, 14:1317 | Cite as

The orf virus inhibitor of apoptosis functions in a Bcl-2-like manner, binding and neutralizing a set of BH3-only proteins and active Bax

  • Dana Westphal
  • Elizabeth C. Ledgerwood
  • Joel D. A. Tyndall
  • Merilyn H. Hibma
  • Norihito Ueda
  • Stephen B. Fleming
  • Andrew A. Mercer
Original Paper


We have previously shown that the Orf virus protein, ORFV125, is a potent inhibitor of the mitochondrial pathway of apoptosis and displays rudimentary sequence similarities to cellular anti-apoptotic Bcl-2 proteins. Here we investigate the proposal that ORFV125 acts in a Bcl-2-like manner to inhibit apoptosis. We show that the viral protein interacted with a range of BH3-only proteins (Bik, Puma, DP5, Noxa and all 3 isoforms of Bim) and neutralized their pro-apoptotic activity. In addition, ORFV125 bound to the active, but not the inactive, form of Bax, and reduced the formation of Bax dimers. Mutation of specific amino acids in ORFV125 that are conserved and functionally important in mammalian Bcl-2 family proteins led to loss of both binding and inhibitory functions. We conclude that ORFV125’s mechanism of action is Bcl-2-like and propose that the viral protein’s combined ability to bind to a range of BH3-only proteins as well as the active form of Bax provides significant protection against apoptosis. Furthermore, we demonstrate that the binding profile of ORFV125 is distinct to that of other poxviral Bcl-2-like proteins.


Apoptosis Mitochondria Bcl-2 family Viral Bcl-2 homolog Poxvirus Orf virus 



B-cell leukemia/lymphoma 2


Bcl-2 homology


Orf virus



This work was supported by the Health Research Council of New Zealand and the University of Otago. We wish to thank David Huang and Jamie Fletcher (The Walter and Eliza Hall Institute of Medical Research (WEHI), Australia) for providing information and a range of plasmids expressing Bcl-2 family members. We are grateful to Jerry Adams (WEHI, Australia) for critical reading of the manuscript, Catherine Day and Fabienne Lecomte (University of Otago, New Zealand) for helpful discussions and Ellena Whelan (University of Otago) for technical support.

Supplementary material

10495_2009_403_MOESM1_ESM.pdf (575 kb)
Supplementary material 1 (PDF 574 kb)


