1887

Abstract

Ubiquitylation is a covalent post-translational modification that regulates protein stability and is involved in many biological functions. Proteins may be modified with mono-ubiquitin or ubiquitin chains. Viruses have evolved multiple mechanisms to perturb the cell ubiquitin system and manipulate it to their own benefit. Here, we report ubiquitylation of vaccinia virus (VACV) protein N1. N1 is an inhibitor of the nuclear factor NF-κB and apoptosis that contributes to virulence, has a Bcl-2-like fold, and is highly conserved amongst orthopoxviruses. The interaction between N1 and ubiquitin occurs at endogenous protein levels during VACV infection and following ectopic expression of N1. Biochemical analysis demonstrated that N1 is covalently ubiquitylated, and heterodimers of ubiquitylated and non-ubiquitylated N1 monomers were identified, suggesting that ubiquitylation does not inhibit N1 dimerization. Studies with other VACV Bcl-2 proteins, such as C6 or B14, revealed that although these proteins also interact with ubiquitin, these interactions are non-covalent. Finally, mutagenesis of N1 showed that ubiquitylation occurs in a conventional lysine-dependent manner at multiple acceptor sites because only an N1 allele devoid of lysine residues remained unmodified. Taken together, we described a previously uncharacterized modification of the VACV protein N1 that provided a new layer of complexity to the biology of this virulence factor, and provided another example of the intricate interplay between poxviruses and the host ubiquitin system.

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2014-09-01
2019-11-22
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References

  1. 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. [CrossRef][PubMed]
    [Google Scholar]
  2. Barrios-Rodiles M., Brown K. R., Ozdamar B., Bose R., Liu Z., Donovan R. S., Shinjo F., Liu Y., Dembowy J.. & other authors ( 2005;). High-throughput mapping of a dynamic signaling network in mammalian cells. . Science 307:, 1621–1625. [CrossRef][PubMed]
    [Google Scholar]
  3. Barry M., van Buuren N., Burles K., Mottet K., Wang Q., Teale A.. ( 2010;). Poxvirus exploitation of the ubiquitin–proteasome system. . Viruses 2:, 2356–2380. [CrossRef][PubMed]
    [Google Scholar]
  4. 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]
  5. Beaudenon S., Huibregtse J. M.. ( 2008;). HPV E6, E6AP and cervical cancer. . BMC Biochem 9: (Suppl 1), S4. [CrossRef][PubMed]
    [Google Scholar]
  6. Bhoj V. G., Chen Z. J.. ( 2009;). Ubiquitylation in innate and adaptive immunity. . Nature 458:, 430–437. [CrossRef][PubMed]
    [Google Scholar]
  7. 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. [CrossRef][PubMed]
    [Google Scholar]
  8. 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. [CrossRef][PubMed]
    [Google Scholar]
  9. Chipuk J. E., Moldoveanu T., Llambi F., Parsons M. J., Green D. R.. ( 2010;). The BCL-2 family reunion. . Mol Cell 37:, 299–310. [CrossRef][PubMed]
    [Google Scholar]
  10. 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. [CrossRef][PubMed]
    [Google Scholar]
  11. Demirov D. G., Ono A., Orenstein J. M., Freed E. O.. ( 2002;). Overexpression of the N-terminal domain of TSG101 inhibits HIV-1 budding by blocking late domain function. . Proc Natl Acad Sci U S A 99:, 955–960. [CrossRef][PubMed]
