletion and specific point mutation. The sequence of the B2 targeting signal peptide (41RTFVISAHAA50) was identified and its presence correlated with loss of mitochondrial membrane potential in fish cells. Protein B2 also was found to dramatically inhibit complex II (succinate dehydrogenase) activity, which impairs ATP synthesis in fish GF-1 cells as well as human embryonic kidney 293T cells. Furthermore, when B2 was injected into zebrafish embryos at the one-cell stage to determine its cytotoxicity and ability to inhibit ATP synthesis, we found that B2 caused massive embryonic cell death and depleted ATP resulting in further embryonic death at 10 and 24 h post-fertilization. Taken together, our results indicate that betanodavirus protein B2-induced cell death is due to direct targeting of the mitochondrial matrix by a specific signal peptide that targets mitochondria and inhibits mitochondrial complex II activity thereby reducing ATP synthesis. IntroductionBetanodaviruses cause viral nervous necrosis, an infectious neuropathological condition in fish that is characterized by necrosis of the central nervous system, including the brain and retina and by clinical signs (e.g. abnormal swimming behavior and development of a darker body color) (1Bovo G. Nishizawa T. Maltese C. Borghesan F. Mutinelli F. Montesi F. De Mas S. Virus Res. 1999; 63: 143-146Crossref PubMed Scopus (93) Google Scholar). This disease can cause mass mortality in larval and juvenile populations of several teleost species and is of global economic importance (2Tan C. Huang B. Chang S.F. Ngoh G.H. Munday B. Chen S.C. Kwang J. J. Gen. Virol. 2001; 82: 647-653Crossref PubMed Scopus (106) Google Scholar).The family Nodaviridae is comprised of the genera Alphanodavirus and Betanodavirus. Alphanodavirus predominantly infects insects, whereas Betanodavirus predominantly infects fish (3Ball L.A. Johnson K.L. Adv. Virus Res. 1999; 53: 229-244Crossref PubMed Google Scholar, 4Schneemann A. Reddy V. Johnson J.E. Adv. Virus Res. 1998; 50: 381-446Crossref PubMed Google Scholar, 5Toffolo V. Negrisolo E. Maltese C. Bovo G. Belvedere P. Colombo L. Valle L.D. Mol. Phylogenet. Evol. 2007; 43: 298-308Crossref PubMed Scopus (101) Google Scholar). Nodaviruses are small, non-enveloped, spherical viruses with bipartite positive-sense RNA genomes (RNA1 and RNA2) that are capped but not polyadenylated (3Ball L.A. Johnson K.L. Adv. Virus Res. 1999; 53: 229-244Crossref PubMed Google Scholar). RNA1 encodes an ∼110-kDa non-structural protein that has been designated RNA-dependent RNA polymerase or protein A. This protein is vital for replication of the viral genome. RNA2 encodes a 42-kDa capsid protein (6Delsert C. Morin N. Comps M. Arch. Virol. 1997; 142: 2359-2371Crossref PubMed Scopus (60) Google Scholar, 7Mori K. Nakai T. Muroga K. Arimoto M. Mushiake K. Furusawa I. Virology. 1992; 187: 368-371Crossref PubMed Scopus (397) Google Scholar), which may also function in the induction of cell death (8Guo Y.X. Wei T. Dallmann K. Kwang J. Virology. 2003; 308: 74-82Crossref PubMed Scopus (52) Google Scholar, 9Wu H.C. Chiu C.S. Wu J.L. Gong H.Y. Chen M.C. Lu M.W. Hong J.R. Fish Shellfish Immunol. 2008; 24: 436-449Crossref PubMed Scopus (36) Google Scholar). Nodaviruses also synthesize RNA3, a sub-genomic RNA species from the 3′ terminus of RNA1. RNA3 contains two putative open reading frames that potentially encode an 111-amino acid protein B1 and a 75-amino acid protein B2 (3Ball L.A. Johnson K.L. Adv. Virus Res. 1999; 53: 229-244Crossref PubMed Google Scholar, 10Chen L.J. Su Y.C. Hong J.R. Virology. 