1887

Abstract

Pestiviruses like bovine viral diarrhoea virus (BVDV) and classical swine fever virus (CSFV) belong to the family . A special feature of the is the importance of nonstructural (NS) proteins for both genome replication and virion morphogenesis. The NS2-3-4A region and its regulated processing by the NS2 autoprotease and the NS3/4A protease plays a central role in the pestiviral life cycle. We report the identification and characterization of a novel internal cleavage in BVDV NS2, which is mediated by the NS3/4A protease. Further mapping using the NS2 of BVDV-1 strain NCP7 showed that cleavage occurs between L188 and G189. This cleavage site represents a novel sequence motif recognized by the NS3/4A protease and is conserved between the pestivirus species A, B and D. Inhibition of this internal NS2 cleavage by mutating the cleavage site did not cause obvious effects on RNA replication or virion morphogenesis in cultured cell lines. Accordingly, this novel internal NS2 cleavage adds an additional layer to the already complex polyprotein processing of Pestiviruses and might further extend the repertoires of the multifunctional NS2. However, unravelling of the functional relevance of this novel processing event in NS2, therefore, awaits future studies.

Funding
This study was supported by the:
  • deutsche forschungsgemeinschaft (Award TA 218 4-2)
    • Principle Award Recipient: ThomasWalther
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/content/journal/jgv/10.1099/jgv.0.001666
2021-10-22
2022-07-01
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References

  1. Simmonds P, Becher P, Bukh J, Gould EA, Meyers G. ICTV Virus Taxonomy Profile: Flaviviridae. J Gen Virol 2017; 98:2–3 [View Article] [PubMed]
    [Google Scholar]
  2. Tautz N, Tews BA, Meyers G. The molecular biology of pestiviruses. In Advances in Virus Research Vol 93 2015 pp 47–160 [View Article] [PubMed]
    [Google Scholar]
  3. Hoff HS, Donis RO. Induction of apoptosis and cleavage of poly(ADP-ribose) polymerase by cytopathic bovine vital diarrhea virus infection. Virus Res 1997; 49:101–113 [View Article] [PubMed]
    [Google Scholar]
  4. Jordan R, Wang L, Graczyk TM, Block TM, Romano PR. Replication of a cytopathic strain of bovine viral diarrhea virus activates PERK and induces endoplasmic reticulum stress-mediated apoptosis of MDBK cells. J Virol 2002; 76:9588–9599 [View Article] [PubMed]
    [Google Scholar]
  5. Schweizer M, Peterhans E. Oxidative stress in cells infected with bovine viral diarrhoea virus: a crucial step in the induction of apoptosis. J Gen Virol 1999; 80:1147–1155 [View Article] [PubMed]
    [Google Scholar]
  6. Yamane D, Kato K, Tohya Y, Akashi H. The double-stranded RNA-induced apoptosis pathway is involved in the cytopathogenicity of cytopathogenic Bovine viral diarrhea virus. J Gen Virol 2006; 87:2961–2970 [View Article] [PubMed]
    [Google Scholar]
  7. Meyers G, Tautz N, Becher P, Thiel HJ, Kümmerer BM. Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs. J Virol 1996; 70:8606–8613 [View Article] [PubMed]
    [Google Scholar]
  8. Lindenbach BD, Murray CL, Thiel HJ, Rice CM. Flaviviridae. Knipe D, Howley P. eds In Fields Virology Philadelphia: Lippincott Williams & Wilkin; 2013 pp 712–746
    [Google Scholar]
  9. Stark R, Meyers G, Rümenapf T, Thiel HJ. Processing of pestivirus polyprotein: cleavage site between autoprotease and nucleocapsid protein of classical swine fever virus. J Virol 1993; 67:7088–7095 [View Article] [PubMed]
    [Google Scholar]
  10. Elbers K, Tautz N, Becher P, Stoll D, Rümenapf T. Processing in the pestivirus E2-NS2 region: identification of proteins p7 and E2p7. J Virol 1996; 70:4131–4135 [View Article] [PubMed]
    [Google Scholar]
  11. Rümenapf T, Unger G, Strauss JH, Thiel HJ. Processing of the envelope glycoproteins of pestiviruses. J Virol 1993; 67:3288–3294 [View Article] [PubMed]
    [Google Scholar]
  12. Lackner T, Müller A, Pankraz A, Becher P, Thiel H-J. Temporal modulation of an autoprotease is crucial for replication and pathogenicity of an RNA virus. J Virol 2004; 78:10765–10775 [View Article] [PubMed]
