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Characterization of the functional domains of nuclear shuttle protein (NSP) of Indian cassava mosaic virus using green fluorescent protein as reporter

Abstract

Indian cassava mosaic virus (ICMV), responsible for the cassava mosaic disease in India, harbours two circular genomic components, DNA-A and DNA-B; the former being responsible for the encapsidation and replication and the latter for intra- and inter-cellular movement of the viral DNA. Two proteins, encoded by DNA-B, the movement protein (MP) and the nuclear shuttle protein (NSP), act in concert on the newly replicated viral DNA to move it from the nucleus to the cell periphery. To map the functional domains of NSP, the intra-cellular localization of its full-length protein and deletion derivatives was studied in the epidermal cells of detached leaves of the laboratory host plant, Nicotiana benthamiana, where the target proteins were transiently expressed as GFP fusions. This analysis revealed domains for nuclear localization at the N-terminus, as well as for localization towards the cell periphery both at the C-terminus and center of the NSP.

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References

  1. Patil BL, Fauquet CM (2009) Cassava mosaic geminiviruses: actual knowledge and perspectives. Mol Plant Pathol 10:685–701

    CAS  Article  Google Scholar 

  2. Rothenstein D, Briddon RW, Haible D, Stanley J, Frischmuth T, Jeske H (2005) Biolistic infection of cassava using cloned components of Indian cassava mosaic virus. Arch Virol 150:1669–1675

    CAS  Article  Google Scholar 

  3. Rothenstein D, Haible D, Dasgupta I, Dutt N, Patil BL, Jeske H (2005) Biodiversity and recombination of cassava-infecting begomoviruses from southern India. Arch Virol 150(8):1669–1675. https://doi.org/10.17007/s00705-005-0520-2

    CAS  Article  PubMed  Google Scholar 

  4. Patil BL, Rajasubramaniam S, Bagchi C, Dasgupta I (2005) Both Indian cassava mosaic virus and Sri Lankan cassava mosaic virus are found in India and exhibit high variability as assessed by PCR-RFLP. Arch Virol 150(2):389–397.  https://doi.org/10.1007/s00705-004-0399-3

    CAS  Article  PubMed  Google Scholar 

  5. Jeske H (2009) Geminiviruses. Curr Top Microbiol Immunol 331:185–226. https://doi.org/10.1007/978-3-540-70972-5_11

    CAS  Article  PubMed  Google Scholar 

  6. Böttcher B, Unseld S, Ceulemans H, Russell RB, Jeske H (2004) Geminate structures of African cassava mosaic virus. J Virol 78:6709–6714

    Article  Google Scholar 

  7. Aberle HJ, Rütz ML, Karayavuz M, Frischmuth S, Wege C, Hülser D, Jeske H (2002) Localizing BC1 movement protein of Abutilon mosaic geminivirus in yeasts by subcellular fractionation and freeze fracture immunolabelling. Arch Virol 147:103–107

    CAS  Article  Google Scholar 

  8. Noueiry AO, Lucas WJ, Gilbertson RL (1994) Two proteins of a plant DNA virus coordinate nuclear and plasmodesmal transport. Cell 76:925–932

    CAS  Article  Google Scholar 

  9. Pascal E, Sanderfoot AA, Ward BM, Medville R, Turgeon R, Lazarowitz SG (1994) The geminivirus BR1 movement protein binds single- stranded DNA and localizes to the cell nucleus. Plant Cell 6:995–1006

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Sanderfoot AA, Ingham DJ, Lazarowitz SG (1996) A viral movement protein as a nuclear shuttle. The geminivirus BR1 movement protein contains domains essential for interaction with BL1 and nuclear localization. Plant Physiol 110:23–33

    CAS  Article  Google Scholar 

  11. Happle A, Jeske H, Kleinow T (2021) Dynamic subcellular distribution of begomoviral nuclear shuttle and movement proteins. Virology 562:158–175. https://doi.org/10.1016/j.virol.2021.07.014

    CAS  Article  PubMed  Google Scholar 

  12. Sanderfoot AA, Lazarowitz SG (1995) Cooperation in viral movement: the geminivirus BL1 movement protein interacts with BR1 and redirects it from the nucleus to the cell periphery. Plant Cell 7:1185–1194

    CAS  Article  Google Scholar 

  13. Ward BM, Lazarowitz SG (1999) Nuclear export in plants. Use of geminivirus movement proteins for a cell-based export assay. Plant Cell 11:1267–1276

    CAS  Article  Google Scholar 

  14. Zhang SC, Ghosh R, Jeske H (2001) Subcellular targeting domains of Abutilon mosaic geminivirus movement protein BC1. Arch Virol 147:2349–2363

    Article  Google Scholar 

  15. Zhang SC, Wege C, Jeske H (2001) Movement proteins of Abutilon mosaic geminivirus are cotransported in and between cells of sink but not of the source leaves as detected by green fluorescent protein tagging. Virology 290:249–260

    CAS  Article  Google Scholar 

  16. Ward BM, Lazarowitz SG (1999) Nuclear export in plants: use of geminivirus movement proteins for a cell-based export assay. Plant Cell 11:1267–1276