2009; 385: 444-454Crossref PubMed Scopus (63) Google Scholar, 11Chen S.P. Wu J.L. Su Y.C. Hong J.R. Apoptosis. 2007; 12: 1043-1060Crossref PubMed Scopus (45) Google Scholar). Recently, betanodavirus B1 was found to play an anti-necrotic death function in the early replication stages (10Chen L.J. Su Y.C. Hong J.R. Virology. 2009; 385: 444-454Crossref PubMed Scopus (63) Google Scholar). In contrast, betanodavirus B2 was found to either be a suppressor of host siRNA silencing (12Fenner B.J. Goh W. Kwang J. J. Virol. 2006; 80: 6822-6833Crossref PubMed Scopus (55) Google Scholar, 13Iwamoto T. Mise K. Takeda A. Okinaka Y. Mori K. Arimoto M. Okuno T. Nakai T. J. Gen. Virol. 2005; 86: 2807-2816Crossref PubMed Scopus (137) Google Scholar) or a necrotic death factor (11Chen S.P. Wu J.L. Su Y.C. Hong J.R. Apoptosis. 2007; 12: 1043-1060Crossref PubMed Scopus (45) Google Scholar, 14Su Y.C. Wu J.L. Hong J.R. Virology. 2009; 385: 143-154Crossref PubMed Scopus (46) Google Scholar).Mitochondria are organelles required for cellular energy production, programmed cell death regulation (15Eisenberg T. Büttner S. Kroemer G. Madeo F. Apoptosis. 2007; 12: 1011-1023Crossref PubMed Scopus (180) Google Scholar), reactive oxygen production (16Boveris A. Oshino N. Chance B. Biochem. J. 1972; 128: 617-630Crossref PubMed Scopus (1207) Google Scholar), and intermediary metabolism (17Jones D.P. Kennedy F.G. Am. J. Physiol. Cell Physiol. 1982; 243: C247-253Crossref PubMed Google Scholar). Changes in mitochondrial function, such as suppression of mitochondrial metabolism, accumulation of reactive oxygen species, loss of mitochondrial membrane potential, and reduced respiration have been shown to play a key role in induction of cell death (18Higuchi Y. J. Cell Mol. Med. 2004; 8: 455-464Crossref PubMed Scopus (188) Google Scholar, 19Zuryn S. Kuang J. Ebert P. Toxicol. Sci. 2008; 102: 179-186Crossref PubMed Scopus (64) Google Scholar). Mitochondria produce the majority of cellular ATP via oxidative phosphorylation. The mitochondrial electron transport chain removes electrons from an electron donor (NADH for Complex I or QH2 for Complex III) and passes them to a terminal electron acceptor (O2) via a series of redox reactions. These reactions are coupled to the creation of a proton gradient across the mitochondrial inner membrane. The resulting transmembrane proton gradient is used to make ATP via ATP synthase (20de Castro I.P. Martins L.M. Tufi R. Expert Rev. Mol. Med. 2010; 12: e12Crossref PubMed Scopus (66) Google Scholar). Mitochondrial disorders are often present as neurological diseases such as Parkinson disease (21Furuya T. Hayakawa H. Yamada M. Yoshimi K. Hisahara S. Miura M. Mizuno Y. Mochizuki H. J. Neurosci. 2004; 24: 1865-1872Crossref PubMed Scopus (116) Google Scholar, 22Kroemer G. Blomgren K. PLoS Biol. 2007; 5: e206Crossref PubMed Scopus (23) Google Scholar), Alzheimer disease (23Manczak M. Anekonda T.S. Henson E. Park B.S. Quinn J. Reddy P.H. Hum. Mol. Genet. 2006; 15: 1437-1449Crossref PubMed Scopus (872) Google Scholar, 24Ohyagi Y. Asahara H. Chui D.H. Tsuruta Y. Sakae N. Miyoshi K. Yamada T. Kikuchi H. Taniwaki T. Murai H. Ikezoe K. Furuya H. Kawarabayashi T. Shoji M. F. T. T. Takeda K. J. T. J. 2005; PubMed Scopus Google Scholar, P.H. 2009; PubMed Google Scholar, P.H. 2009; PubMed Scopus Google Scholar, P.H. S. 2006; PubMed Scopus Google Scholar), and disease P.H. P. M. Res. Rev. 2009; PubMed Scopus Google viruses and viral can the death in cells L. C. E. Kroemer G. PLoS 2008; PubMed Scopus Google Scholar). may be death which when into mitochondria loss of mitochondrial membrane potential used membrane yellow