1887

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Volume 95, Issue 12

Analysis of the anti-apoptotic activity of four vaccinia virus proteins demonstrates that B13 is the most potent inhibitor in isolation and during viral infection Open Access

Abstract

Vaccinia virus (VACV) is a large dsDNA virus encoding ~200 proteins, several of which inhibit apoptosis. Here, a comparative study of anti-apoptotic proteins N1, F1, B13 and Golgi anti-apoptotic protein (GAAP) in isolation and during viral infection is presented. VACVs strains engineered to lack each gene separately still blocked apoptosis to some degree because of functional redundancy provided by the other anti-apoptotic proteins. To overcome this redundancy, we inserted each gene separately into a VACV strain (vv811) that lacked all these anti-apoptotic proteins and that induced apoptosis efficiently during infection. Each protein was also expressed in cells using lentivirus vectors. In isolation, each VACV protein showed anti-apoptotic activity in response to specific stimuli, as measured by immunoblotting for cleaved poly(ADP ribose) polymerase-1 and caspase-3 activation. Of the proteins tested, B13 was the most potent inhibitor, blocking both intrinsic and extrinsic stimuli, whilst the activity of the other proteins was largely restricted to inhibition of intrinsic stimuli. In addition, B13 and F1 were effective blockers of apoptosis induced by vv811 infection. Finally, whilst differences in induction of apoptosis were barely detectable during infection with VACV strain Western Reserve compared with derivative viruses lacking individual anti-apoptotic genes, several of these proteins reduced activation of caspase-3 during infection by vv811 strains expressing these proteins. These results illustrated that vv811 was a useful tool to determine the role of VACV proteins during infection and that whilst all of these proteins have some anti-apoptotic activity, B13 was the most potent.

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© 2014 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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2014-12-01
2024-04-19
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References

  1. Alcamí A., Khanna A., Paul N. L., Smith G. L. 1999; Vaccinia virus strains Lister, USSR and Evans express soluble and cell-surface tumour necrosis factor receptors. J Gen Virol 80:949–959[PubMed]
     [Google Scholar]
  2. Ali A. N., Turner P. C., Brooks M. A., Moyer R. W. 1994; The SPI-1 gene of rabbitpox virus determines host range and is required for hemorrhagic pock formation. Virology 202:305–314 [View Article][PubMed]
     [Google Scholar]
  3. Aoyagi M., Zhai D., Jin C., Aleshin A. E., Stec B., Reed J. C., Liddington R. C. 2007; Vaccinia virus N1L protein resembles a B cell lymphoma-2 (Bcl-2) family protein. Protein Sci 16:118–124 [View Article][PubMed]
     [Google Scholar]
  4. 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 83:7085–7098 [View Article][PubMed]
     [Google Scholar]
  5. Bartlett N., Symons J. A., Tscharke D. C., Smith G. L. 2002; The vaccinia virus N1L protein is an intracellular homodimer that promotes virulence. J Gen Virol 83:1965–1976[PubMed]
     [Google Scholar]
  6. Brooks M. A., Ali A. N., Turner P. C., Moyer R. W. 1995; A rabbitpox virus serpin gene controls host range by inhibiting apoptosis in restrictive cells. J Virol 69:7688–7698[PubMed]
     [Google Scholar]
