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Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, Groundnut bud necrosis virus

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Abstract

The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 5′ α phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.

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References

  1. Wang X, Lee WM, Watanabe T, Schwartz M, Janda M, Ahlquist P (2005) Brome mosaic virus 1a nucleoside triphosphatase/helicase domain plays crucial roles in recruiting RNA replication templates. J Virol 79:13747–13758

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. 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  PubMed Central  CAS  PubMed  Google Scholar 

  3. Kovalev N, Pogany J, Nagy PD (2012) A co-opted DEAD-box RNA helicase enhances tombusvirus plus-strand synthesis. PLoS Pathog 8:e1002537

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Huang T-S, Wei T, Laliberté J-F, Wang A (2010) A host RNA helicase-like protein, AtRH8, interacts with the potyviral genome-linked protein, VPg, associates with the virus accumulation complex, and is essential for infection. Plant Physiol 152:255–266

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Mir MA, Panganiban AT (2006) The bunyavirus nucleocapsid protein is an RNA chaperone: possible roles in viral RNA panhandle formation and genome replication. RNA 12:272–282

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Kinsella E, Martin SG, Grolla A, Czub M, Feldmann H, Flick R (2004) Sequence determination of the Crimean-Congo hemorrhagic fever virus L segment. Virology 321:23–28

    Article  CAS  PubMed  Google Scholar 

  7. Ranji A, Boris-Lawrie K (2010) RNA helicases: emerging roles in viral replication and the host innate response. RNA Biol 7:775–787

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Kadare G, Haenni AL (1997) Virus-encoded RNA helicases. J Virol 71:2583–2590

    PubMed Central  CAS  PubMed  Google Scholar 

  9. Frick DN, Banik S, Rypma RS (2007) Role of divalent metal cations in ATP hydrolysis catalyzed by the hepatitis C virus NS3 helicase: magnesium provides a bridge for ATP to fuel unwinding. J Mol Biol J 365:1017–1032

    Article  CAS  Google Scholar 

  10. de la Cruz J, Kressler D, Linder P (1999) Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families. Trends Biochem Sci 24:192–198

    Article  PubMed  Google Scholar 

  11. Singleton MR, Dillingham MS, Wigley DB (2007) Structure and mechanism of helicases and nucleic acid translocases. Annu Rev Biochem 7:23–50

    Article  Google Scholar 

  12. Jankowsky E, Fairman ME (2007) RNA helicases–one fold for many functions. Curr Opin Struct Biol 17:316–324

    Article  CAS  PubMed  Google Scholar 

  13. Tuteja N, Huang NW, Skopac D, Tuteja R, Hrvatic S, Zhang J, Pongor S, Joseph G, Faucher C, Amalric F et al (1995) Human DNA helicase IV is nucleolin, an RNA helicase modulated by phosphorylation. Gene 160:143–148

    Article  CAS  PubMed  Google Scholar 

  14. Tuteja N, Tuteja R, Ochem A, Taneja P, Huang NW, Simoncsits A, Susic S, Rahman K, Marusic L, Chen J et al (1994) Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen. EMBO J 13:4991–5001

    PubMed Central  CAS  PubMed  Google Scholar 

  15. Costa M, Ochem A, Staub A, Falaschi A (1999) Human DNA helicase VIII: a DNA and RNA helicase corresponding to the G3BP protein, an element of the ras transduction pathway. Nucleic Acids Res 27:817–821

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Prins M, Goldbach R (1998) The emerging problem of tospovirus infection and nonconventional methods of control. Trends Microbiol 6:31–35

    Article  CAS  PubMed  Google Scholar 

  17. Lokesh B, Rashmi PR, Amruta BS, Srisathiyanarayanan D, Murthy MR, Savithri HS (2010) NSs encoded by groundnut bud necrosis virus is a bifunctional enzyme. PLoS ONE 5:e9757

    Article  PubMed Central  PubMed  Google Scholar 

  18. Schnettler E, Hemmes H, Huismann R, Goldbach R, Prins M, Kormelink R (2010) Diverging affinity of tospovirus RNA silencing suppressor proteins, NSs, for various RNA duplex molecules. J Virol 84:11542–11554

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Bridgen A, Weber F, Fazakerley JK, Elliott RM (2001) Bunyamwera bunyavirus nonstructural protein NSs is a nonessential gene product that contributes to viral pathogenesis. Proc Natl Acad Sci 98:664–669

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Le May N, Dubaele S, Proietti De Santis L, Billecocq A, Bouloy M, Egly JM (2004) TFIIH transcription factor, a target for the Rift Valley hemorrhagic fever virus. Cell 116:541–550

    Article  PubMed  Google Scholar 

  21. Kalveram B, Lihoradova O, Ikegami T (2011) NSs protein of rift valley fever virus promotes posttranslational downregulation of the TFIIH subunit p62. J Virol 85:6234–6243

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Copeland AM, Altamura LA, Van Deusen NM, Schmaljohn CS (2013) Nuclear relocalization of polyadenylate binding protein during rift valley fever virus infection involves expression of the NSs gene. J Virol 87:11659–11669

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. Takeda A, Sugiyama K, Nagano H, Mori M, Kaido M, Mise K, Tsuda S, Okuno T (2002) Identification of a novel RNA silencing suppressor, NSs protein of Tomato spotted wilt virus. FEBS Lett 532:75–79

    Article  CAS  PubMed  Google Scholar 

  24. Goswami S, Sahana N, Pandey V, Doblas P, Jain RK, Palukaitis P, Canto T, Praveen S (2012) Interference in plant defense and development by non-structural protein NSs of Groundnut bud necrosis virus. Virus Res 163:368–373