    [Google Scholar]
  13. Lackner T, Müller A, König M, Thiel H-J, Tautz N. Persistence of bovine viral diarrhea virus is determined by a cellular cofactor of a viral autoprotease. J Virol 2005; 79:9746–9755 [View Article] [PubMed]
    [Google Scholar]
  14. Wiskerchen M, Collett MS. Pestivirus gene expression: Protein p80 of bovine viral diarrhea virus is a proteinase involved in polyprotein processing. Virology 1991; 184:341–350 [View Article] [PubMed]
    [Google Scholar]
  15. Tautz N, Elbers K, Stoll D, Meyers G, Thiel HJ. Serine protease of pestiviruses: determination of cleavage sites. J Virol 1997; 71:5415–5422 [View Article] [PubMed]
    [Google Scholar]
  16. Xu J, Mendez E, Caron PR, Lin C, Murcko MA. Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication. J Virol 1997; 71:5312–5322 [View Article] [PubMed]
    [Google Scholar]
  17. Tautz N, Kaiser A, Thiel H-J. NS3 serine protease of bovine viral diarrhea virus: characterization of active site residues, NS4A cofactor domain, and protease–cofactor interactions. Virology 2000; 273:351–363 [View Article] [PubMed]
    [Google Scholar]
  18. Behrens S-E, Grassmann CW, Thiel H-J, Meyers G, Tautz N. Characterization of an autonomous subgenomic pestivirus RNA replicon. J Virol 1998; 72:2364–2372 [View Article] [PubMed]
    [Google Scholar]
  19. De Moerlooze L, Desport M, Renard A, Lecomte C, Brownlie J et al. The coding region for the 54-kDa protein of several pestiviruses lacks host insertions but reveals a “zinc finger-like” domain. Virology 1990; 177:812–815 [View Article] [PubMed]
    [Google Scholar]
  20. Lackner T, Thiel H-J, Tautz N. Dissection of a viral autoprotease elucidates a function of a cellular chaperone in proteolysis. Proc Natl Acad Sci U S A 2006; 103:1510–1515 [View Article] [PubMed]
    [Google Scholar]
  21. Walther T, Fellenberg J, Klemens O, Isken O, Tautz N. Membrane topology of pestiviral nonstructural protein 2 and determination of the minimal autoprotease domain. J Virol 2021; 95:e00154-21 [View Article] [PubMed]
    [Google Scholar]
  22. Moulin HR, Seuberlich T, Bauhofer O, Bennett LC, Tratschin J-D. Nonstructural proteins NS2-3 and NS4A of classical swine fever virus: Essential features for infectious particle formation. Virology 2007; 365:376–389 [View Article] [PubMed]
    [Google Scholar]
  23. Brownlie J, Clarke MC, Howard CJ. Experimental infection of cattle in early pregnancy with a cytopathic strain of bovine virus diarrhoea virus. Res Vet Sci 1989; 46:307–311 [View Article] [PubMed]
    [Google Scholar]
  24. Isken O, Postel A, Bruhn B, Lattwein E, Becher P. CRISPR/Cas9-mediated knockout of DNAJC14 verifies this chaperone as a pivotal host factor for RNA replication of pestiviruses. J Virol 2019; 93:e01714-18 [View Article] [PubMed]
    [Google Scholar]
  25. Tautz N, Meyers G, Stark R, Dubovi EJ, Thiel HJ. Cytopathogenicity of a pestivirus correlates with a 27-nucleotide insertion. J Virol 1996; 70:7851–7858 [View Article] [PubMed]
    [Google Scholar]
  26. Agapov EV, Murray CL, Frolov I, Qu L, Myers TM et al. Uncleaved NS2-3 is required for production of infectious bovine viral diarrhea virus. J Virol 2004; 78:2414–2425 [View Article] [PubMed]
    [Google Scholar]
  27. Tautz N, Meyers G, Thiel H-J. Processing of poly-ubiquitin in the polyprotein of an RNA virus. Virology 1993; 197:74–85 [View Article] [PubMed]
    [Google Scholar]
  28. Gorbalenya AE, Koonin E, Donchenko AP, Blinov VM. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res 1989; 17:4713–4730 [View Article] [PubMed]
    [Google Scholar]
  29. Warrener P, Collett MS. Pestivirus NS3 (p80) protein possesses RNA helicase activity. J Virol 1995; 69:1720–1726 [View Article] [PubMed]
    [Google Scholar]
  30. Dubrau D, Tortorici MA, Rey FA, Tautz N. A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis. PLoS Pathog 2017; 13:1–30 [View Article] [PubMed]
    [Google Scholar]
  31. Schechter I, Berger A. On the size of the active site in proteases. Biochem Biophys Res Commun 1967; 27:157–162 [View Article] [PubMed]
    [Google Scholar]
