Betanodavirus B2 causes ATP depletion-induced cell death via mitochondrial targeting and complex II inhibition in vitro and in vivo.
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 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.
Su
Yu-Chin
YC
Laboratory of Molecular Virology and Biotechnology, Institute of Biotechnology, National Cheng Kung University, Tainan 701, Taiwan.
Hong
Jiann-Ruey
JR
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D016923:Cell Death / Q000502:physiology; D002460:Cell Line; D042963:Electron Transport Complex II / Q000378:metabolism*; D005399:Fishes / Q000378:metabolism / Q000821:virology; D015967:Gene Expression Regulation, Viral; D006801:Humans; D008928:Mitochondria / Q000378:metabolism* / Q000821:virology; D030381:Nodaviridae / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D012327:RNA Virus Infections / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis*
D021382:Protein Sorting Signals; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D042963:Electron Transport Complex II