Prediction of putative regulatory elements in the subgenomic promoters of cucumber green mottle mosaic virus and their interactions with the RNA dependent RNA polymerase domain

Abstract

Characterization of the subgenomic RNA (sgRNA) promoter of many plant viruses is important to understand the expression of downstream genes and also to configure their genome into a suitable virus gene-vector system. Cucumber green mottle mosaic virus (CGMMV, genus Tobamovirus) is one of the RNA viruses, which is extensively being exploited as the suitable gene silencing and protein expression vector. Even though, characters of the sgRNA promoters (SGPs) of CGMMV are yet to be addressed. In the present study, we predicted the SGP for the movement protein (MP) and coat protein (CP) of CGMMV. Further, we identified the key regulatory elements in the SGP regions of MP and CP, and their interactions with the core RNA dependent RNA polymerase (RdRp) domain of CGMMV was deciphered. The modeled structure of core RdRp contains two palm (1–41 aa, and 63–109 aa), one finger (42–62 aa) subdomains with three conserved RdRp motifs that played important role in binding to the SGP nucleic acids. RdRp strongly preferred the double helix form of the stem region in the stem and loop (SL) structures, and the internal bulge elements. In MP-SGP, a total of six elements was identified; of them, the affinity of binding to − 26 nt to − 17 nt site (CGCGGAAAAG) was higher through the formation of strong hydrogen bonds with LYS16, TYR17, LYS19, SER20, etc. of the motif A in the palm subdomain of RdRp. Similar strong interactions were noticed in the internal bulge (CAACUUU) located at + 33 to + 39 nt adjacent to the translation start site (TLSS) (+ 1). These could be proposed as the putative core promoter elements in MP-SGP. Likewise, total five elements were predicted within − 114 nt to + 144 nt region of CP-SGP with respect to CP-TLSS. Of them, RdRp preferred to bind at the small hairpin located at − 60 nt to − 43 nt (UUGGAGGUUUAGCCUCCA) in the upstream region, and at the complex duplex structure spanning between + 99 and + 114 nt in the downstream region, thus indicating the distribution of core promoter within − 60 nt to + 114 nt region of CP-SGP with respect to TLSS (+ 1) of the CP; whereas, the − 114 nt to + 144 nt region of CP-SGP might be necessary for the full activity of the CP-SGP. Our in silico prediction certifies the gravity of these nucleotide stretches as the RNA regulatory elements and identifies their potentiality for binding with of palm and finger sub-domain of RdRp. Identification of such elements will be helpful to anticipate the critical length of the SGPs. Our finding will not only be helpful to delineate the SGPs of CGMMV but also their subsequent application in the efficient construction of virus gene-vector for the expression of foreign protein in plant.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. 1.

    Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE. The protein data bank. Nucleic Acids Res. 2000;28:235–42.

    CAS  Article  Google Scholar 

  2. 2.

    Biesiada M, Purzycka KJ, Szachniuk M, Blazewicz J, Adamiak RW. Automated RNA 3D structure prediction with RNAComposer. Methods Mol Biol. 2016;1490:199–215. https://doi.org/10.1007/978-1-4939-6433-8_13.

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Chen MH, Roossinck MJ, Kao CC. Efficient and specific initiation of subgenomic RNA synthesis by cucumber mosaic virus replicase in vitro requires an upstream RNA stem-loop1. J Virol. 2000;74(23):11201–9.

    CAS  Article  Google Scholar 

  4. 4.

    Colovos C, Yeates TO. Verification of protein structures: patterns of nonbonded atomic interactions. Protein Sci. 1993;2(9):1511–9.

    CAS  Article  Google Scholar 

  5. 5.

    Darty K, Denise A, Ponty Y. VARNA: interactive drawing and editing of the RNA secondary structure. Bioinformatics. 2009;25(15):1974–5.

    CAS  Article  Google Scholar 

  6. 6.

    Dorokhov YL, Ivanov PA, Komarova TV, Skulachev MV, Atabekov JG. An internal ribosome entry site located upstream of the crucifer-infecting tobamovirus coat protein (CP) gene can be used for CP synthesis in vivo. J Gen Virol. 2006;87(9):2693–7.

    CAS  Article  Google Scholar 

  7. 7.

