Aquatic birnavirus capsid protein, VP3, induces apoptosis via the Bad-mediated mitochondria pathway in fish and mouse cells.
Aquatic birnavirus induces post-apoptotic necrotic cell death via a newly synthesized protein-dependent pathway. However, the involvement of viral genome-encoded protein(s) in this death process remains unknown. In the present study, we demonstrated that the submajor capsid protein, VP3, up-regulates the pro-apoptotic protein, Bad, in fish and mouse cells. Western blot analysis revealed that VP3 was expressed in CHSE-214 cells at 4 h post-infection (pi), indicating an early role during viral replication. We cloned the VP3 gene and tested its function in fish and mouse cells; VP3 overexpression induced apoptotic cell death by TUNEL assay. In addition, it up-regulated Bad gene expression in zebrafish ZLE cells by threefold at 12 h post-transfection (pt) and in mouse NIH3T3 cells by tenfold at 24 h pt. VP3 up-regulation of Bad expression altered mitochondria function, inducing mitochondrial membrane potential (MMP) loss and activating initiator caspase-9 and effector caspase-3. Furthermore, reduced Bad expression (65% reduction), MMP loss (up to 40%), and enhanced cell viability (up to 60%) upon expression of VP3 antisense RNA in CHSE-214 cells at 24 h post-IPNV infection was observed. Finally, overexpression of the anti-apoptotic gene, zfBcl-xL, reduced VP3-induced apoptotic cell death and caspase-3 activation at 24 h in fish cells. Taken together, these results suggest that aquatic birnavirus VP3 induces apoptosis via up-regulation of Bad expression and mitochondrial disruption, which activates a downstream caspase-3-mediated death pathway that is blocked by zfBcl-xL.
Chiu
Chien-Li
CL
Institute of Biotechnology, National Cheng Kung University, Tainan, Taiwan, ROC.
Wu
Jen-Leih
JL
Her
Guor-Mour
GM
Chou
Yi-Li
YL
Hong
Jiann-Ruey
JR
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D018175:Birnaviridae Infections / Q000378:metabolism / Q000503:physiopathology / Q000662:veterinary* / Q000821:virology; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002460:Cell Line; D005393:Fish Diseases / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology* / Q000821:virology; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism*; D005399:Fishes; D015967:Gene Expression Regulation, Viral; D018061:Infectious pancreatic necrosis virus / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D015398:Signal Transduction; D015854:Up-Regulation; D051025:bcl-Associated Death Protein / Q000235:genetics / Q000378:metabolism*
D036022:Capsid Proteins; D029941:Fish Proteins; D051025:bcl-Associated Death Protein; D053148:Caspase 3; D053453:Caspase 9