fluorescent fluorescent grouper nervous necrosis of or loss of of host or death have sequence to of the family or inhibit cell death via L. C. E. Kroemer G. PLoS 2008; PubMed Scopus Google Scholar). Mitochondrial membrane in the loss of mitochondrial transmembrane potential an of mitochondrial bioenergetic and in the of mitochondrial C. L. M. C. Kroemer G. Cell 2006; PubMed Scopus Google Scholar, J. R. 86: PubMed Scopus Google Scholar) and C. M. Y. L. 102: PubMed Scopus Google Scholar), factor N. F. Madeo F. Kroemer G. Cell Biol. 2006; PubMed Scopus Google Scholar, N. I. J. E. P. M. N. R. D.P. Kroemer G. 1999; PubMed Scopus Google Scholar), and 2001; PubMed Scopus Google into the and in to death G. L. C. Physiol. Rev. 2007; PubMed Scopus Google Scholar, A. Rev. Mol. Cell Biol. 2008; PubMed Scopus Google our of host cell the red-spotted grouper nervous necrosis virus and necrosis in a grouper cell S.P. H.Y. Wu J.L. Hong J.R. Virology. 2006; PubMed Scopus (45) Google Scholar). The also loss of the which was by the S.P. H.Y. Wu J.L. Hong J.R. Virology. 2006; PubMed Scopus (45) Google Scholar) as well as the protein T. Mise K. Takeda A. Okinaka Y. Mori K. Arimoto M. Okuno T. Nakai T. J. Gen. Virol. 2005; 86: 2807-2816Crossref PubMed Scopus (137) Google Scholar). In B2 protein necrotic cell death from via a Y.C. Wu J.L. Hong J.R. Virology. 2009; 385: 143-154Crossref PubMed Scopus (46) Google Scholar) and is from via of the of (11Chen S.P. Wu J.L. Su Y.C. Hong J.R. Apoptosis. 2007; 12: 1043-1060Crossref PubMed Scopus (45) Google Scholar, 14Su Y.C. Wu J.L. Hong J.R. Virology. 2009; 385: 143-154Crossref PubMed Scopus (46) Google Scholar). the of protein B2 induction of necrotic cell death is still and may into the of RNA virus the present study, we that B2 targets mitochondria using a specific signal peptide that B2 to the mitochondrial mitochondrial and necrotic cell death in fish cells. Furthermore, we to determine B2 necrotic cell viral nervous necrosis and the fish to its or as in or to the cellular and necrotic in the central nervous and retina S. I. Nakai T. T. J. Fish 2003; PubMed Scopus Google Scholar). The in the of disease are still the present study, the sequence (41RTFVISAHAA50) in protein B2 was identified as the signal used to the mitochondrial Protein B2 loss in fish and human by mitochondrial ATP Furthermore, necrotic death factor ATP depletion in embryos and embryonic cell These results may be the to the of in the central nervous and retina S. I. Nakai T. T. J. Fish 2003; PubMed Scopus Google of the B2 Protein for Mitochondrial the of 75-amino acid protein from T. Mise K. Takeda A. Okinaka Y. Mori K. Arimoto M. Okuno T. Nakai T. J. Gen. Virol. 2005; 86: 2807-2816Crossref PubMed Scopus (137) Google Scholar) may either be a suppressor of host siRNA silencing (12Fenner B.J. Goh W. Kwang J. J. Virol. 2006; 80: 6822-6833Crossref PubMed Scopus (55) Google Scholar, 13Iwamoto T. Mise K. Takeda A. Okinaka Y. Mori K. Arimoto M. Okuno T. Nakai T. J. Gen. Virol. 2005; 86: 2807-2816Crossref PubMed Scopus (137) Google Scholar) or a necrotic death factor (11Chen S.P. Wu J.L. Su Y.C. Hong J.R. Apoptosis. 2007; 12: 1043-1060Crossref PubMed Scopus (45) Google Scholar, 14Su Y.C. Wu J.L. Hong J.R. Virology. 2009; 385: 143-154Crossref PubMed Scopus (46) Google Scholar). Mitochondria to be the targets of In our system, B2 was early replication at 24 h that B2 targets and and to in the at h may as a host siRNA silencing suppressor by and and and protein B2 was shown to Furthermore, we to B2 in the membrane or inner membrane of mitochondria protein function them L. C. E. Kroemer G. PLoS 2008; PubMed Scopus Google Scholar). that protein B2 and in the mitochondrial matrix by mitochondrial are in the as which are into either the or inner membrane of the mitochondria W. Rev. Biochem. 