    Dorokhov YL, Sheshukova EV, Komarova TV. Tobamovirus 3′-terminal gene overlap may be a mechanism for within-host fitness improvement. Front Microbiol. 2017;8:851.

    Article  Google Scholar 

  8. 8.

    Feklistov A. RNA polymerase: in search of promoters. Ann N Y Acad Sci. 2013;1293:25–32. https://doi.org/10.1111/nyas.12197.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Grdzelishvili VZ, Chapman SN, Dawson WO, Lewandowski DJ. Mapping of the Tobacco mosaic virus movement protein and coat protein subgenomic RNA promoters in vivo. Virology. 2000;275(1):177–92. https://doi.org/10.1006/viro.2000.0511(PMID: 11017798).

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Gupta D, Ranjan R. In silico comparative analysis of promoters derived from plant para retroviruses. Virus Dis. 2017;28(4):416–21. https://doi.org/10.1007/s13337-017-0410-8.

    Article  Google Scholar 

  11. 11.

    Haasnoot PC, Olsthoorn RC, Bol JF. The Brome mosaic virus subgenomic promoter hairpin is structurally similar to the iron-responsive element and functionally equivalent to the minus-strand core promoter stem-loop C. RNA. 2002;8(1):110–22.

    CAS  Article  Google Scholar 

  12. 12.

    Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Sympos Ser. 1999;41:95–8.

    CAS  Google Scholar 

  13. 13.

    Hernandez-Garcia CM, Finer JJ. Identification and validation of promoters and cis-acting regulatory elements. Plant Sci. 2014;217:109–19.

    Article  Google Scholar 

  14. 14.

    Hofmann MA, Sethna PB, Brian DA. Bovine coronavirus mRNA replication continues throughout persistent infection in cell culture. J Virol. 1990;64(9):4108–14.

    CAS  Article  Google Scholar 

  15. 15.

    Jailani AA, Solanki V, Roy A, Sivasudha T, Mandal B. A CGMMV genome-replicon vector with partial sequences of coat protein gene efficiently expresses GFP in Nicotiana benthamiana. Virus Res. 2017;233:77–85.

    CAS  Article  Google Scholar 

  16. 16.

    Koramutla MK, Bhatt D, Negi M, Venkatachalam P, Jain PK, Bhattacharya R. Strength, stability, and cis-motifs of in silico identified phloem-specific promoters in Brassica juncea (L.). Front Plant Sci. 2016;7:457.

    Article  Google Scholar 

  17. 17.

    Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 70 for bigger datasets. Mol Biol Evol. 2015;33:1870–4.

    Article  Google Scholar 

  18. 18.

    Kumari S, Ware D. Genome-wide computational prediction and analysis of core promoter elements across plant monocots and dicots. PLoS ONE. 2013;8(10):e79011. https://doi.org/10.1371/journal.pone.0079011.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002;30(1):325–7.

    CAS  Article  Google Scholar 

  20. 20.

    Li L, Wang M, Chen Y, Hu T, Yang Y, Zhang Y, Bi G, Wang W, Liu E, Han J, Lu T, Su D. Structure of the enterovirus D68 RNA-dependent RNA polymerase in complex with NADPH implicates an inhibitor binding site in the RNA template tunnel. J Struct Biol. 2020;211(1):107510.

    CAS  Article  Google Scholar 

  21. 21.

    Li R, Zheng Y, Fei Z, Ling KS. First complete genome sequence of an emerging cucumber green mottle mosaic virus isolate in North America. Genome Announce. 2015;3(3):e00452–515. https://doi.org/10.1128/genomeA.00452-15.

    Article  Google Scholar 

  22. 22.

    Liu M, Liu L, Wu H, Kang B, Gu Q. Mapping subgenomic promoter of coat protein gene of Cucumber green mottle mosaic virus. J Integr Agric. 2020;19(1):153–63. https://doi.org/10.1016/S2095-3119(19)62647-X.

    CAS  Article  Google Scholar 

  23. 23.

    Lovell SC, Davis IW, Arendall WB, DeBakker PI, Word JM, Prisant MG, Richardson JS, Richardson DC. Structure validation by Cα geometry: ϕ, ψ and Cβ deviation. Proteins: Struct, Funct, Bioinf. 2003;50(3):437–50.

    CAS  Article  Google Scholar 

  24. 24.