  1. 1.
    Youle RJ, Strasser A (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9:47–59PubMedCrossRefGoogle Scholar
  2. 2.
    Griffiths GJ, Dubrez L, Morgan CP et al (1999) Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis. J Cell Biol 144:903–914PubMedCrossRefGoogle Scholar
  3. 3.
    Er E, Oliver L, Cartron PF, Juin P, Manon S, Vallette FM (2006) Mitochondria as the target of the pro-apoptotic protein Bax. Biochim Biophys Acta 1757:1301–1311PubMedCrossRefGoogle Scholar
  4. 4.
    Adams JM, Cory S (2007) Bcl-2-regulated apoptosis: mechanism and therapeutic potential. Curr Opin Immunol 19:488–496PubMedCrossRefGoogle Scholar
  5. 5.
    Chen L, Willis SN, Wei A et al (2005) Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol Cell 17:393–403PubMedCrossRefGoogle Scholar
  6. 6.
    Willis SN, Fletcher JI, Kaufmann T et al (2007) Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak. Science 315:856–859PubMedCrossRefGoogle Scholar
  7. 7.
    Letai A, Bassik MC, Walensky LD, Sorcinelli MD, Weiler S, Korsmeyer SJ (2002) Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics. Cancer Cell 2:183–192PubMedCrossRefGoogle Scholar
  8. 8.
    Kim H, Rafiuddin-Shah M, Tu HC et al (2006) Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies. Nat Cell Biol 8:1348–1358PubMedCrossRefGoogle Scholar
  9. 9.
    Sattler M, Liang H, Nettesheim D et al (1997) Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science 275:983–986PubMedCrossRefGoogle Scholar
  10. 10.
    Denisov AY, Chen G, Sprules T, Moldoveanu T, Beauparlant P, Gehring K (2006) Structural model of the BCL-w-BID peptide complex and its interactions with phospholipid micelles. Biochemistry 45:2250–2256PubMedCrossRefGoogle Scholar
  11. 11.
    Czabotar PE, Lee EF, van Delft MF et al (2007) Structural insights into the degradation of Mcl-1 induced by BH3 domains. Proc Natl Acad Sci USA 104:6217–6222PubMedCrossRefGoogle Scholar
  12. 12.
    Yin XM, Oltvai ZN, Korsmeyer SJ (1994) BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 369:321–323PubMedCrossRefGoogle Scholar
  13. 13.
    Cheng EH, Levine B, Boise LH, Thompson CB, Hardwick JM (1996) Bax-independent inhibition of apoptosis by Bcl-XL. Nature 379:554–556PubMedCrossRefGoogle Scholar
  14. 14.
    Oda E, Ohki R, Murasawa H et al (2000) Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 288:1053–1058PubMedCrossRefGoogle Scholar
  15. 15.
    Zha J, Harada H, Osipov K, Jockel J, Waksman G, Korsmeyer SJ (1997) BH3 domain of BAD is required for heterodimerization with BCL-XL and pro-apoptotic activity. J Biol Chem 272:24101–24104PubMedCrossRefGoogle Scholar
  16. 16.
    Wang K, Gross A, Waksman G, Korsmeyer SJ (1998) Mutagenesis of the BH3 domain of BAX identifies residues critical for dimerization and killing. Mol Cell Biol 18:6083–6089PubMedGoogle Scholar
  17. 17.
    Cuconati A, White E (2002) Viral homologs of BCL-2: role of apoptosis in the regulation of virus infection. Genes Dev 16:2465–2478PubMedCrossRefGoogle Scholar
  18. 18.
    Galluzzi L, Brenner C, Morselli E, Touat Z, Kroemer G (2008) Viral control of mitochondrial apoptosis. PLoS Pathog 4:e1000018PubMedCrossRefGoogle Scholar
  19. 19.
    Afonso CL, Tulman ER, Lu Z, Zsak L, Kutish GF, Rock DL (2000) The genome of fowlpox virus. J Virol 74:3815–3831PubMedCrossRefGoogle Scholar
  20. 20.
    Wasilenko ST, Stewart TL, Meyers AF, Barry M (2003) Vaccinia virus encodes a previously uncharacterized mitochondrial-associated inhibitor of apoptosis. Proc Natl Acad Sci USA 100:14345–14350PubMedCrossRefGoogle Scholar
  21. 21.
    Aoyagi M, Zhai D, Jin C et al (2007) Vaccinia virus N1L protein resembles a B cell lymphoma-2 (Bcl-2) family protein. Protein Sci 16:118–124PubMedCrossRefGoogle Scholar
  22. 22.
    Cooray S, Bahar MW, Abrescia NG et al (2007) Functional and structural studies of the vaccinia virus virulence factor N1 reveal a Bcl-2-like anti-apoptotic protein. J Gen Virol 88:1656–1666PubMedCrossRefGoogle Scholar
  23. 23.
    Everett H, Barry M, Lee SF et al (2000) M11L: a novel mitochondria-localized protein of myxoma virus that blocks apoptosis of infected leukocytes. J Exp Med 191:1487–1498PubMedCrossRefGoogle Scholar
  24. 24.
    Westphal D, Ledgerwood EC, Hibma MH, Fleming SB, Whelan EM, Mercer AA (2007) A novel Bcl-2-like inhibitor of apoptosis is encoded by the parapoxvirus ORF virus. J Virol 81:7178–7188PubMedCrossRefGoogle Scholar
  25. 25.
    O’Reilly LA, Huang DC, Strasser A (1996) The cell death inhibitor Bcl-2 and its homologues influence control of cell cycle entry. EMBO J 15:6979–6990PubMedGoogle Scholar
  26. 26.
    Huang DC, Cory S, Strasser A (1997) Bcl-2, Bcl-XL and adenovirus protein E1B19kD are functionally equivalent in their ability to inhibit cell death. Oncogene 14:405–414PubMedCrossRefGoogle Scholar
  27. 27.
    Moriishi K, Huang DC, Cory S, Adams JM (1999) Bcl-2 family members do not inhibit apoptosis by binding the caspase activator Apaf-1. Proc Natl Acad Sci USA 96:9683–9688PubMedCrossRefGoogle Scholar
  28. 28.
    Konagurthu AS, Whisstock JC, Stuckey PJ, Lesk AM (2006) MUSTANG: a multiple structural alignment algorithm. Proteins 64:559–574PubMedCrossRefGoogle Scholar