    [Google Scholar]
  12. 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. [CrossRef][PubMed]
    [Google Scholar]
  13. 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. [CrossRef][PubMed]
    [Google Scholar]
  14. Fedosyuk S., Grishkovskaya I., de Almeida Ribeiro E. Jr, Skern T.. ( 2014;). Characterization and structure of the vaccinia virus NF-κB antagonist A46. . J Biol Chem 289:, 3749–3762. [CrossRef][PubMed]
    [Google Scholar]
  15. Garrus J. E., von Schwedler U. K., Pornillos O. W., Morham S. G., Zavitz K. H., Wang H. E., Wettstein D. A., Stray K. M., Côté M.. & other authors ( 2001;). Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. . Cell 107:, 55–65. [CrossRef][PubMed]
    [Google Scholar]
  16. Gerlach B., Cordier S. M., Schmukle A. C., Emmerich C. H., Rieser E., Haas T. L., Webb A. I., Rickard J. A., Anderton H.. & other authors ( 2011;). Linear ubiquitination prevents inflammation and regulates immune signalling. . Nature 471:, 591–596. [CrossRef][PubMed]
    [Google Scholar]
  17. 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. [CrossRef][PubMed]
    [Google Scholar]
  18. González J. M., Esteban M.. ( 2010;). A poxvirus Bcl-2-like gene family involved in regulation of host immune response: sequence similarity and evolutionary history. . Virol J 7:, 59. [CrossRef][PubMed]
    [Google Scholar]
  19. González-Santamaría J., Campagna M., García M. A., Marcos-Villar L., González D., Gallego P., Lopitz-Otsoa F., Guerra S., Rodríguez M. S.. & other authors ( 2011;). Regulation of vaccinia virus E3 protein by small ubiquitin-like modifier proteins. . J Virol 85:, 12890–12900. [CrossRef][PubMed]
    [Google Scholar]
  20. 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. [CrossRef][PubMed]
    [Google Scholar]
  21. Guerin J. L., Gelfi J., Boullier S., Delverdier M., Bellanger F. A., Bertagnoli S., Drexler I., Sutter G., Messud-Petit F.. ( 2002;). Myxoma virus leukemia-associated protein is responsible for major histocompatibility complex class I and Fas-CD95 down-regulation and defines scrapins, a new group of surface cellular receptor abductor proteins. . J Virol 76:, 2912–2923. [CrossRef][PubMed]
    [Google Scholar]
  22. Gustin J. K., Moses A. V., Früh K., Douglas J. L.. ( 2011;). Viral takeover of the host ubiquitin system. . Front Microbiol 2:, 161. [CrossRef][PubMed]
    [Google Scholar]
  23. Kalverda A. P., Thompson G. S., Vogel A., Schröder M., Bowie A. G., Khan A. R., Homans S. W.. ( 2009;). Poxvirus K7 protein adopts a Bcl-2 fold: biochemical mapping of its interactions with human DEAD box RNA helicase DDX3. . J Mol Biol 385:, 843–853. [CrossRef][PubMed]
    [Google Scholar]
  24. Kamio M., Yoshida T., Ogata H., Douchi T., Nagata Y., Inoue M., Hasegawa M., Yonemitsu Y., Yoshimura A.. ( 2004;). SOCS1 [corrected] inhibits HPV-E7-mediated transformation by inducing degradation of E7 protein. . Oncogene 23:, 3107–3115. [CrossRef][PubMed]
    [Google Scholar]
  25. Komander D.. ( 2009;). The emerging complexity of protein ubiquitination. . Biochem Soc Trans 37:, 937–953. [CrossRef][PubMed]
    [Google Scholar]
  26. Komander D., Rape M.. ( 2012;). The ubiquitin code. . Annu Rev Biochem 81:, 203–229. [CrossRef][PubMed]
    [Google Scholar]
