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A positively charged surface patch on the pestivirus NS3 protease module plays an important role in modulating NS3 helicase activity and virus production

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Abstract

Pestivirus nonstructural protein 3 (NS3) is a multifunctional protein with protease and helicase activities that are essential for virus replication. In this study, we used a combination of biochemical and genetic approaches to investigate the relationship between a positively charged patch on the protease module and NS3 function. The surface patch is composed of four basic residues, R50, K74 and K94 in the NS3 protease domain and H24 in the structurally integrated cofactor NS4APCS. Single-residue or simultaneous four-residue substitutions in the patch to alanine or aspartic acid had little effect on ATPase activity. However, single substitutions of R50, K94 or H24 or a simultaneous four-residue substitution resulted in apparent changes in the helicase activity and RNA-binding ability of NS3. When these mutations were introduced into a classical swine fever virus (CSFV) cDNA clone, a single substitution at K94 or a simultaneous four-residue substitution (Qua_A or Qua_D) impaired the production of infectious virus. Furthermore, the replication efficiency of the CSFV variants was partially correlated with the helicase activity of NS3 in vitro. Our results suggest that the conserved positively charged patch on NS3 plays an important role in modulating the NS3 helicase activity in vitro and CSFV production.

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References

  1. Aydin C, Mukherjee S, Hanson AM, Frick DN, Schiffer CA (2013) The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities. Protein Sci 22:1786–1798

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Becher P, Orlich M, Thiel HJ (1998) Complete genomic sequence of border disease virus, a pestivirus from sheep. J Virol 72:5165–5173

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Beran RK, Serebrov V, Pyle AM (2007) The serine protease domain of hepatitis C viral NS3 activates RNA helicase activity by promoting the binding of RNA substrate. J Bio Chem 282:34913–34920

    Article  CAS  Google Scholar 

  4. Beran RK, Pyle AM (2008) Hepatitis C viral NS3-4A protease activity is enhanced by the NS3 helicase. J Bio Chem 283:29929–29937

    Article  CAS  Google Scholar 

  5. Bintintan I, Meyers G (2010) A new type of signal peptidase cleavage site identified in an RNA virus polyprotein. J Bio Chem 285:8572–8584

    Article  CAS  Google Scholar 

  6. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  7. Chernov AV, Shiryaev SA, Aleshin AE, Ratnikov BI, Smith JW, Liddington RC, Strongin AY (2008) The two-component NS2B-NS3 proteinase represses DNA unwinding activity of the West Nile virus NS3 helicase. J Bio Chem 283:17270–17278

    Article  CAS  Google Scholar 

  8. Colett MS, Larson R, Gold C, Strick D, Anderson DK, Purchio AF (1988) Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology 165:191–199

    Article  CAS  PubMed  Google Scholar 

  9. Dubrau D, Tortorici MA, Rey FA, Tautz N (2017) 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 13:e1006134

    Article  PubMed  PubMed Central  Google Scholar 

  10. Edwards S, Fukusho A, Lefevre PC, Lipowski A, Pejsak Z, Roehe P, Westergaard J (2000) Classical swine fever: the global situation. Vet Microbiol 73:103–119

    Article  CAS  PubMed  Google Scholar 

  11. Frick DN, Rypma RS, Lam AMI, Gu BH (2004) The nonstructural protein 3 protease/helicase requires an intact protease domain to unwind duplex RNA efficiently. J Bio Chem 279:1269–1280

    Article  CAS  Google Scholar 

  12. Gladue DP, Holinka LG, Largo E, Fernandez Sainz I, Carrillo C, O’Donnell V, Baker-Branstetter R, Lu Z, Ambroggio X, Risatti GR, Nieva JL, Borca MV (2012) Classical swine fever virus p7 protein is a viroporin involved in virulence in swine. J Virol 86:6778–6791

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gu B, Liu C, Lin-Goerke J, Maley DR, Gutshall LL, Feltenberger CA, Del Vecchio AM (2000) The RNA helicase and nucleotide triphosphatase activities of the bovine viral diarrhea virus NS3 protein are essential for viral replication. J Virol 74:1794–1800

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Harada T, Tautz N, Thiel HJ (2000) E2–p7 region of the bovine viral diarrhea virus polyprotein: processing and functional studies. J Virol 74:9498–9506

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Houe H (1999) Epidemiological features and economical importance of bovine virus diarrhoea virus (BVDV) infections. Vet Microbiol 64:89–107

    Article  CAS  PubMed  Google Scholar 

  16. Lackner T, Muller A, Pankraz A, Becher P, Thiel HJ, Gorbalenya AE, Tautz N (2004) Temporal modulation of an autoprotease is crucial for replication and pathogenicity of an RNA virus. J Virol 78:10765–10775