1997; PubMed Scopus Google Scholar, W. H. T. N. 1999; PubMed Scopus Google Scholar, W. 2008; PubMed Scopus Google Scholar). These have an sequence that is either or and an T. S. C. R. G. K. J. 1998; PubMed Scopus (137) Google Scholar, G. J. L. J. 1999; PubMed Scopus Google Scholar, M. J. Biol. 2001; PubMed Scopus Google Scholar, P. N. C. J. Mol. Biol. 2003; PubMed Scopus Google Scholar, G. J. Biol. PubMed Scopus Google Scholar). to mitochondrial membrane often an or targeting sequence and signal sequence W. Rev. Biochem. 1997; PubMed Scopus Google Scholar, J. 1997; PubMed Scopus Google Scholar), but the of the B2 targeting is not well In our study, the targeting (41RTFVISAHAA50) of protein B2 10 acid and a function early of sequence in loss of and loss of activity of in fish GF-1 cells. The and the signal peptide are for mitochondrial targeting W. Rev. Biochem. 1997; PubMed Scopus Google Scholar, G. J. Biol. PubMed Scopus Google Scholar, H. R. Y. N. H. 2004; PubMed Scopus Google Scholar). the and and the two and the signal sequence to targeting to the was that and of protein B2 are for targeting and necrosis induction B2 and a Mitochondrial mitochondria are vital cellular for cellular energy and oxygen to produce ATP a as oxidative (16Boveris A. Oshino N. Chance B. Biochem. J. 1972; 128: 617-630Crossref PubMed Scopus (1207) Google Scholar). The inner mitochondrial membrane contains a chain of protein that energy used to across membrane. The gradient of and resulting mitochondria membrane potential ATP from and (16Boveris A. Oshino N. Chance B. Biochem. J. 1972; 128: 617-630Crossref PubMed Scopus (1207) Google Scholar). to mitochondria an role in a of human diseases (20de Castro I.P. Martins L.M. Tufi R. Expert Rev. Mol. Med. 2010; 12: e12Crossref PubMed Scopus (66) Google Scholar, N. S. Ebert 2008; PubMed Scopus Google Scholar). Mitochondrial disorders often present as neurological disorders such as Parkinson disease (21Furuya T. Hayakawa H. Yamada M. Yoshimi K. Hisahara S. Miura M. Mizuno Y. Mochizuki H. J. Neurosci. 2004; 24: 1865-1872Crossref PubMed Scopus (116) Google Scholar, 22Kroemer G. Blomgren K. PLoS Biol. 2007; 5: e206Crossref PubMed Scopus (23) Google Scholar), Alzheimer disease (23Manczak M. Anekonda T.S. Henson E. Park B.S. Quinn J. Reddy P.H. Hum. Mol. Genet. 2006; 15: 1437-1449Crossref PubMed Scopus (872) Google Scholar, 24Ohyagi Y. Asahara H. Chui D.H. Tsuruta Y. Sakae N. Miyoshi K. Yamada T. Kikuchi H. Taniwaki T. Murai H. Ikezoe K. Furuya H. Kawarabayashi T. Shoji M. F. T. T. Takeda K. J. T. J. 2005; PubMed Scopus Google Scholar, P.H. 2009; PubMed Google Scholar, P.H. 2009; PubMed Scopus Google Scholar, P.H. S. 2006; PubMed Scopus Google Scholar), and disease P.H. P. M. Res. Rev. 2009; PubMed Scopus Google Scholar). of have that cell death be due to mitochondrial energy Cell death be by loss of of and depletion of ATP B. Res. 2006; PubMed Scopus Google Scholar). In our system, protein B2 mitochondria to inhibit the activity of complex II but not complex I and thereby ATP and the of complex II as and In fish and human depletion of ATP in early zebrafish embryos used as a J.R. G.H. Wu J.L. 2004; PubMed Scopus Google Scholar), which in embryonic death and These in vitro and in results that protein B2 a mitochondrial energy Mitochondrial and well and RNA viruses have been shown to cause cell of viral by either or of the mitochondrial membrane or by of the cell L. C. E. Kroemer G. PLoS 2008; PubMed Scopus Google Scholar). In contrast, a viral (e.g. Y. M. L. S. S. K. H. J. Gen. Virol. 