  32. Lamp B, Riedel C, Wentz E, Tortorici M-A, Rümenapf T. Autocatalytic cleavage within classical swine fever virus NS3 leads to a functional separation of protease and helicase. J Virol 2013; 87:11872–11883 [View Article] [PubMed]
    [Google Scholar]
  33. Nestorowicz A, Chambers TJ, Rice CM. Mutagenesis of the Yellow Fever virus NS2A/2B cleavage site: Effects on proteolytic processing, viral replication, and evidence for alternative processing of the NS2A protein. Virology 1994; 199:114–123 [View Article] [PubMed]
    [Google Scholar]
  34. Kümmerer BM, Rice CM. Mutations in the yellow fever virus nonstructural protein NS2A selectively block production of infectious particles. J Virol 2002; 76:4773–4784 [View Article] [PubMed]
    [Google Scholar]
  35. Jirasko V, Montserret R, Appel N, Janvier A, Eustachi L et al. Structural and functional characterization of nonstructural protein 2 for its role in hepatitis C virus assembly. J Biol Chem 2008; 283:28546–28562 [View Article] [PubMed]
    [Google Scholar]
  36. Corapi W, Donis RO, Dubovi EJ. Monoclonal antibody analyses of cytopathic and noncytopathic viruses from fatal bovine viral diarrhea virus infections. J Virol 1988; 62:2823–2827 [View Article] [PubMed]
    [Google Scholar]
  37. Sutter G, Ohlmann M, Erfle V. Non-replicating vaccinia vector efficiently expresses bacteriophage T7 RNA polymerase. FEBS Lett 1995; 371:9–12 [View Article] [PubMed]
    [Google Scholar]
  38. Backes P, Quinkert D, Reiss S, Binder M, Zayas M et al. Role of annexin A2 in the production of infectious hepatitis C virus particles. J Virol 2010; 84:5775–5789 [View Article] [PubMed]
    [Google Scholar]
  39. Meyers G, Rümenapf T, Thiel H-J. Molecular cloning and nucleotide sequence of the genome of hog cholera virus. Virology 1989; 171:555–567 [View Article] [PubMed]
    [Google Scholar]
  40. Tautz N, Harada T, Kaiser A, Rinck G, Behrens S-E. Establishment and characterization of cytopathogenic and noncytopathogenic pestivirus replicons. J Virol 1999; 73:9422–9432 [View Article] [PubMed]
    [Google Scholar]
  41. Schägger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 1987; 166:368–379 [View Article] [PubMed]
    [Google Scholar]
  42. Yang S-H, Lee CG, Song MK, Sung YC. Internal cleavage of hepatitis C virus NS3 protein is dependent on the activity of NS34A protease. Virology 2000; 268:132–140 [View Article]
    [Google Scholar]
  43. Amberg SM, Nestorowicz A, McCourt DW, Rice CM. NS2B-3 proteinase-mediated processing in the yellow fever virus structural region: in vitro and in vivo studies. J Virol 1994; 68:3794–3802 [View Article] [PubMed]
    [Google Scholar]
  44. Lamp B, Riedel C, Roman-Sosa G, Heimann M, Jacobi S et al. Biosynthesis of classical swine fever virus nonstructural proteins. J Virol 2011; 85:3607–3620 [View Article] [PubMed]
    [Google Scholar]
  45. Bálint Á, Pálfi V, Belák S, Baule C. Viral sequence insertions and a novel cellular insertion in the NS2 gene of cytopathic isolates of bovine viral diarrhea virus as potential cytopathogenicity markers. Virus Genes 2005; 30:49–58 [View Article] [PubMed]
    [Google Scholar]
  46. Bálint Á, Baule C, Pálfi V, Dencsö L, Hornyák Á. A 45-nucleotide insertion in the NS2 gene is responsible for the cytopathogenicity of a bovine viral diarrhoea virus strain. Virus Genes 2005; 31:135–144 [View Article] [PubMed]
    [Google Scholar]
  47. Isken O, Langerwisch U, Schönherr R, Lamp B, Schröder K et al. Functional characterization of bovine viral diarrhea virus nonstructural protein 5A by reverse genetic analysis and live cell imaging. J Virol 2014; 88:82–98 [View Article] [PubMed]
    [Google Scholar]
  48. Klemens O, Dubrau D, Tautz N. Characterization of the determinants of NS2-3-independent virion morphogenesis of pestiviruses. J Virol 2015; 89:11668–11680 [View Article] [PubMed]
    [Google Scholar]
  49. Smith DB, Meyers G, Bukh J, Gould EA, Monath T. Proposed revision to the taxonomy of the genus Pestivirus, family Flaviviridae . J Gen Virol 2017; 98:2106–2112 [View Article] [PubMed]
    [Google Scholar]
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