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Lamp B, Riedel C, Roman-Sosa G, Heimann M, Jacobi S, Becher P, Thiel HJ, Rumenapf T (2011) Biosynthesis of classical swine fever virus nonstructural proteins. J Virol 85:3607–3620

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lamp B, Riedel C, Wentz E, Tortorici MA, Rumenapf T (2013) Autocatalytic cleavage within classical swine fever virus NS3 leads to a functional separation of protease and helicase. J Virol 87:11872–11883

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Li L, Wu R, Zheng F, Zhao C, Pan Z (2015) The N-terminus of classical swine fever virus (CSFV) nonstructural protein 2 modulates viral genome RNA replication. Virus Res 210:90–99

    Article  CAS  PubMed  Google Scholar 

  20. Li L, Pang H, Wu R, Zhang Y, Tan Y, Pan Z (2016) Development of a novel single-step reverse genetics system for the generation of classical swine fever virus. Arch Virol 161:1831–1838

    Article  CAS  PubMed  Google Scholar 

  21. Lindenbach BD, Thiel H-J, Rice CM (2007) Flaviviridae: the viruses and their replication. In: Knipe DM, Howley PM (ed) Fields virology, 5th edn, vol 1. Lippincott, Williams & Wilkins, Philadelphia, pp 1101–1152

  22. Lu G, Gong P (2013) Crystal Structure of the full-length Japanese encephalitis virus NS5 reveals a conserved methyltransferase-polymerase interface. PLoS Pathog 9:e1003549

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Luo D, Wei N, Doan DN, Paradkar PN, Chong Y, Davidson AD, Kotaka M, Lescar J, Vasudevan SG (2010) Flexibility between the protease and helicase domains of the dengue virus NS3 protein conferred by the linker region and its functional implications. J Bio Chem 285:18817–18827

    Article  CAS  Google Scholar 

  24. Ma Y, Anantpadma M, Timpe JM, Shanmugam S, Singh SM, Lemon SM, Yi M (2011) Hepatitis C virus NS2 protein serves as a scaffold for virus assembly by interacting with both structural and nonstructural proteins. J Virol 85:86–97

    Article  CAS  PubMed  Google Scholar 

  25. Meyers G, Rumenapf T, Thiel HJ (1989) Molecular cloning and nucleotide sequence of the genome of hog cholera virus. Virology 171:555–567

    Article  CAS  PubMed  Google Scholar 

  26. Morgenstern KA, Landro JA, Hsiao K, Lin C, Gu Y, Su MS, Thomson JA (1997) Polynucleotide modulation of the protease, nucleoside triphosphatase, and helicase activities of a hepatitis C virus NS3-NS4A complex isolated from transfected COS cells. J Virol 71:3767–3775

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Moulin HR, Seuberlich T, Bauhofer O, Bennett LC, Tratschin JD, Hofmann MA, Ruggli N (2007) Nonstructural proteins NS2-3 and NS4A of classical swine fever virus: essential features for infectious particle formation. Virology 365:376–389

    Article  CAS  PubMed  Google Scholar 

  28. Mukherjee S, Hanson AM, Shadrick WR, Ndjomou J, Sweeney NL, Hernandez JJ, Bartczak D, Li K, Frankowski KJ, Heck JA, Arnold LA, Schoenen FJ, Frick DN (2012) Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays. Nucleic acids Res 40:8607–8621

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Pang PS, Jankowsky E, Planet PJ, Pyle AM (2002) The hepatitis C viral NS3 protein is a processive DNA helicase with cofactor enhanced RNA unwinding. EMBO J 21:1168–1176

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Pei J, Zhao M, Ye Z, Gou H, Wang J, Yi L, Dong X, Liu W, Luo Y, Liao M, Chen J (2014) Autophagy enhances the replication of classical swine fever virus in vitro. Autophagy 10:93–110

    Article  CAS  PubMed  Google Scholar 

  31. Provazzi PJ, Mukherjee S, Hanson AM, Nogueira ML, Carneiro BM, Frick DN, Rahal P (2015) Analysis of the enzymatic activity of an NS3 helicase genotype 3a variant sequence obtained from a relapse patient. PLoS ONE 10:e0144638

    Article  PubMed  PubMed Central  Google Scholar 

  32. Reed LJ, Muench H (1938) A simple method of estimating fifty per cent endpoints. Am J Epidemol 27:493–497

    Article  Google Scholar 

  33. Sheng C, Xiao M, Geng X, Liu J, Wang Y, Gu F (2007) Characterization of interaction of classical swine fever virus NS3 helicase with 3’ untranslated region. Virus Res 129:43–53

    Article  CAS  PubMed  Google Scholar 

  34. Stapleford KA, Lindenbach BD (2011) Hepatitis C virus NS2 coordinates virus particle assembly through physical interactions with the E1–E2 glycoprotein and NS3-NS4A enzyme complexes. J Virol 85:1706–1717

