Betanodavirus non-structural protein B1: A novel anti-necrotic death factor that modulates cell death in early replication cycle in fish cells

Chen, Lei Jia; Su, Yu Chin; Hong, Jiann Ruey

doi:10.1016/j.virol.2008.11.048

Cited by 78 publications

(68 citation statements)

References 51 publications

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“…RNA3, a subgenome of RNA1, encodes the B1 and B2 proteins (12). The B1 protein is expressed at the early stage of infection, exhibiting an antinecrotic cell death function by reducing mitochondrial membrane potential (MMP) loss and, thus, enhancing cell viability (13). The B2 protein binds to NNV intermediate double-stranded RNA (dsRNA) and prevents host RNA interference (RNAi)-mediated cleavage (14).…”

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confidence: 99%

GHSC70 Is Involved in the Cellular Entry of Nervous Necrosis Virus

Chang

Chi

2015

J Virol

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Nervous necrosis virus (NNV) is a devastating pathogen of cultured marine fish and has affected more than 40 fish species. NNV belongs to the betanodaviruses of Nodaviridae and is a nonenveloped icosahedral particle with 2 single-stranded positive-sense RNAs. To date, knowledge regarding NNV entry into the host cell remains limited, and no NNV-specific receptor protein has been published. Using grouper fin cell line GF-1 and purified NNV capsid protein in a virus overlay protein binding assay (VOPBA), grouper heat shock cognate protein 70 (GHSC70) and grouper voltage-dependent anion selective channel protein 2 (GVDAC2) were investigated as NNV receptor protein candidates. We cloned and sequenced the genes for GHSC70 and GVDAC2 and expressed them in Escherichia coli for antiserum preparation. Knockdown of the expression of GHSC70 and GVDAC2 genes with specific short interfering RNAs (siRNAs) significantly downregulated viral RNA expression in NNV-infected GF-1 cells. By performing an immunoprecipitation assay, we confirmed that GHSC70 interacted with NNV capsid protein, while VDAC2 did not. Immunofluorescence staining and flow cytometry analysis revealed the presence of the GHSC70 protein on the cell surface. After a blocking assay, we detected the NNV RNA2 levels after 1 h of adsorption to GF-1 cells; the level was significantly lower in the cells pretreated with the GHSC70 antiserum than in nontreated cells. Therefore, we suggest that GHSC70 participates in the NNV entry of GF-1 cells, likely functioning as an NNV receptor or coreceptor protein. IMPORTANCEFish nodavirus has caused mass mortality of more than 40 fish species worldwide and resulted in huge economic losses in the past 20 years. Among the four genotypes of fish nodaviruses, the red-spotted grouper nervous necrosis virus (RGNNV) genotype exhibits the widest host range. In our previous study, we developed monoclonal antibodies with high neutralizing efficiency against grouper NNV in GF-1 cells, indicating that NNV-specific receptor(s) may exist on the GF-1 cell membrane. However, no NNV receptor protein has been published. In this study, we found GHSC70 to be an NNV receptor (or coreceptor) candidate through VOBPA and provided several lines of evidence demonstrating that GHSC70 protein has a role in the NNV entry step of GF-1 cells. To the best of our knowledge, this is the first report identifying grouper HSC70 and its role in NNV entry into GF-1 cells.

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confidence: 99%

GHSC70 Is Involved in the Cellular Entry of Nervous Necrosis Virus

Chang

Chi

2015

J Virol

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“…Nodaviruses also synthesize RNA3, a sub-genomic RNA species from the 3Ј 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 (3,10,11). Recently, betanodavirus B1 was found to play an anti-necrotic death function in the early replication stages (10).…”

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confidence: 99%

“…RNA3 contains two putative open reading frames that potentially encode an 111-amino acid protein B1 and a 75-amino acid protein B2 (3,10,11). Recently, betanodavirus B1 was found to play an anti-necrotic death function in the early replication stages (10). In contrast, betanodavirus B2 was found to either be a suppressor of host siRNA silencing (12,13) or a necrotic death factor (11,14).…”

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

Hong

2010

Journal of Biological Chemistry

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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 ( 41 RTFVISAHAA 50 ) 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 postfertilization. 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.Betanodaviruses 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) (1). This disease can cause mass mortality in larval and juvenile populations of several teleost species and is of global economic importance (2).The family Nodaviridae is comprised of the genera Alphanodavirus and Betanodavirus. Alphanodavirus predominantly infects insects, whereas Betanodavirus predominantly infects fish (3-5). Nodaviruses are small, non-enveloped, spherical viruses with bipartite positive-sense RNA genomes (RNA1 and RNA2) that are capped but not polyadenylated (3). RNA1 encodes an ϳ110-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 (6, 7), which may also function in the induction of cell death (8, 9). Nodaviruses also synthesize RNA3, a sub-genomic RNA species from the 3Ј 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 (3, 10, 11). Recently, betanodavirus B1 was found to play an anti-necrotic death function in the early replication stages (10). In contrast, betanodaviru...

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“…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 a 111 amino-acid protein B1 and a 75 amino-acid protein B2 [3,10,11] . Recently, the betanodavirus B1 protein has been shown to have an anti-necrotic death function during the early replication stages [10] .…”

Section: Betanodavirusmentioning

confidence: 99%

“…RNA3 contains two putative open reading frames that potentially encode a 111 amino-acid protein B1 and a 75 amino-acid protein B2 [3,10,11] . Recently, the betanodavirus B1 protein has been shown to have an anti-necrotic death function during the early replication stages [10] . In contrast, the betanodavirus B2 protein appears to function as a suppressor of host siRNA silencing [12,13] or as a EDITORIAL necrotic death factor [14,15] .…”

Section: Betanodavirusmentioning

confidence: 99%

Betanodavirus: Mitochondrial disruption and necrotic cell death

Hong¹

2013

WJV

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Betanodaviruses 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. This disease can cause mass mortality in larval and juvenile populations of several teleost species and is of global economic importance. The mechanism of brain and retina damage during betanodavirus infection is poorly understood. In this review, we will focus recent results that highlight betanodavirus infection-induced molecular death mechanisms in vitro . Betanodavirus can induce host cellular death and post-apoptotic necrosis in fish cells. Betanodavirus-induced necrotic cell death is also correlated with loss of mitochondrial membrane potential in fish cells, as this necrotic cell death is blocked by the mitochondrial membrane permeability transition pore inhibitor bongkrekic acid and the expression of the antiapoptotic Bcl-2 family member zfBcl-xL. Moreover, this mitochondria-mediated necrotic cell death may require a caspase-independent pathway. A possible cellular death pathway involving mitochondrial function and the modulator zfBcl-xs is discussed which may provide new insights into the necrotic pathogenesis of betanodavirus.

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Betanodavirus non-structural protein B1: A novel anti-necrotic death factor that modulates cell death in early replication cycle in fish cells

Cited by 78 publications

References 51 publications

GHSC70 Is Involved in the Cellular Entry of Nervous Necrosis Virus

GHSC70 Is Involved in the Cellular Entry of Nervous Necrosis Virus

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

Betanodavirus: Mitochondrial disruption and necrotic cell death

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