, Volume 15, Issue 6, pp 653–668

Aquatic birnavirus capsid protein, VP3, induces apoptosis via the Bad-mediated mitochondria pathway in fish and mouse cells

  • Chien-Li Chiu
  • Jen-Leih Wu
  • Guor-Mour Her
  • Yi-Li Chou
  • Jiann-Ruey Hong
Original Paper


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.


Infectious pancreatic necrosis virus Submajor capsid VP3 Pro-apoptotic Bad Mitochondrial membrane potential loss zfBcl-xL Antisense RNA 


  1. 1.
    Wyllie A, Kerr J, Currie A (1980) Cell death: the significance of apoptosis. Int Rev Cytol 68:251–306CrossRefPubMedGoogle Scholar
  2. 2.
    Newton K, Strasser A (1998) The Bcl-2 family and cell death regulation. Curr Opin Gene Develop 8:68–75CrossRefGoogle Scholar
  3. 3.
    Farrow S, Brown R (1996) New members of the Bcl-2 family and their protein partners. Curr Opin Gene Develop 6:45–49CrossRefGoogle Scholar
  4. 4.
    Oltvai Z, Milliman C, Korsmeyer S (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74:609–619CrossRefPubMedGoogle Scholar
  5. 5.
    Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ (1995) Bad, a heterodimeric partner for Bcl-xL and Bcl-2, displaces bax and promotes cell death. Cell 80:285–291CrossRefPubMedGoogle Scholar
  6. 6.
    Zha J, Harada H, Yang E, Jockel J, Korsmeyer S (1996) Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-XL. Cell 87:619–628CrossRefPubMedGoogle Scholar
  7. 7.
    Cartier A, Komai T, Masucci MG (2003) The Us3 protein kinase of herpes simplex virus 1 blocks apoptosis and induces phosporylation of the Bcl-2 family member Bad. Exp Cell Res 291:242–250CrossRefPubMedGoogle Scholar
  8. 8.
    Tudor G, Aguilera A, Halverson DO, Laing ND, Sausville EA (2000) Susceptibility to drug-induced apoptosis correlates with differential modulation of Bad, Bcl-2 and Bcl-xL protein levels. Cell Death Differ 7:574–586CrossRefPubMedGoogle Scholar
  9. 9.
    Zamzami N, Kroemer G (2001) The mitochondrion in apoptosis: how Pandora’s box opens. Nat Rev Mol Cell Biol 2:67–71CrossRefPubMedGoogle Scholar
  10. 10.
    Dobos P, Hill B, Hallett R, Kells D, Becht H, Teninges D (1979) Biophysical and biochemical characterization of five animal viruses with bisegmented double-stranded RNA genomes. J Virol 32:593–605PubMedGoogle Scholar
  11. 11.
    Dobos P (1995) The molecular biology of infectious pancreatic necrosis virus (IPNV). Annu Rev Fish Dis 5:25–54CrossRefGoogle Scholar
  12. 12.
    Wu JL, Chang CY, Hsu YL (1987) Characteristics of an infectious pancreatic necrosis like virus isolated from Japanese eel (Anguilla japonina). Bull Inst Zoo Acad Sinica 26:201–214Google Scholar
  13. 13.
    Hjalmarsson A, Carlemalm E, Everitt E (1999) Infectious pancreatic necrosis: identification of a VP3-containing ribonucleoprotein core structure and evidence for O-linked glycosylation of the capsid protein VP2. J Virol 73:3484–3490PubMedGoogle Scholar
  14. 14.
    Pedersen T, Skjesol A, Jorgensen JB (2007) VP3, a structural protein of infectious pancreatic virus interacts with RNA-dependent RNA polymerase VP1 and with double-stranded RNA. J Virol 81:6652–6663CrossRefPubMedGoogle Scholar
  15. 15.
    Hong JR, Gong HY, Wu JL (2002) IPNV VP5, a novel anti-apoptosis gene of the Bcl-2 family, regulates Mcl-1 and viral protein expression. Virology 295:217–229CrossRefPubMedGoogle Scholar
  16. 16.
    Hong JR, Lin TL, Hsu YL, Wu JL (1998) Apoptosis precedes necrosis of fish cell line with infectious pancreatic necrosis virus infection. Virology 250:76–84CrossRefPubMedGoogle Scholar
  17. 17.
    Hong JR, Hsu YL, Wu JL (1999) Infectious pancreatic necrosis virus induces apoptosis due to down-regulation of survival factor MCL-1 protein expression in a fish cell line. Virus Res 63:75–83CrossRefPubMedGoogle Scholar
  18. 18.
    Hong JR, Wu JL (2002) Induction of apoptotic death in cells via Bad gene expression by Infectious pancreatic necrosis virus infection. Cell Death Differ 9:113–124CrossRefPubMedGoogle Scholar