    Article  CAS  PubMed  Google Scholar 

  35. Stoscheck CM (1990) Quantitation of protein. Methods Enzymol 182:50–68

    Article  CAS  PubMed  Google Scholar 

  36. Tautz N, Kaiser A, Thiel HJ (2000) NS3 serine protease of bovine viral diarrhea virus: characterization of active site residues, NS4A cofactor domain, and protease-cofactor interactions. Virology 273:351–363

    Article  CAS  PubMed  Google Scholar 

  37. Tautz N, Tews BA, Meyers G (2015) The molecular biology of pestiviruses. Adv Virus Res 93:47–160

    Article  CAS  PubMed  Google Scholar 

  38. Thiel HJ, Stark R, Weiland E, Rumenapf T, Meyers G (1991) Hog cholera virus: molecular composition of virions from a pestivirus. J Virol 65:4705–4712

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Tortorici MA, Duquerroy S, Kwok J, Vonrhein C, Perez J, Lamp B, Bricogne G, Rumenapf T, Vachette P, Rey FA (2015) X-ray structure of the pestivirus NS3 helicase and its conformation in solution. J Virol 89:4356–4371

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Tratschin JD, Moser C, Ruggli N, Hofmann MA (1998) Classical swine fever virus leader proteinase Npro is not required for viral replication in cell culture. J Virol 72:7681–7684

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Weiland F, Weiland E, Unger G, Saalmuller A, Thiel HJ (1999) Localization of pestiviral envelope proteins E(rns) and E2 at the cell surface and on isolated particles. J Gen Virol 80(Pt 5):1157–1165

    Article  CAS  PubMed  Google Scholar 

  42. Wen G, Chen C, Luo X, Wang Y, Zhang C, Pan Z (2007) Identification and characterization of the NTPase activity of classical swine fever virus (CSFV) nonstructural protein 3 (NS3) expressed in bacteria. Arch Virol 152:1565–1573

    Article  CAS  PubMed  Google Scholar 

  43. Wen G, Xue J, Shen Y, Zhang C, Pan Z (2009) Characterization of classical swine fever virus (CSFV) nonstructural protein 3 (NS3) helicase activity and its modulation by CSFV RNA-dependent RNA polymerase. Virus Res 141:63–70

    Article  CAS  PubMed  Google Scholar 

  44. Wu J, Bera AK, Kuhn RJ, Smith JL (2005) Structure of the Flavivirus helicase: implications for catalytic activity, protein interactions, and proteolytic processing. J Virol 79:10268–10277

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Wu R, Li L, Zhao Y, Tu J, Pan Z (2016) Identification of two amino acids within E2 important for the pathogenicity of chimeric classical swine fever virus. Virus Res 211:79–85

    Article  CAS  PubMed  Google Scholar 

  46. Yang ZH, Wu R, Li RW, Li L, Xiong ZL, Zhao HZ, Guo DY, Pan ZS (2012) Chimeric classical swine fever (CSF)-Japanese encephalitis (JE) viral replicon as a non-transmissible vaccine candidate against CSF and JE infections. Virus Res 165:61–70

    Article  CAS  PubMed  Google Scholar 

  47. Yi M, Ma Y, Yates J, Lemon SM (2007) Compensatory mutations in E1, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus. J Virol 81:629–638

    Article  CAS  PubMed  Google Scholar 

  48. Zhang C, Cai Z, Kim YC, Kumar R, Yuan F, Shi PY, Kao C, Luo G (2005) Stimulation of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase activity by the NS3 protease domain and by HCV RNA-dependent RNA polymerase. J Virol 79:8687–8697

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Zheng F, Lu G, Li L, Gong P, Pan Z (2017) Uncoupling of protease trans-cleavage and helicase activities in pestivirus NS3. J Virol 91:e1904-1917

    Article  Google Scholar 

  50. Zheng L, Baumann U, Reymond JL (2004) An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res 32:e115

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This study was supported by the National Key Research and Development Program of China (2018YFD0500104) and the National Natural Science Foundation of China (31570152 and 31670154).

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  1. Fengwei Zheng and Weicheng Yi contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China

    Fengwei Zheng, Weicheng Yi, Hongchang Zhu & Zishu Pan

  2. Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China

    Weichi Liu & Peng Gong

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  1. Fengwei Zheng
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Correspondence to Peng Gong or Zishu Pan.

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Zheng, F., Yi, W., Liu, W. et al. A positively charged surface patch on the pestivirus NS3 protease module plays an important role in modulating NS3 helicase activity and virus production. Arch Virol 166, 1633–1642 (2021). https://doi.org/10.1007/s00705-021-05055-5

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