2006; PubMed Scopus Google Scholar) and C. V. PubMed Scopus Google in and cell S.P. H.Y. Wu J.L. Hong J.R. Virology. 2006; PubMed Scopus (45) Google Scholar) that the and necrosis in a grouper cell in fish cells loss of which was by the mitochondrial membrane S.P. H.Y. Wu J.L. Hong J.R. Virology. 2006; PubMed Scopus (45) Google Scholar) as well as the protein (11Chen S.P. Wu J.L. Su Y.C. Hong J.R. Apoptosis. 2007; 12: 1043-1060Crossref PubMed Scopus (45) Google Scholar). that cell death was viral RNA the for the of the viral death protein be and be in mitochondria H.C. Wu J.L. Su Y.C. Hong J.R. Fish Shellfish 2010; PubMed Scopus Google Scholar). Furthermore, in the viral capsid protein (8Guo Y.X. Wei T. Dallmann K. Kwang J. Virology. 2003; 308: 74-82Crossref PubMed Scopus (52) Google Scholar, 9Wu H.C. Chiu C.S. Wu J.L. Gong H.Y. Chen M.C. Lu M.W. Hong J.R. Fish Shellfish Immunol. 2008; 24: 436-449Crossref PubMed Scopus (36) Google Scholar) and non-structural protein B2 (11Chen S.P. Wu J.L. Su Y.C. Hong J.R. Apoptosis. 2007; 12: 1043-1060Crossref PubMed Scopus (45) Google Scholar, 14Su Y.C. Wu J.L. Hong J.R. Virology. 2009; 385: 143-154Crossref PubMed Scopus (46) Google Scholar) loss of and Protein and and necrosis thereby cells (8Guo Y.X. Wei T. Dallmann K. Kwang J. Virology. 2003; 308: 74-82Crossref PubMed Scopus (52) Google Scholar, 9Wu H.C. Chiu C.S. Wu J.L. Gong H.Y. Chen M.C. Lu M.W. Hong J.R. Fish Shellfish Immunol. 2008; 24: 436-449Crossref PubMed Scopus (36) Google Scholar). the protein B2 the to but not of and of (11Chen S.P. Wu J.L. Su Y.C. Hong J.R. Apoptosis. 2007; 12: 1043-1060Crossref PubMed Scopus (45) Google Scholar). necrosis can be by siRNA and the protein (11Chen S.P. Wu J.L. Su Y.C. Hong J.R. Apoptosis. 2007; 12: 1043-1060Crossref PubMed Scopus (45) Google Scholar, 14Su Y.C. Wu J.L. Hong J.R. Virology. 2009; 385: 143-154Crossref PubMed Scopus (46) Google Scholar). In the present study, protein B2 mitochondria via targeting and inhibition of complex II function and The resulting loss and ATP depletion and to necrosis which is a used by viruses to the of cell in protein B2 is a non-structural protein early in the in the mitochondria and in the targeting signal sequence of 10 in the central of the B2 protein B2 to the mitochondrial matrix mitochondrial function. Protein B2 inhibits complex II activity to induction of loss and ATP depletion in a bioenergetic crisis and These may into the of RNA viruses and clinical IntroductionBetanodaviruses cause viral nervous necrosis, an infectious neuropathological condition in fish that is characterized by necrosis of the central nervous system, including the brain and retina and by clinical signs (e.g. abnormal swimming behavior and development of a darker body color) (1Bovo G. Nishizawa T. Maltese C. Borghesan F. Mutinelli F. Montesi F. De Mas S. Virus Res. 1999; 63: 143-146Crossref PubMed Scopus (93) Google Scholar). This disease can cause mass mortality in larval and juvenile populations of several teleost species and is of global economic importance (2Tan C. Huang B. Chang S.F. Ngoh G.H. Munday B. Chen S.C. Kwang J. J. Gen. Virol. 