  27. Kvansakul M., van Delft M. F., Lee E. F., Gulbis J. M., Fairlie W. D., Huang D. C., Colman P. M.. ( 2007;). A structural viral mimic of prosurvival Bcl-2: a pivotal role for sequestering proapoptotic Bax and Bak. . Mol Cell 25:, 933–942. [CrossRef][PubMed]
    [Google Scholar]
  28. Liao T. L., Wu C. Y., Su W. C., Jeng K. S., Lai M. M.. ( 2010;). Ubiquitination and deubiquitination of NP protein regulates influenza A virus RNA replication. . EMBO J 29:, 3879–3890. [CrossRef][PubMed]
    [Google Scholar]
  29. 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. [CrossRef][PubMed]
    [Google Scholar]
  30. Mammas I. N., Sourvinos G., Giannoudis A., Spandidos D. A.. ( 2008;). Human papilloma virus (HPV) and host cellular interactions. . Pathol Oncol Res 14:, 345–354. [CrossRef][PubMed]
    [Google Scholar]
  31. 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. [CrossRef][PubMed]
    [Google Scholar]
  32. Martin-Serrano J.. ( 2007;). The role of ubiquitin in retroviral egress. . Traffic 8:, 1297–1303. [CrossRef][PubMed]
    [Google Scholar]
  33. Mercer J., Snijder B., Sacher R., Burkard C., Bleck C. K., Stahlberg H., Pelkmans L., Helenius A.. ( 2012;). RNAi screening reveals proteasome- and Cullin3-dependent stages in vaccinia virus infection. . Cell Rep 2:, 1036–1047. [CrossRef][PubMed]
    [Google Scholar]
  34. Mo M., Fleming S. B., Mercer A. A.. ( 2009;). Cell cycle deregulation by a poxvirus partial mimic of anaphase-promoting complex subunit 11. . Proc Natl Acad Sci U S A 106:, 19527–19532. [CrossRef][PubMed]
    [Google Scholar]
  35. Mohamed M. R., Rahman M. M., Lanchbury J. S., Shattuck D., Neff C., Dufford M., van Buuren N., Fagan K., Barry M.. & other authors ( 2009;). Proteomic screening of variola virus reveals a unique NF-kappaB inhibitor that is highly conserved among pathogenic orthopoxviruses. . Proc Natl Acad Sci U S A 106:, 9045–9050. [CrossRef][PubMed]
    [Google Scholar]
  36. Moss B.. ( 2007;). Poxviridae: the viruses and their replicaton. . In Fields Virology, , 5th edn., pp. 2905–2946. Edited by Knipe D. M., Howley P. M., Griffin D. E., Lamb R. A., Martin M. A., Roizman B., Straus S. E... Philadelphia, PA:: Lippincott Williams & Wilkins;.
    [Google Scholar]
  37. Nerenberg B. T., Taylor J., Bartee E., Gouveia K., Barry M., Früh K.. ( 2005;). The poxviral RING protein p28 is a ubiquitin ligase that targets ubiquitin to viral replication factories. . J Virol 79:, 597–601. [CrossRef][PubMed]
    [Google Scholar]
  38. Parkinson J. E., Smith G. L.. ( 1994;). Vaccinia virus gene A36R encodes a Mr 43–50 K protein on the surface of extracellular enveloped virus. . Virology 204:, 376–390. [CrossRef][PubMed]
    [Google Scholar]
  39. Peters N. E., Ferguson B. J., Mazzon M., Fahy A. S., Krysztofinska E., Arribas-Bosacoma R., Pearl L. H., Ren H., Smith G. L.. ( 2013;). A mechanism for the inhibition of DNA-PK-mediated DNA sensing by a virus. . PLoS Pathog 9:, e1003649. [CrossRef][PubMed]
    [Google Scholar]
  40. Pickart C. M.. ( 2001;). Mechanisms underlying ubiquitination. . Annu Rev Biochem 70:, 503–533. [CrossRef][PubMed]
    [Google Scholar]
  41. Postigo A., Way M.. ( 2012;). The vaccinia virus-encoded Bcl-2 homologues do not act as direct Bax inhibitors. . J Virol 86:, 203–213. [CrossRef][PubMed]
    [Google Scholar]
  42. Randow F., Lehner P. J.. ( 2009;). Viral avoidance and exploitation of the ubiquitin system. . Nat Cell Biol 11:, 527–534. [CrossRef][PubMed]
    [Google Scholar]