    Man M, Epel BL. Characterization of regulatory elements within the coat protein (CP) coding region of Tobacco mosaic virus affecting subgenomic transcription and green fluorescent protein expression from the CP subgenomic RNA promoter. J Gen Virol. 2004;85(6):1727–38. https://doi.org/10.1099/vir.0.79838-0.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Marks H, Ren XY, Sandbrink H, van Hulten MC, Vlak JM. In silico identification of putative promoter motifs of white spot syndrome virus. BMC Bioinf. 2006;7(1):309. https://doi.org/10.1186/1471-2105-7-309.

    CAS  Article  Google Scholar 

  26. 26.

    Marsh LE, Dreher TW, Hall TC. Mutational analysis of the core and modulator sequences of the BMV RNA3 subgenomlc promoter. Nucleic Acids Res. 1988;16(3):981–95.

    CAS  Article  Google Scholar 

  27. 27.

    Mashiach E, Schneidman-Duhovny D, Andrusier N, Nussinov R, Wolfson HJ. FireDock: a web server for fast interaction refinement in molecular docking. Nucleic Acids Res. 2008;36:W229–32.

    CAS  Article  Google Scholar 

  28. 28.

    Miller WA, Koev G. Synthesis of subgenomic RNAs by positive-strand RNA viruses. Virology. 2000;273(1):1–8. https://doi.org/10.1006/viro.2000.0421.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Nain V, Sahi S, Kumar PA. In silico identification of regulatory elements in promoters. In: Lopes H, editor. Computational biology and applied bioinformatics. In Tech; 2011. pp. 47–66. https://doi.org/10.5772/22230.

  30. 30.

    Olsthoorn RC, Haasnoot PJ, Bol JF. Similarities and differences between the subgenomic and minus-strand promoters of an RNA plant virus. J Virol. 2004;78(8):4048–53.

    CAS  Article  Google Scholar 

  31. 31.

    Ooi A, Tan S, Mohamed R, Rahman NA, Othman RY. The full-length clone of cucumber green mottle mosaic virus and its application as an expression system for Hepatitis B surface antigen. J Biotechnol. 2006;121(4):471–81.

    CAS  Article  Google Scholar 

  32. 32.

    O’Reilly EK, Kao CC. Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure. Virology. 1998;252(2):287–303. https://doi.org/10.1006/viro.1998.9463.

    Article  PubMed  Google Scholar 

  33. 33.

    Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem. 2004;25(13):1605–12.

    CAS  Article  Google Scholar 

  34. 34.

    Popenda M, Szachniuk M, Antczak M, Purzycka KJ, Lukasiak P, Bartol N, Blazewicz J, Adamiak RW. Automated 3D structure composition for large RNAs. Nucleic Acids Res. 2012;40(14):e112.

    CAS  Article  Google Scholar 

  35. 35.

    Rhee SJ, Jang YJ, Lee GP. Identification of the subgenomic promoter of the coat protein gene of cucumber fruit mottle mosaic virus and development of a heterologous expression vector. Arch Virol. 2016;161(6):1527–38.

    CAS  Article  Google Scholar 

  36. 36.

    Schirawski J, Voyatzakis A, Zaccomer B, Bernardi F, Haenni AL. Identification and functional analysis of the turnip yellow mosaic tymovirus subgenomic promoter. J Virol. 2000;74(23):11073–80. https://doi.org/10.1128/jvi.74.23.11073-11080.2000.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Schneidman-Duhovny D, Inbar Y, Nussinov R, Wolfson HJ. PatchDock and SymmDock: servers for rigid and symmetric docking. Nucl Acids Res. 2005;33:W363–7.

    CAS  Article  Google Scholar 

  38. 38.

    Sethna PB, Hung SL, Brian DA. Coronavirus subgenomic minus-strand RNAs and the potential for mRNA replicons. Proc Natl Acad Sci. 1989;86(14):5626–30.

    CAS  Article  Google Scholar 

  39. 39.

    Sztuba-Solińska J, Stollar V, Bujarski JJ. Subgenomic messenger RNAs: mastering regulation of (+)-strand RNA virus life cycle. Virology. 2011;412(2):245–55.

    Article  Google Scholar 

  40. 40.