  7. Carrara G., Saraiva N., Gubser C., Johnson B. F., Smith G. L. 2012; Six-transmembrane topology for Golgi anti-apoptotic protein (GAAP) and Bax inhibitor 1 (BI-1) provides model for the transmembrane Bax inhibitor-containing motif (TMBIM) family. J Biol Chem 287:15896–15905 [View Article][PubMed]
     [Google Scholar]
  8. Chen R. A., Jacobs N., Smith G. L. 2006; Vaccinia virus strain Western Reserve protein B14 is an intracellular virulence factor. J Gen Virol 87:1451–1458 [View Article][PubMed]
     [Google Scholar]
  9. Chen R. A., Ryzhakov G., Cooray S., Randow F., Smith G. L. 2008; Inhibition of IkappaB kinase by vaccinia virus virulence factor B14. PLoS Pathog 4:e22 [View Article][PubMed]
     [Google Scholar]
  10. Cho Y. S., Challa S., Moquin D., Genga R., Ray T. D., Guildford M., Chan F. K. 2009; Phosphorylation-driven assembly of the RIP1–RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 137:1112–1123 [View Article][PubMed]
     [Google Scholar]
  11. Cooray S., Bahar M. W., Abrescia N. G., McVey C. E., Bartlett N. W., Chen R. A., Stuart D. I., Grimes J. M., Smith G. L. 2007; Functional and structural studies of the vaccinia virus virulence factor N1 reveal a Bcl-2-like anti-apoptotic protein. J Gen Virol 88:1656–1666 [View Article][PubMed]
     [Google Scholar]
  12. Dai P., Wang W., Cao H., Avogadri F., Dai L., Drexler I., Joyce J. A., Li X. D., Chen Z.other authors 2014; Modified vaccinia virus Ankara triggers type I IFN production in murine conventional dendritic cells via a cGAS/STING-mediated cytosolic DNA-sensing pathway. PLoS Pathog 10:e1003989 [View Article][PubMed]
     [Google Scholar]
  13. de Mattia F., Gubser C., van Dommelen M. M., Visch H. J., Distelmaier F., Postigo A., Luyten T., Parys J. B., de Smedt H.other authors 2009; Human Golgi antiapoptotic protein modulates intracellular calcium fluxes. Mol Biol Cell 20:3638–3645 [View Article][PubMed]
     [Google Scholar]
  14. DiPerna G., Stack J., Bowie A. G., Boyd A., Kotwal G., Zhang Z., Arvikar S., Latz E., Fitzgerald K. A., Marshall W. L. 2004; Poxvirus protein N1L targets the I-kappaB kinase complex, inhibits signaling to NF-kappaB by the tumor necrosis factor superfamily of receptors, and inhibits NF-kappaB and IRF3 signaling by toll-like receptors. J Biol Chem 279:36570–36578 [View Article][PubMed]
     [Google Scholar]
  15. Dobbelstein M., Shenk T. 1996; Protection against apoptosis by the vaccinia virus SPI-2 (B13R) gene product. J Virol 70:6479–6485[PubMed]
     [Google Scholar]
  16. Doceul V., Hollinshead M., van der Linden L., Smith G. L. 2010; Repulsion of superinfecting virions: a mechanism for rapid virus spread. Science 327:873–876 [View Article][PubMed]
     [Google Scholar]
  17. Ember S. W., Ren H., Ferguson B. J., Smith G. L. 2012; Vaccinia virus protein C4 inhibits NF-κB activation and promotes virus virulence. J Gen Virol 93:2098–2108 [View Article][PubMed]
     [Google Scholar]
  18. Enari M., Hug H., Nagata S. 1995; Involvement of an ICE-like protease in Fas-mediated apoptosis. Nature 375:78–81 [View Article][PubMed]
     [Google Scholar]
  19. Falkner F. G., Moss B. 1990; Transient dominant selection of recombinant vaccinia viruses. J Virol 64:3108–3111[PubMed]
     [Google Scholar]
  20. Ferguson B. J., Benfield C. T., Ren H., Lee V. H., Frazer G. L., Strnadova P., Sumner R. P., Smith G. L. 2013; Vaccinia virus protein N2 is a nuclear IRF3 inhibitor that promotes virulence. J Gen Virol 94:2070–2081 [View Article][PubMed]
     [Google Scholar]
  21. García M. A., Guerra S., Gil J., Jimenez V., Esteban M. 2002; Anti-apoptotic and oncogenic properties of the dsRNA-binding protein of vaccinia virus, E3L. Oncogene 21:8379–8387 [View Article][PubMed]
     [Google Scholar]