2001; 82: 647-653Crossref PubMed Scopus (106) Google Scholar).The family Nodaviridae is comprised of the genera Alphanodavirus and Betanodavirus. Alphanodavirus predominantly infects insects, whereas Betanodavirus predominantly infects fish (3Ball L.A. Johnson K.L. Adv. Virus Res. 1999; 53: 229-244Crossref PubMed Google Scholar, 4Schneemann A. Reddy V. Johnson J.E. Adv. Virus Res. 1998; 50: 381-446Crossref PubMed Google Scholar, 5Toffolo V. Negrisolo E. Maltese C. Bovo G. Belvedere P. Colombo L. Valle L.D. Mol. Phylogenet. Evol. 2007; 43: 298-308Crossref PubMed Scopus (101) Google Scholar). Nodaviruses are small, non-enveloped, spherical viruses with bipartite positive-sense RNA genomes (RNA1 and RNA2) that are capped but not polyadenylated (3Ball L.A. Johnson K.L. Adv. Virus Res. 1999; 53: 229-244Crossref PubMed Google Scholar). RNA1 encodes an ∼110-kDa non-structural protein that has been designated RNA-dependent RNA polymerase or protein A. This protein is vital for replication of the viral genome. RNA2 encodes a 42-kDa capsid protein (6Delsert C. Morin N. Comps M. Arch. Virol. 1997; 142: 2359-2371Crossref PubMed Scopus (60) Google Scholar, 7Mori K. Nakai T. Muroga K. Arimoto M. Mushiake K. Furusawa I. Virology. 1992; 187: 368-371Crossref PubMed Scopus (397) Google Scholar), which may also function in the induction of cell death (8Guo Y.X. Wei T. Dallmann K. Kwang J. Virology. 2003; 308: 74-82Crossref PubMed Scopus (52) Google Scholar, 9Wu H.C. Chiu C.S. Wu J.L. Gong H.Y. Chen M.C. Lu M.W. Hong J.R. Fish Shellfish Immunol. 2008; 24: 436-449Crossref PubMed Scopus (36) Google Scholar). Nodaviruses also synthesize RNA3, a sub-genomic RNA species from the 3′ terminus of RNA1. RNA3 contains two putative open reading frames that potentially encode an 111-amino acid protein B1 and a 75-amino acid protein B2 (3Ball L.A. Johnson K.L. Adv. Virus Res. 1999; 53: 229-244Crossref PubMed Google Scholar, 10Chen L.J. Su Y.C. Hong J.R. Virology. 2009; 385: 444-454Crossref PubMed Scopus (63) Google Scholar, 11Chen S.P. Wu J.L. Su Y.C. Hong J.R. Apoptosis. 2007; 12: 1043-1060Crossref PubMed Scopus (45) Google Scholar). Recently, betanodavirus B1 was found to play an anti-necrotic death function in the early replication stages (10Chen L.J. Su Y.C. Hong J.R. Virology. 2009; 385: 444-454Crossref PubMed Scopus (63) Google Scholar). In contrast, betanodavirus B2 was found to either be a suppressor of host siRNA silencing (12Fenner B.J. Goh W. Kwang J. J. Virol. 2006; 80: 6822-6833Crossref PubMed Scopus (55) Google Scholar, 13Iwamoto T. Mise K. Takeda A. Okinaka Y. Mori K. Arimoto M. Okuno T. Nakai T. J. Gen. Virol. 2005; 86: 2807-2816Crossref PubMed Scopus (137) Google Scholar) or a necrotic death factor (11Chen S.P. Wu J.L. Su Y.C. Hong J.R. Apoptosis. 2007; 12: 1043-1060Crossref PubMed Scopus (45) Google Scholar, 14Su Y.C. Wu J.L. Hong J.R. Virology. 2009; 385: 143-154Crossref PubMed Scopus (46) Google Scholar).Mitochondria are organelles required for cellular energy production, programmed cell death regulation (15Eisenberg T. Büttner S. Kroemer G. Madeo F. Apoptosis. 2007; 12: 1011-1023Crossref PubMed Scopus (180) Google Scholar), reactive oxygen production (16Boveris A. Oshino N. Chance B. Biochem. J. 1972; 128: 617-630Crossref PubMed Scopus (1207) Google Scholar), and intermediary metabolism (17Jones D.P. Kennedy F.G. Am. J. Physiol. Cell Physiol. 1982; 243: C247-253Crossref PubMed Google Scholar). Changes in mitochondrial function, such as suppression of mitochondrial metabolism, accumulation of reactive oxygen species, loss of mitochondrial membrane potential, and reduced respiration have been shown to play a key role in induction of cell death (18Higuchi Y. J. Cell Mol. Med. 2004; 8: 455-464Crossref PubMed Scopus (188) Google Scholar, 19Zuryn S. Kuang J. Ebert P. Toxicol. Sci. 2008; 102: 179-186Crossref PubMed Scopus (64) Google Scholar). Mitochondria produce the majority of cellular ATP via