  43. Satheshkumar P. S., Anton L. C., Sanz P., Moss B.. ( 2009;). Inhibition of the ubiquitin–proteasome system prevents vaccinia virus DNA replication and expression of intermediate and late genes. . J Virol 83:, 2469–2479. [CrossRef][PubMed]
    [Google Scholar]
  44. Shchelkunov S. N.. ( 2010;). Interaction of orthopoxviruses with the cellular ubiquitin–ligase system. . Virus Genes 41:, 309–318. [CrossRef][PubMed]
    [Google Scholar]
  45. 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. [CrossRef][PubMed]
    [Google Scholar]
  46. Sonnberg S., Seet B. T., Pawson T., Fleming S. B., Mercer A. A.. ( 2008;). Poxvirus ankyrin repeat proteins are a unique class of F-box proteins that associate with cellular SCF1 ubiquitin ligase complexes. . Proc Natl Acad Sci U S A 105:, 10955–10960. [CrossRef][PubMed]
    [Google Scholar]
  47. Sperling K. M., Schwantes A., Schnierle B. S., Sutter G.. ( 2008;). The highly conserved orthopoxvirus 68k ankyrin-like protein is part of a cellular SCF ubiquitin ligase complex. . Virology 374:, 234–239. [CrossRef][PubMed]
    [Google Scholar]
  48. Taylor R. T., Lubick K. J., Robertson S. J., Broughton J. P., Bloom M. E., Bresnahan W. A., Best S. M.. ( 2011;). TRIM79α, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral RNA polymerase. . Cell Host Microbe 10:, 185–196. [CrossRef][PubMed]
    [Google Scholar]
  49. Teale A., Campbell S., Van Buuren N., Magee W. C., Watmough K., Couturier B., Shipclark R., Barry M.. ( 2009;). Orthopoxviruses require a functional ubiquitin–proteasome system for productive replication. . J Virol 83:, 2099–2108. [CrossRef][PubMed]
    [Google Scholar]
  50. Thurston T. L., Ryzhakov G., Bloor S., von Muhlinen N., Randow F.. ( 2009;). The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria. . Nat Immunol 10:, 1215–1221. [CrossRef][PubMed]
    [Google Scholar]
  51. 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. [CrossRef][PubMed]
    [Google Scholar]
  52. van Buuren N., Couturier B., Xiong Y., Barry M.. ( 2008;). Ectromelia virus encodes a novel family of F-box proteins that interact with the SCF complex. . J Virol 82:, 9917–9927. [CrossRef][PubMed]
    [Google Scholar]
  53. Welchman R. L., Gordon C., Mayer R. J.. ( 2005;). Ubiquitin and ubiquitin-like proteins as multifunctional signals. . Nat Rev Mol Cell Biol 6:, 599–609. [CrossRef][PubMed]
    [Google Scholar]
  54. Wilton B. A., Campbell S., Van Buuren N., Garneau R., Furukawa M., Xiong Y., Barry M.. ( 2008;). Ectromelia virus BTB/kelch proteins, EVM150 and EVM167, interact with cullin-3-based ubiquitin ligases. . Virology 374:, 82–99. [CrossRef][PubMed]
    [Google Scholar]
  55. Yamazaki K., Gohda J., Kanayama A., Miyamoto Y., Sakurai H., Yamamoto M., Akira S., Hayashi H., Su B., Inoue J.. ( 2009;). Two mechanistically and temporally distinct NF-kappaB activation pathways in IL-1 signaling. . Sci Signal 2:, ra66. [CrossRef][PubMed]
    [Google Scholar]
  56. Yasuda J., Nakao M., Kawaoka Y., Shida H.. ( 2003;). Nedd4 regulates egress of Ebola virus-like particles from host cells. . J Virol 77:, 9987–9992. [CrossRef][PubMed]
    [Google Scholar]
  57. Zhang L., Villa N. Y., McFadden G.. ( 2009;). Interplay between poxviruses and the cellular ubiquitin/ubiquitin-like pathways. . FEBS Lett 583:, 607–614. [CrossRef][PubMed]
    [Google Scholar]
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