  22. Garcia-Calvo M., Peterson E. P., Leiting B., Ruel R., Nicholson D. W., Thornberry N. A. 1998; Inhibition of human caspases by peptide-based and macromolecular inhibitors. J Biol Chem 273:32608–32613 [View Article][PubMed]
     [Google Scholar]
  23. Gerlic M., Faustin B., Postigo A., Yu E. C., Proell M., Gombosuren N., Krajewska M., Flynn R., Croft M.other authors 2013; Vaccinia virus F1L protein promotes virulence by inhibiting inflammasome activation. Proc Natl Acad Sci U S A 110:7808–7813 [View Article][PubMed]
     [Google Scholar]
  24. Gloeckner C. J., Boldt K., Schumacher A., Roepman R., Ueffing M. 2007; A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes. Proteomics 7:4228–4234 [View Article][PubMed]
     [Google Scholar]
  25. Goebel S. J., Johnson G. P., Perkus M. E., Davis S. W., Winslow J. P., Paoletti E. 1990; The complete DNA sequence of vaccinia virus. Virology 179:247–266 [View Article][PubMed]
     [Google Scholar]
  26. Graham S. C., Bahar M. W., Cooray S., Chen R. A., Whalen D. M., Abrescia N. G., Alderton D., Owens R. J., Stuart D. I.other authors 2008; Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-kappaB rather than apoptosis. PLoS Pathog 4:e1000128 [View Article][PubMed]
     [Google Scholar]
  27. Griffith T. S., Ferguson T. A. 2011; Cell death in the maintenance and abrogation of tolerance: the five Ws of dying cells. Immunity 35:456–466 [View Article][PubMed]
     [Google Scholar]
  28. Gubser C., Smith G. L. 2002; The sequence of camelpox virus shows it is most closely related to variola virus, the cause of smallpox. J Gen Virol 83:855–872[PubMed]
     [Google Scholar]
  29. Gubser C., Hué S., Kellam P., Smith G. L. 2004; Poxvirus genomes: a phylogenetic analysis. J Gen Virol 85:105–117 [View Article][PubMed]
     [Google Scholar]
  30. Gubser C., Bergamaschi D., Hollinshead M., Lu X., van Kuppeveld F. J., Smith G. L. 2007; A new inhibitor of apoptosis from vaccinia virus and eukaryotes. PLoS Pathog 3:e17 [View Article][PubMed]
     [Google Scholar]
  31. Han J., Zhong C. Q., Zhang D. W. 2011; Programmed necrosis: backup to and competitor with apoptosis in the immune system. Nat Immunol 12:1143–1149 [View Article][PubMed]
     [Google Scholar]
  32. Heinkelein M., Pilz S., Jassoy C. 1996; Inhibition of CD95 (Fas/Apo1)-mediated apoptosis by vaccinia virus WR. Clin Exp Immunol 103:8–14 [View Article][PubMed]
     [Google Scholar]
  33. Holler N., Zaru R., Micheau O., Thome M., Attinger A., Valitutti S., Bodmer J. L., Schneider P., Seed B., Tschopp J. 2000; Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol 1:489–495 [View Article][PubMed]
     [Google Scholar]
  34. Hughes S. J., Johnston L. H., de Carlos A., Smith G. L. 1991; Vaccinia virus encodes an active thymidylate kinase that complements a cdc8 mutant of Saccharomyces cerevisiae. J Biol Chem 266:20103–20109[PubMed]
     [Google Scholar]
  35. Kettle S., Blake N. W., Law K. M., Smith G. L. 1995; Vaccinia virus serpins B13R (SPI-2) and B22R (SPI-1) encode Mr 38.5 and 40K, intracellular polypeptides that do not affect virus virulence in a murine intranasal model. Virology 206:136–147 [View Article][PubMed]
     [Google Scholar]
  36. Kettle S., Alcamí A., Khanna A., Ehret R., Jassoy C., Smith G. L. 1997; Vaccinia virus serpin B13R (SPI-2) inhibits interleukin-1beta-converting enzyme and protects virus-infected cells from TNF- and Fas-mediated apoptosis, but does not prevent IL-1beta-induced fever. J Gen Virol 78:677–685[PubMed]
     [Google Scholar]