oxidative phosphorylation. The mitochondrial electron transport chain removes electrons from an electron donor (NADH for Complex I or QH2 for Complex III) and passes them to a terminal electron acceptor (O2) via a series of redox reactions. These reactions are coupled to the creation of a proton gradient across the mitochondrial inner membrane. The resulting transmembrane proton gradient is used to make ATP via ATP synthase (20de Castro I.P. Martins L.M. Tufi R. Expert Rev. Mol. Med. 2010; 12: e12Crossref PubMed Scopus (66) Google Scholar). Mitochondrial disorders are often present as neurological diseases such as Parkinson disease (21Furuya T. Hayakawa H. Yamada M. Yoshimi K. Hisahara S. Miura M. Mizuno Y. Mochizuki H. J. Neurosci. 2004; 24: 1865-1872Crossref PubMed Scopus (116) Google Scholar, 22Kroemer G. Blomgren K. PLoS Biol. 2007; 5: e206Crossref PubMed Scopus (23) Google Scholar), Alzheimer disease (23Manczak M. Anekonda T.S. Henson E. Park B.S. Quinn J. Reddy P.H. Hum. Mol. Genet. 2006; 15: 1437-1449Crossref PubMed Scopus (872) Google Scholar, 24Ohyagi Y. Asahara H. Chui D.H. Tsuruta Y. Sakae N. Miyoshi K. Yamada T. Kikuchi H. Taniwaki T. Murai H. Ikezoe K. Furuya H. Kawarabayashi T. Shoji M. F. T. T. Takeda K. J. T. J. 2005; PubMed Scopus Google Scholar, P.H. 2009; PubMed Google Scholar, P.H. 2009; PubMed Scopus Google Scholar, P.H. S. 2006; PubMed Scopus Google Scholar), and disease P.H. P. M. Res. Rev. 2009; PubMed Scopus Google viruses and viral can the death in cells L. C. E. Kroemer G. PLoS 2008; PubMed Scopus Google Scholar). may be death which when into mitochondria loss of mitochondrial membrane potential used membrane yellow fluorescent fluorescent grouper nervous necrosis of or loss of of host or death have sequence to of the family or inhibit cell death via L. C. E. Kroemer G. PLoS 2008; PubMed Scopus Google Scholar). Mitochondrial membrane in the loss of mitochondrial transmembrane potential an of mitochondrial bioenergetic and in the of mitochondrial C. L. M. C. Kroemer G. Cell 2006; PubMed Scopus Google Scholar, J. R. 86: PubMed Scopus Google Scholar) and C. M. Y. L. 102: PubMed Scopus Google Scholar), factor N. F. Madeo F. Kroemer G. Cell Biol. 2006; PubMed Scopus Google Scholar, N. I. J. E. P. M. N. R. D.P. Kroemer G. 1999; PubMed Scopus Google Scholar), and 2001; PubMed Scopus Google into the and in to death G. L. C. Physiol. Rev. 2007; PubMed Scopus Google Scholar, A. Rev. Mol. Cell Biol. 2008; PubMed Scopus Google our of host cell the red-spotted grouper nervous necrosis virus and necrosis in a grouper cell S.P. H.Y. Wu J.L. Hong J.R. Virology. 2006; PubMed Scopus (45) Google Scholar). The also loss of the which was by the S.P. H.Y. Wu J.L. Hong J.R. Virology. 2006; PubMed Scopus (45) Google Scholar) as well as the protein T. Mise K. Takeda A. Okinaka Y. Mori K. Arimoto M. Okuno T. Nakai T. J. Gen. Virol. 2005; 86: 2807-2816Crossref PubMed Scopus (137) Google Scholar). In B2 protein necrotic cell death from via a Y.C. Wu J.L. Hong J.R. Virology. 2009; 385: 143-154Crossref PubMed Scopus (46) Google Scholar) and is from via of the of (11Chen S.P. Wu J.L. Su Y.C. Hong J.R. Apoptosis. 2007; 12: 1043-1060Crossref PubMed Scopus (45) Google Scholar, 14Su Y.C. Wu J.L. Hong J.R. Virology. 2009; 385: 143-154Crossref PubMed Scopus (46) Google Scholar). the of protein B2 induction of necrotic cell death is still and may into the of RNA virus the present study, we that B2 targets mitochondria using a specific signal peptide that B2 to the mitochondrial mitochondrial and necrotic cell death in fish cells. Furthermore, we to determine B2 necrotic cell