    Article  CAS  PubMed  Google Scholar 

  25. Zhai Y, Bag S, Mitter N, Turina M, Pappu H (2013) Mutational analysis of two highly conserved motifs in the silencing suppressor encoded by tomato spotted wilt virus (genus Tospovirus, family Bunyaviridae). Arch Virol 159:1499–1504

    Article  PubMed  Google Scholar 

  26. Geerts-Dimitriadou C, Lu Y-Y, Geertsema C, Goldbach R, Kormelink R (2012) Analysis of the Tomato spotted wilt virus ambisense S RNA-Encoded hairpin structure in translation. PLoS ONE 7:e31013

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Margaria P, Ciuffo M, Pacifico D, Turina M (2007) Evidence that the nonstructural protein of Tomato spotted wilt virus is the avirulence determinant in the Interaction with resistant pepper carrying the Tsw gene. Mol Plant Microbe Interact 20:547–558

    Article  CAS  PubMed  Google Scholar 

  28. Margaria P, Bosco L, Vallino M, Ciuffo M, Mautino GC, Tavella L, Turina M (2014) The NSs protein of Tomato spotted wilt virus is required for persistent infection and transmission by Frankliniella occidentalis. J Virol 88:5788–5802

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Choudhury NR, Malik PS, Singh DK, Islam MN, Kaliappan K, Mukherjee SK (2006) The oligomeric Rep protein of Mungbean yellow mosaic India virus (MYMIV) is a likely replicative helicase. Nucleic Acids Res 34:6362–6377

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Eskelin K, Suntio T, Hyvarinen S, Hafren A, Makinen K (2010) Renilla luciferase-based quantitation of Potato virus A infection initiated with Agrobacterium infiltration of N. benthamiana leaves. J Virol Methods 64:101–110

    Article  Google Scholar 

  31. Pall GS, Hamilton AJ (2008) Improved northern blot method for enhanced detection of small RNA. Nat Protoc 3:1077–1084

    Article  CAS  PubMed  Google Scholar 

  32. Kalinina NO, Rakitina DV, Solovyev AG, Schiemann J, Morozov SY (2002) RNA helicase activity of the plant virus movement proteins encoded by the first gene of the triple gene block. Virology 296:321–329

    Article  CAS  PubMed  Google Scholar 

  33. Howard AR, Heppler ML, Ju HJ, Krishnamurthy K, Payton ME, Verchot-Lubicz J (2004) Potato virus X TGBp1 induces plasmodesmata gating and moves between cells in several host species whereas CP moves only in N. benthamiana leaves. Virology 328:185–197

    Article  CAS  PubMed  Google Scholar 

  34. Kang W-H, Seo J-K, Chung BN, Kim K-H, Kang B-C (2012) Helicase domain encoded by Cucumber mosaic virus RNA1 determines systemic infection of Cmr1 in pepper. PLoS ONE 7:e43136

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Wang X, Kelman Z, Culver JN (2010) Helicase ATPase activity of the Tobacco mosaic virus 126-kDa protein modulates replicase complex assembly. Virology 402:292–302

    Article  CAS  PubMed  Google Scholar 

  36. Tuteja N, Tarique M, Tuteja R (2014) Rice SUV3 is a bidirectional helicase that binds both DNA and RNA. BMC Plant Biol 14:283. doi:10.1186/s12870-014-0283-6

    Article  PubMed Central  PubMed  Google Scholar 

  37. Wessel R, Schweizer J, Stahl H (1992) Simian virus 40 T-antigen DNA helicase is a hexamer which forms a binary complex during bidirectional unwinding from the viral origin of DNA replication. J Virol 66:804–815

    PubMed Central  CAS  PubMed  Google Scholar 

  38. Mansuroglu Z, Josse T, Gilleron J, Billecocq A, Leger P, Bouloy M, Bonnefoy E (2010) Nonstructural NSs protein of rift valley fever virus interacts with pericentromeric DNA sequences of the host cell, inducing chromosome cohesion and segregation defects. J Virol 84:928–939

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank Sir David Baulcombe (Cambridge University, London) for the gift of N. benthamiana 16c seeds, and Dr. Kazuyuki Mise (Kyoto University, Japan) for the pBIC vectors expressing GFP, dsGFP and P19. We also thank the Department of Science and Technology for the J. C. Bose fellowship to Prof. H. S. Savithri, Department of Biotechnology, and Indian Institute of Science for financial support.

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Author notes

  1. Lokesh Bhushan

    Present address: Cincinnati Children’s Hospital Medical Center, Cincinnati, USA

  2. Vipin Singh Rana

    Present address: Department of Zoology, University of Delhi, New Delhi, 110 007, India

  3. Sunil Kumar Mukherjee

    Present address: Department of Genetics, University of Delhi, New Delhi, 110 0012, India

Authors and Affiliations

  1. Department of Biochemistry, New Biological Sciences, Indian Institute of Science, Bangalore, 560012, Karnataka, India

    Lokesh Bhushan, Ambily Abraham & Handanahal Subbarao Savithri

  2. International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067, India

    Nirupam Roy Choudhury, Vipin Singh Rana & Sunil Kumar Mukherjee

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  1. Lokesh Bhushan
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Correspondence to Handanahal Subbarao Savithri.

Additional information

L. Bhushan and A. Abraham contributed equally.

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Bhushan, L., Abraham, A., Choudhury, N.R. et al. Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, Groundnut bud necrosis virus. Arch Virol 160, 959–967 (2015). https://doi.org/10.1007/s00705-014-2331-9

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