  37. Kibler K. V., Shors T., Perkins K. B., Zeman C. C., Banaszak M. P., Biesterfeldt J., Langland J. O., Jacobs B. L. 1997; Double-stranded RNA is a trigger for apoptosis in vaccinia virus-infected cells. J Virol 71:1992–2003[PubMed]
     [Google Scholar]
  38. Kotwal G. J., Moss B. 1989; Vaccinia virus encodes two proteins that are structurally related to members of the plasma serine protease inhibitor superfamily. J Virol 63:600–606[PubMed]
     [Google Scholar]
  39. Kvansakul M., Yang H., Fairlie W. D., Czabotar P. E., Fischer S. F., Perugini M. A., Huang D. C., Colman P. M. 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 15:1564–1571 [View Article][PubMed]
     [Google Scholar]
  40. Lee S. B., Esteban M. 1994; The interferon-induced double-stranded RNA-activated protein kinase induces apoptosis. Virology 199:491–496 [View Article][PubMed]
     [Google Scholar]
  41. Los M., Van de Craen M., Penning L. C., Schenk H., Westendorp M., Baeuerle P. A., Dröge W., Krammer P. H., Fiers W., Schulze-Osthoff K. 1995; Requirement of an ICE/CED-3 protease for Fas/APO-1-mediated apoptosis. Nature 375:81–83 [View Article][PubMed]
     [Google Scholar]
  42. Mackett M., Smith G. L., Moss B. 1984; General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. J Virol 49:857–864[PubMed]
     [Google Scholar]
  43. Maluquer de Motes C., Cooray S., Ren H., Almeida G. M. F., McGourty K., Bahar M. W., Stuart D. I., Grimes J. M., Graham S. C., Smith G. L. 2011; Inhibition of apoptosis and NF-κB activation by vaccinia protein N1 occur via distinct binding surfaces and make different contributions to virulence. PLoS Pathog 7:e1002430 [View Article][PubMed]
     [Google Scholar]
  44. Mansur D. S., Maluquer de Motes C., Unterholzner L., Sumner R. P., Ferguson B. J., Ren H., Strnadova P., Bowie A. G., Smith G. L. 2013; Poxvirus targeting of E3 ligase β-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence. PLoS Pathog 9:e1003183 [View Article][PubMed]
     [Google Scholar]
  45. Miura M., Friedlander R. M., Yuan J. 1995; Tumor necrosis factor-induced apoptosis is mediated by a CrmA-sensitive cell death pathway. Proc Natl Acad Sci U S A 92:8318–8322 [View Article][PubMed]
     [Google Scholar]
  46. Perkus M. E., Goebel S. J., Davis S. W., Johnson G. P., Norton E. K., Paoletti E. 1991; Deletion of 55 open reading frames from the termini of vaccinia virus. Virology 180:406–410 [View Article][PubMed]
     [Google Scholar]
  47. Postigo A., Way M. 2012; The vaccinia virus-encoded Bcl-2 homologues do not act as direct Bax inhibitors. J Virol 86:203–213 [View Article][PubMed]
     [Google Scholar]
  48. Postigo A., Cross J. R., Downward J., Way M. 2006; Interaction of F1L with the BH3 domain of Bak is responsible for inhibiting vaccinia-induced apoptosis. Cell Death Differ 13:1651–1662 [View Article][PubMed]
     [Google Scholar]
  49. Ray C. A., Black R. A., Kronheim S. R., Greenstreet T. A., Sleath P. R., Salvesen G. S., Pickup D. J. 1992; Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzyme. Cell 69:597–604 [View Article][PubMed]
     [Google Scholar]
  50. Saraiva N., Prole D. L., Carrara G., Johnson B. F., Taylor C. W., Parsons M., Smith G. L. 2013a; hGAAP promotes cell adhesion and migration via the stimulation of store-operated Ca2+ entry and calpain 2. J Cell Biol 202:699–713 [View Article][PubMed]
     [Google Scholar]
  51. Saraiva N., Prole D. L., Carrara G., Maluquer de Motes C., Johnson B. F., Byrne B., Taylor C. W., Smith G. L. 2013b; Human and viral Golgi anti-apoptotic proteins (GAAPs) oligomerize via different mechanisms and monomeric GAAP inhibits apoptosis and modulates calcium. J Biol Chem 288:13057–13067 [View Article][PubMed]