  19. 19.
    Hong JR, Huang LJ, Wu JL (2005) Aquatic birnavirus induces apoptosis through activated caspase-8 and -3 in a zebrafish cell line. J Fish Dis 28:133–140CrossRefPubMedGoogle Scholar
  20. 20.
    Hong JR, Guan BJ, Her GM, Evensen O, Santi N, Wu JL (2008) Aquatic birnavirus infection activates the transcription factor NF-κB via tyrosine kinase signalling leading to cell death. J Fish Dis 31:451–460CrossRefPubMedGoogle Scholar
  21. 21.
    Hwang HJ, Moon CH, Kim HG, Kim JY, Lee JM, Park JW, Chung DK (2007) Identification and functional analysis of salmon annexin 1 induced by a virus infection in a fish cell line. J Virol 81:13816–13824CrossRefPubMedGoogle Scholar
  22. 22.
    Chen PC, Wu JL, Her GM, Hong JR (2009) Aquatic birnavirus induces necrotic cell death via the mitochondria-mediated caspase pathway. Fish Shellfish Immunol. doi:10.1016/j.fsi.2009.11.014 (in press)
  23. 23.
    Nicholson B, Dunn J (1974) Homologous viral interference in trout and Atlantic salmon cell cultures infected with infectious pancreatic necrosis virus. Am Soc Microbiol 14:180–182Google Scholar
  24. 24.
    Hong JR, Lin TL, Yang JY, Hsu YL, Wu JL (1999) Dynamics of nontypical apoptotic morphological changes visualized by green fluorescent protein in living cells with infectious pancreatic necrosis virus infection. J Virol 73:5056–5063PubMedGoogle Scholar
  25. 25.
    Laemmli U (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  26. 26.
    Kain S, Mai K, Sinai P (1994) Human multiple tissue western blots: a new immunological tool for the analysis of tissue-specific protein expression. Biotechniques 17:982–987PubMedGoogle Scholar
  27. 27.
    Chen SP, Yang HL, Her GM, Lin HY, Jeng MF, Wu JL, Hong JR (2006) Betanodavirus induces phosphatidylserine exposure and loss of mitochondrial membrane potential in secondary necrotic cells, both of which are blocked by bongkrekic acid. Virology 347:379–391CrossRefPubMedGoogle Scholar
  28. 28.
    Chen LJ, Su YC, Hong JR (2009) Betanodavirus non-structural protein B1: a novel anti-necrotic death factor that modulates cell death in early replication cycle in fish cells. Virology 385:444–454CrossRefPubMedGoogle Scholar
  29. 29.
    Falquet L, Pagni M, Bucher P, Hulo N, Sigrist CJ, Hofmann K, Bairoch A (2004) The PROSITE database, its status in 2002. Nucleic Acids Res 30:235–238CrossRefGoogle Scholar
  30. 30.
    An S, Knox K (1996) Ligation of CD40 rescues Ramos–Burkitt lymphoma B cells from calcium ionophore-and antigen receptor-triggered apoptosis by inhibiting activation of the cysteine protease CPP32/Yama and cleavage of its substrate PARP. FEBS Lett 386:15–122CrossRefGoogle Scholar
  31. 31.
    Chalfie M, Tu Y, Euskirchen G, Ward W, Prasher D (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805CrossRefPubMedGoogle Scholar
  32. 32.
    Maniak M, Rauchenberger R, Albrecht R, Murphy J, Gerisch G (1995) Coronin involved in phagocytosis: dynamics of particle-induced relocalization visualized by a green fluorescent protein tag. Cell 83:915–924CrossRefPubMedGoogle Scholar
  33. 33.
    Oparka K, Roberts A, Santa Cruz S, Boevink P, Prior D, Smallcombe A (1997) Using GFP to study virus invasion and spread in plant tissues. Nature 388:401–402CrossRefGoogle Scholar
  34. 34.
    Espinoza J, Hjalmarsson A, Everitt E, Kuznar J (2000) Temporal and subcellular localization of infectious pancreatic necrosis virus structural proteins. Arch Virol 145:739–748CrossRefPubMedGoogle Scholar
  35. 35.
    Yoshida M (2001) Multiple viral strategies of HTLV-1 for dysregulation of cell growth control. Annu Rev Immunol 19:475–497CrossRefPubMedGoogle Scholar
  36. 36.
    Seet BT, Hohnston JB, Brunetti CD, Barrett JW, Everett H, Cameron C, Sypula J, Nazarian SH, Lucas A, McFadden G (2003) Poxviruses and immune evasion. Annu Rev Immunol 21:377–423CrossRefPubMedGoogle Scholar
