Zebrafish anti-apoptotic protein zfBcl-XL can block betanodavirus protein α-induced mitochondria-mediated secondary necrosis cell death

Betanodavirus protein a 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 24 h 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 a 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-X-L on cell death, RNA2-transfected cells were co-transfected with zfBcl-X-L. Co-transfection of GF-1 cells prevented toss 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. (c) 2008 Elsevier Ltd. All rights reserved.