     [Google Scholar]
  52. Shisler J. L., Moss B. 2001; Immunology 102 at poxvirus U: avoiding apoptosis. Semin Immunol 13:67–72 [View Article][PubMed]
     [Google Scholar]
  53. Skaletskaya A., Bartle L. M., Chittenden T., McCormick A. L., Mocarski E. S., Goldmacher V. S. 2001; A cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation. Proc Natl Acad Sci U S A 98:7829–7834 [View Article][PubMed]
     [Google Scholar]
  54. Smith G. L., de Carlos A., Chan Y. S. 1989a; Vaccinia virus encodes a thymidylate kinase gene: sequence and transcriptional mapping. Nucleic Acids Res 17:7581–7590 [View Article][PubMed]
     [Google Scholar]
  55. Smith G. L., Howard S. T., Chan Y. S. 1989b; Vaccinia virus encodes a family of genes with homology to serine proteinase inhibitors. J Gen Virol 70:2333–2343 [View Article][PubMed]
     [Google Scholar]
  56. Smith G. L., Chan Y. S., Howard S. T. 1991; Nucleotide sequence of 42 kbp of vaccinia virus strain WR from near the right inverted terminal repeat. J Gen Virol 72:1349–1376 [View Article][PubMed]
     [Google Scholar]
  57. Smith G. L., Benfield C. T., Maluquer de Motes C., Mazzon M., Ember S. W., Ferguson B. J., Sumner R. P. 2013; Vaccinia virus immune evasion: mechanisms, virulence and immunogenicity. J Gen Virol 94:2367–2392 [View Article][PubMed]
     [Google Scholar]
  58. Tait S. W., Green D. R. 2010; Mitochondria and cell death: outer membrane permeabilization and beyond. Nat Rev Mol Cell Biol 11:621–632 [View Article][PubMed]
     [Google Scholar]
  59. Taylor J. M., Barry M. 2006; Near death experiences: poxvirus regulation of apoptotic death. Virology 344:139–150 [View Article][PubMed]
     [Google Scholar]
  60. Tengelsen L. A., Slabaugh M. B., Bibler J. K., Hruby D. E. 1988; Nucleotide sequence and molecular genetic analysis of the large subunit of ribonucleotide reductase encoded by vaccinia virus. Virology 164:121–131 [View Article][PubMed]
     [Google Scholar]
  61. Tewari M., Dixit V. M. 1995; Fas- and tumor necrosis factor-induced apoptosis is inhibited by the poxvirus crmA gene product. J Biol Chem 270:3255–3260 [View Article][PubMed]
     [Google Scholar]
  62. Unterholzner L., Sumner R. P., Baran M., Ren H., Mansur D. S., Bourke N. M., Randow F., Smith G. L., Bowie A. G. 2011; Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7. PLoS Pathog 7:e1002247 [View Article][PubMed]
     [Google Scholar]
  63. Wasilenko S. T., Meyers A. F., Vander Helm K., Barry M. 2001; Vaccinia virus infection disarms the mitochondrion-mediated pathway of the apoptotic cascade by modulating the permeability transition pore. J Virol 75:11437–11448 [View Article][PubMed]
     [Google Scholar]
  64. Wasilenko S. T., Stewart T. L., Meyers A. F., Barry M. 2003; Vaccinia virus encodes a previously uncharacterized mitochondrial-associated inhibitor of apoptosis. Proc Natl Acad Sci U S A 100:14345–14350 [View Article][PubMed]
     [Google Scholar]
  65. Wasilenko S. T., Banadyga L., Bond D., Barry M. 2005; The vaccinia virus F1L protein interacts with the proapoptotic protein Bak and inhibits Bak activation. J Virol 79:14031–14043 [View Article][PubMed]
     [Google Scholar]
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Analysis of the anti-apoptotic activity of four vaccinia virus proteins demonstrates that B13 is the most potent inhibitor in isolation and during viral infection
J. Gen. Virol. 95, 2757 (2014); https://doi.org/10.1099/vir.0.068833-0
/content/journal/jgv/10.1099/vir.0.068833-0
/content/journal/jgv/10.1099/vir.0.068833-0
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