Zebrafish anti-apoptotic protein zfBcl-xL can block betanodavirus protein alpha-induced mitochondria-mediated secondary necrosis cell death.
Betanodavirus protein alpha induces cell apoptosis or secondary necrosis by a poorly understood process. In the present work, red spotted grouper nervous necrosis virus (RGNNV) RNA 2 was cloned and transfected into tissue culture cells (GF-1) which then underwent apoptosis or post-apoptotic necrosis. In the early apoptotic stage, progressive phosphatidylserine externalization was evident at 24h post-transfection (p.t.) by Annexin V-FLUOS staining. TUNEL assay revealed apoptotic cells at 24-72 h p.t, after which post-apoptotic necrotic cells were identified by acridine orange/ethidium bromide dual dye staining from 48 to 72 h p.t. Protein alpha induced progressive loss of mitochondrial membrane potential (MMP) which was detected in RNA2-transfected GF-1 cells at 24, 48, and 72 h p.t., which correlated with cytochrome c release, especially at 72 h p.t. To assess the effect of zfBcl-xL on cell death, RNA2-transfected cells were co-transfected with zfBcl-x(L). Co-transfection of GF-1 cells prevented loss of MMP at 24 h and 48 h p.t. and blocked initiator caspase-8 and effector caspase-3 activation at 48 h p.t. We conclude that RGNNV protein alpha induces apoptosis followed by secondary necrotic cell death through a mitochondria-mediated death pathway and activation of caspases-8 and -3.
Wu
Horng-Cherng
HC
Laboratory of Molecular Virology and Biotechnology, Institute of Biotechnology, National Cheng-Kung University, Tainan 701, Taiwan.
Chiu
Chuan-Sheng
CS
Wu
Jen-Leih
JL
Gong
Hong-Yi
HY
Chen
Ming-Chyuan
MC
Lu
Ming-Wei
MW
Hong
Jiann-Ruey
JR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D045304:Cytochromes c / Q000378:metabolism; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009336:Necrosis / Q000473:pathology / Q000821:virology; D030381:Nodaviridae / Q000502:physiology*; D014764:Viral Proteins / Q000378:metabolism; D015027:Zebrafish / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism*
D036022:Capsid Proteins; D014764:Viral Proteins; D029961:Zebrafish Proteins; D051020:bcl-X Protein; C494810:bcl2l1 protein, zebrafish; D045304:Cytochromes c; D053148:Caspase 3; D053181:Caspase 8