    Teoh PG, Ooi AS, AbuBakar S, Othman RY. Virus-specific read-through codon preference affects infectivity of chimeric cucumber green mottle mosaic viruses displaying a dengue virus epitope. J Biomed Biotechnol. 2009. https://doi.org/10.1155/2009/781712.

    Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22(22):4673–80. https://doi.org/10.1093/nar/22.22.4673.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Tran HH, Chen B, Chen H, Menassa R, Hao X, Bernards M, Hüner NP, Wang A. Development of a cucumber green mottle mosaic virus-based expression vector for the production in cucumber of neutralizing epitopes against a devastating animal virus. J Virol Methods. 2019;269:18–25.

    CAS  Article  Google Scholar 

  43. 43.

    Ugaki M, Tomiyama M, Kakutani T, Hidaka S, Kiguchi T, Nagata R, Sato T, Motoyoshi F, Nishiguchi M. The complete nucleotide sequence of cucumber green mottle mosaic virus (SH strain) genomic RNA. J Gen Virol. 1991;72(7):1487–95. https://doi.org/10.1099/0022-1317-72-7-1487.

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Venkataraman S, Prasad B, Selvarajan R. RNA dependent RNA polymerases: insights from structure, function and evolution. Viruses. 2018;10(2):76. https://doi.org/10.3390/v10020076.

    CAS  Article  PubMed Central  Google Scholar 

  45. 45.

    Watanabe Y, Meshi T, Okada Y. Infection of tobacco protoplasts with in vitro transcribed tobacco mosaic virus RNA using an improved electroporation method. FEBS Lett. 1987;219:65–9.

    CAS  Article  Google Scholar 

  46. 46.

    Wiederstein M, Sippl MJ. ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res. 2007;35(suppl_2):W407-10.

    PubMed  Google Scholar 

  47. 47.

    Wierzchoslawski R, Dzianott A, Bujarski J. Dissecting the requirement for subgenomic promoter sequences by RNA recombination of brome mosaic virus in vivo: evidence for functional separation of transcription and recombination. J Virol. 2004;78(16):8552–64.

    CAS  Article  Google Scholar 

  48. 48.

    Wierzchoslawski R, Urbanowicz A, Dzianott A, Figlerowicz M, Bujarski JJ. Characterization of a novel 5′ subgenomic RNA3a derived from RNA3 of Brome mosaic bromovirus. J Virol. 2006;80(24):12357–66.

    CAS  Article  Google Scholar 

  49. 49.

    Williams CJ, Headd JJ, Moriarty NW, Prisant MG, Videau LL, Deis LN, Verma V, Keedy DA, Hintze BJ, Chen VB, Jain S. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 2018;27(1):293–315.

    CAS  Article  Google Scholar 

  50. 50.

    Young ND, Zaitlin M. An analysis of tobacco mosaic virus replicative structures synthesized in vitro. Plant Mol Biol. 1986;6:455–65. https://doi.org/10.1007/BF00027137.

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Zheng H, Xiao C, Han K, Peng J, Lin L, Lu Y, Xie L, Wu X, Xu P, Li G, Chen J. Development of an agroinoculation system for full-length and GFP-tagged cDNA clones of cucumber green mottle mosaic virus. Arch Virol. 2015;160(11):2867–72. https://doi.org/10.1007/s00705-015-2584-y.

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Zok T, Antczak M, Zurkowski M, Popenda M, Blazewicz J, Adamiak RW, Szachniuk M. RNApdbee 20: multifunctional tool for RNA structure annotation. Nucleic Acids Res. 2018;46(W1):W30–5.

    CAS  Article  Google Scholar 

  53. 53.

    Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003;31(13):3406–15.

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The financial support as PhD scholarship to A. Chottopadhyay from the PG School, Indian Agricultural Research Institute, New Delhi is thankfully acknowledged.

Author information

Affiliations

Authors

Corresponding authors

Correspondence to Anirban Roy or Bikash Mandal.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chattopadhyay, A., Abdul Kader Jailani, A., Roy, A. et al. Prediction of putative regulatory elements in the subgenomic promoters of cucumber green mottle mosaic virus and their interactions with the RNA dependent RNA polymerase domain. VirusDis. 31, 503–516 (2020). https://doi.org/10.1007/s13337-020-00640-9

Download citation

Keywords

  • CGMMV
  • Tobamovirus
  • sgRNA promoters
  • Regulatory elements
  • RdRp
  • In-silico