title: Anti-Bcl-2 family members, zfBcl-x(L) and zfMcl-1a, prevent cytochrome c release from cells undergoing betanodavirus-induced secondary necrotic cell death.
abstract: Nervous necrosis virus (NNV)-induced, host cell apoptosis mediates secondary necrosis by an ill-understood process. In this study, redspotted grouper nervous necrosis virus (RGNNV) is shown to induce mitochondria-mediated necrotic cell death in GL-av cells (fish cells) via cytochrome c release, and anti-apoptotic proteins are shown to protect these cells from death. Western blots revealed that cytochrome c release coincided with disruption of mitochondrial ultrastructure and preceded necrosis, but did not correlate with caspases activation. To identify the mediator(s) of this necrotic process, a protein synthesis inhibitor (cycloheximide; CHX; 0.33 microg/ml) was used to block cytochrome c release as well as PS exposure and mitochondrial membrane permeability transition pore (MMP) loss. CHX (0.33 microg/ml) completely blocked viral protein B2 expression, and partly blocked protein A, protein alpha, and a pro-apoptotic death protein (Bad) expression. Overexpression of B2 gene increased necrotic-like cell death up to 30% at 48 h post-transfection, suggesting that newly synthesized protein (B2) may be involved in this necrotic process. Finally, necrotic death was prevented by overexpression of Bcl-2 family proteins, zfBcl-x(L) and xfMcl-1a. Thus, new protein synthesis and release of cytochrome c are required for RGNNV-induced necrotic cell death, which can be blocked by anti-apoptotic Bcl-2 members.
authors.affiliation: Laboratory of Molecular Virology and Biotechnology, Institute of Biotechnology, National Cheng Kung University, Tainan 701, Taiwan.
authors.forename: Shi-Ping
authors.initials: SP
authors.lastname: Chen
authors.forename: Jen-Leih
authors.initials: JL
authors.lastname: Wu
authors.forename: Yu-Chin
authors.initials: YC
authors.lastname: Su
authors.forename: Jiann-Ruey
authors.initials: JR
authors.lastname: Hong
mesh_terms: D000818:Animals; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005399:Fishes / Q000821:virology; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000648:ultrastructure; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D008954:Models, Biological; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009336:Necrosis / Q000473:pathology*; D009363:Neoplasm Proteins / Q000378:metabolism*; D030381:Nodaviridae / Q000502:physiology*; D010718:Phosphatidylserines / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism; D014777:Virus Diseases; D051020:bcl-X Protein / Q000378:metabolism*
chemical_list: D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D010718:Phosphatidylserines; D019253:Proto-Oncogene Proteins c-bcl-2; D014764:Viral Proteins; D051020:bcl-X Protein; D045304:Cytochromes c; D003513:Cycloheximide; D020169:Caspases