  29. 29.
    Soding J (2005) Protein homology detection by HMM-HMM comparison. Bioinformatics 21:951–960PubMedCrossRefGoogle Scholar
  30. 30.
    Hinds MG, Smits C, Fredericks-Short R et al (2007) Bim, Bad and Bmf: intrinsically unstructured BH3-only proteins that undergo a localized conformational change upon binding to prosurvival Bcl-2 targets. Cell Death Differ 14:128–136PubMedCrossRefGoogle Scholar
  31. 31.
    Petros AM, Olejniczak ET, Fesik SW (2004) Structural biology of the Bcl-2 family of proteins. Biochim Biophys Acta 1644:83–94PubMedCrossRefGoogle Scholar
  32. 32.
    Willis SN, Chen L, Dewson G et al (2005) Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. Genes Dev 19:1294–1305PubMedCrossRefGoogle Scholar
  33. 33.
    Subramanian T, Vijayalingam S, Lomonosova E, Zhao LJ, Chinnadurai G (2007) Evidence for involvement of BH3-only proapoptotic members in adenovirus-induced apoptosis. J Virol 81:10486–10495PubMedCrossRefGoogle Scholar
  34. 34.
    Sun Y, Leaman DW (2005) Involvement of Noxa in cellular apoptotic responses to interferon, double-stranded RNA, and virus infection. J Biol Chem 280:15561–15568PubMedCrossRefGoogle Scholar
  35. 35.
    Belov GA, Romanova LI, Tolskaya EA, Kolesnikova MS, Lazebnik YA, Agol VI (2003) The major apoptotic pathway activated and suppressed by poliovirus. J Virol 77:45–56PubMedCrossRefGoogle Scholar
  36. 36.
    Fleming SB, McCaughan CA, Andrews AE, Nash AD, Mercer AA (1997) A homolog of interleukin-10 is encoded by the poxvirus orf virus. J Virol 71:4857–4861PubMedGoogle Scholar
  37. 37.
    Becherel PA, LeGoff L, Frances C et al (1997) Induction of IL-10 synthesis by human keratinocytes through CD23 ligation: a cyclic adenosine 3′,5′-monophosphate-dependent mechanism. J Immunol 159:5761–5765PubMedGoogle Scholar
  38. 38.
    Perez D, White E (2000) TNF-alpha signals apoptosis through a bid-dependent conformational change in Bax that is inhibited by E1B 19K. Mol Cell 6:53–63PubMedCrossRefGoogle Scholar
  39. 39.
    Zhai D, Jin C, Huang Z, Satterthwait AC, Reed JC (2008) Differential regulation of Bax and Bak by anti-apoptotic Bcl-2-family proteins, Bcl-B and Mcl-1. J Biol Chem 283:9580–9586PubMedCrossRefGoogle Scholar
  40. 40.
    Kulms D, Zeise E, Poppelmann B, Schwarz T (2002) DNA damage, death receptor activation and reactive oxygen species contribute to ultraviolet radiation-induced apoptosis in an essential and independent way. Oncogene 21:5844–5851PubMedCrossRefGoogle Scholar
  41. 41.
    Douglas AE, Corbett KD, Berger JM, McFadden G, Handel TM (2007) Structure of M11L: a myxoma virus structural homolog of the apoptosis inhibitor, Bcl-2. Protein Sci 16:695–703PubMedCrossRefGoogle Scholar
  42. 42.
    Kvansakul M, van Delft MF, Lee EF et al (2007) A structural viral mimic of prosurvival Bcl-2: a pivotal role for sequestering proapoptotic Bax and Bak. Mol Cell 25:933–942PubMedCrossRefGoogle Scholar
  43. 43.
    Kvansakul M, Yang H, Fairlie WD et al (2008) Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds a highly selective subset of BH3-containing death ligands. Cell Death Differ 15:1564–1571PubMedCrossRefGoogle Scholar
  44. 44.
    Banadyga L, Gerig J, Stewart T, Barry M (2007) Fowlpox virus encodes a Bcl-2 homologue that protects cells from apoptotic death through interaction with the proapoptotic protein Bak. J Virol 81:11032–11045PubMedCrossRefGoogle Scholar
  45. 45.
    Wang G, Barrett JW, Nazarian SH et al (2004) Myxoma virus M11L prevents apoptosis through constitutive interaction with Bak. J Virol 78:7097–7111PubMedCrossRefGoogle Scholar
  46. 46.
    Su J, Wang G, Barrett JW, Irvine TS, Gao X, McFadden G (2006) Myxoma virus M11L blocks apoptosis through inhibition of conformational activation of Bax at the mitochondria. J Virol 80:1140–1151PubMedCrossRefGoogle Scholar
  47. 47.
    Wasilenko ST, Banadyga L, Bond D, Barry M (2005) The vaccinia virus F1L protein interacts with the proapoptotic protein Bak and inhibits Bak activation. J Virol 79:14031–14043PubMedCrossRefGoogle Scholar
  48. 48.
    Banadyga L, Veugelers K, Campbell S, Barry M (2009) The fowlpox virus BCL-2 homologue, FPV039, interacts with activated Bax and a discrete subset of BH3-only proteins to inhibit apoptosis. J Virol 83:7085–7098PubMedCrossRefGoogle Scholar
  49. 49.
    Taylor JM, Barry M (2006) Near death experiences: poxvirus regulation of apoptotic death. Virology 344:139–150PubMedCrossRefGoogle Scholar
  50. 50.
    Taylor JM, Quilty D, Banadyga L, Barry M (2006) The vaccinia virus protein F1L interacts with Bim and inhibits activation of the pro-apoptotic protein Bax. J Biol Chem 281:39728–39739PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Dana Westphal
    • 1
  • Elizabeth C. Ledgerwood
    • 2
  • Joel D. A. Tyndall
    • 3
  • Merilyn H. Hibma
    • 1
  • Norihito Ueda
    • 1
  • Stephen B. Fleming
    • 1
  • Andrew A. Mercer
    • 1
  1. 1.Virus Research Unit, Department of Microbiology and ImmunologyUniversity of OtagoDunedinNew Zealand
  2. 2.Department of BiochemistryUniversity of OtagoDunedinNew Zealand
  3. 3.The National School of PharmacyUniversity of OtagoDunedinNew Zealand

Personalised recommendations