  37. 37.
    Evertt H, McFadden G (1999) Apoptosis: an innate immune response to virus infection. Trends Microbiol 7:160–165CrossRefGoogle Scholar
  38. 38.
    Benedict CA, Norris PS, Ware CF (2002) To kill or be killed: viral evasion of apoptosis. Nat Immunol 3:1013–1018CrossRefPubMedGoogle Scholar
  39. 39.
    Wu HC, Chiu CS, Wu JL, Gong HY, Chen MC, Lu MW, Hong JR (2008) Zebrafish anti-apoptotic protein zfBcl-xL can block betnodavirus protein α-induced mitochondria-mediated secondary necrosis cell death. Fish Shell Immunol 24:436–449CrossRefGoogle Scholar
  40. 40.
    Su YC, Wu JL, Hong JR (2008) Betanodavirus non-structural protein B2: a novel necrotic death factor that induces mitochondria-mediated cell death in fish cells. Virology 385:143–154CrossRefPubMedGoogle Scholar
  41. 41.
    Datta S, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg M (1997) Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91:231–242CrossRefPubMedGoogle Scholar
  42. 42.
    del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G (1997) Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 278:687–689CrossRefPubMedGoogle Scholar
  43. 43.
    Freilinger A, Rosner M, Krupitza G, Nishino M, Lubec G, Korsmeyer S, Hengstschlager M (2006) Tuberin activates the proapoptotic molecule BAD. Oncogene 25:6467–6479CrossRefPubMedGoogle Scholar
  44. 44.
    Hayakawa J, Ohmichi M, Kurachi H, Kanda Y, Hisamoto K, Nishio Y, Adachi K, Tasaka K, Kanzaki T, Murata Y (2000) Inhibition of BAD phosphorylation either at serine 112 via extracellular signal-regulated protein kinase cascade or at serine 136 via Akt cascade sensitizes human ovarian cancer cells to cisplatin. Cancer Res 60:5988–5994PubMedGoogle Scholar
  45. 45.
    Chen SP, Wu JL, Su YC, Hong JR (2007) Anti-Bcl-2 family members, zfBcl-x L and zfMcl-1a, prevent cytochrome c release from cells undergoing betanodavirus-induced secondary necrotic cell death. Apoptosis 12:1043–1060CrossRefPubMedGoogle Scholar
  46. 46.
    Zander K, Sherman M, Tessmer U, Bruns K, Wray V, Prechtel A, Schubert E, Henklein P, Luban J, Neidleman J, Greene WC, Schubert U (2003) Cyclophilin A interacts with HIV-1 Vpr and is required for its functional expression. J Biol Chem 278:43202–43213CrossRefPubMedGoogle Scholar
  47. 47.
    Takada S, Shirakata Y, Kaneniwa N, Koike K (1999) Association of hepatitis B virus X protein with mitochondria causes mitochondrial aggregation at the nuclear periphery, leading to cell death. Oncogene 18:6965–6973CrossRefPubMedGoogle Scholar
  48. 48.
    Boyce M, Degterev A, Yuan J (2004) Caspases: an ancient cellular sword of Damocles. Cell Death Differ 11:29–37CrossRefPubMedGoogle Scholar
  49. 49.
    Sato A, Hiramoto A, Uchikubo Y, Miyazaki E, Satake A, Naito T, Hiraoka O, Miyake T, Kim HS, Wataya Y (2008) Gene expression profiles of necrosis and apoptosis induced by 5-fluoro-2′-deoxyuridine. Genomics 92:9–17CrossRefPubMedGoogle Scholar
  50. 50.
    Hitomi J, Christofferson DE, Ng A, Yao J, Degterev A, Xavier RJ, Yuan J (2008) Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell 135:1311–1323CrossRefPubMedGoogle Scholar
  51. 51.
    Hong JR, Wu JL (2002) Molecular regulation of cellular apoptosis by fish infectious pancreatic necrosis virus (IPNV) infection. Curr Top Virol 2:151–160Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Chien-Li Chiu
    • 1
  • Jen-Leih Wu
    • 2
  • Guor-Mour Her
    • 3
  • Yi-Li Chou
    • 1
  • Jiann-Ruey Hong
    • 1
  1. 1.Laboratory of Molecular Virology and Biotechnology, Institute of BiotechnologyNational Cheng Kung UniversityTainanTaiwan, ROC
  2. 2.Laboratory of Marine Molecular Biology and Biotechnology, Institute of Cellular and Organismic BiologyAcademia SinicaNankang, TaipeiTaiwan, ROC
  3. 3.Institute of Bioscience and BiotechnologyNational Taiwan Ocean UniversityKeelungTaiwan, ROC

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