Abstract
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.
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Acknowledgments
The authors are grateful to Dr. Wu (Biotechnology, Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei 115, Taiwan) for providing the zfBcl-xL and IPNV VP5 expression plasmids and anti-IPNV E1S VP3 monoclonal antibodies (MAb, 8-42-E7). This work was supported by grants from the National Science Council, Taiwan, Republic of China awarded to Dr. Jainn-Ruey Hong (NSC-92-2313-B006-005 and NSC 96-2313-B-004-MY3).
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Chiu, CL., Wu, JL., Her, GM. et al. Aquatic birnavirus capsid protein, VP3, induces apoptosis via the Bad-mediated mitochondria pathway in fish and mouse cells. Apoptosis 15, 653–668 (2010). https://doi.org/10.1007/s10495-010-0468-x
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DOI: https://doi.org/10.1007/s10495-010-0468-x