    CAS  Article  Google Scholar 

  17. Zhang SC, Ghosh R, Jeske H (2002) Subcellular targeting domains of Abutilon mosaic geminivirus movement protein BC1. Arch Virol 147:2349–2363

    CAS  Article  Google Scholar 

  18. Zhang SC, Wege C, Jeske H (2001) Movement proteins (BC1 and BV1) of Abutilon mosaic geminivirus are cotransported in and between cells of sink but not of source leaves as detected by green fluorescent protein tagging. Virology 290:249–260

    CAS  Article  Google Scholar 

  19. Kalderon D, Roberts BL, Richardson WD, Smith AE (1984) A short amino acid sequence able to specify nuclear localization. Cell 39:499–509

    CAS  Article  Google Scholar 

  20. Robbins J, Dilworth SM, Laskey RA, Digwall C (1991) Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell 64:615–623

    CAS  Article  Google Scholar 

  21. Lazarowitz SG, Beachy RN (1999) Viral movement proteins as probes for intracellular and intercellular trafficking in plants. Plant Cell 11:535–548

    CAS  Article  Google Scholar 

  22. Lazarowitz SG (1999) Probing plant cell structure and function with viral movement proteins. Curr Opin Plant Biol 2:332–338

    CAS  Article  Google Scholar 

  23. Unseld S, Höhnle M, Ringel M, Frischmuth T (2001) Subcellular targeting of the coat protein of African cassava mosaic geminivirus. Virology 286:373–383

    CAS  Article  Google Scholar 

  24. Kunik T, Palanichelvam K, Czosnek H, Citovsky V, Gafni Y (1998) Nuclear import of the capsid protein of tomato yellow leaf curl virus (TYLCV) in plant and insect cells. Plant J 13(3):393–399

    CAS  Article  Google Scholar 

  25. Unseld S, Frischmuth T, Jeske H (2004) Short deletions in nuclear targeting sequences of African cassava mosaic virus coat protein prevent geminivirus twinned particle formation. Virology 318:89–100

    Article  Google Scholar 

  26. Kikuno R, Toh H, Hayashida H, Miyata T (1984) Sequence similaritiy between putative gene products of geminiviral DNAs. Nature 308:562

    CAS  Article  Google Scholar 

  27. Qin SW, Ward BM, Lazarowitz SG (1998) The bipartite geminivirus coat protein aids BR1 function in viral movement by affecting the accumulation of viral single-stranded DNA. J Virol 72:9247–9256

    CAS  Article  Google Scholar 

  28. Saunders K, Salim N, Mali VR, Malathi VG, Briddon R, Markham PG, Stanley J (2002) Characterisation of Sri Lankan cassava mosaic virus and Indian cassava mosaic virus: evidence for acquisition of a DNA B component by a monopartite begomovirus. Virology 293:63–74

    CAS  Article  Google Scholar 

  29. Haseloff J, Siemering KR, Prasher DC, Hodge S (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci USA 94:2122–2127

    CAS  Article  Google Scholar 

  30. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Press, Cold Spring Harbor

    Google Scholar 

  31. Martins LGC, Raimundo GAS, Ribeiro NGA, Silva JCF, Euclydes NC, Loriato VAP, Duarte CEM, Fontes EPB (2020) A begomovirus nuclear shuttle protein-interacting immune hub: hijacking host transport activities and suppressing incompatible functions. Front Plant Sci 11:398. https://doi.org/10.3389/fpls.2020.00398

    Article  PubMed  PubMed Central  Google Scholar 

  32. Mariano AC, Andrade MO, Santos AA, Carolino SM, Oliveira ML, Baracat-Pereira MC, Brommonshenkel SH, Fontes EP (2004) Identification of a novel receptor-like protein kinase that interacts with a geminivirus nuclear shuttle protein. Virology 318:24–31

    CAS  Article  Google Scholar 

  33. Rojas MR, Noueiry AO, Lucas WJ, Gilbertson RL (1998) Bean dwarf mosaic geminivirus movement proteins recognize DNA in a form-and size- specific manner. Cell 95:105–113

    CAS  Article  Google Scholar 

  34. Whittaker GR, Helenius A (1998) Nuclear import and export of viruses and virus genomes. Virology 246:1–23

    CAS  Article  Google Scholar 

  35. Fontes EP, Santos AA, Luz DF, Waclawovsky AJ, Chory J (2004) The geminivirus nuclear shuttle protein is a virulence factor that suppresses transmembrane receptor kinase activity. Genes Dev 18:2545–2556

    CAS  Article  Google Scholar 

  36. Krapp S, Greiner E, Amin B, Sonnewald U, Krenz B (2017) The stress granule component G3BP is a novel interaction partner for the nuclear shuttle proteins of the nanovirus pea necrotic yellow dwarf virus and geminivirus abutilon mosaic virus. Virus Res 227:6–14. https://doi.org/10.1016/j.virusres.2016.09.021

    CAS  Article  PubMed  Google Scholar 

  37. Ward BM, Medville R, Lazarowitz SG, Turgeon R (1997) The geminivirus BL1 movement protein is associated with endoplasmic reticulum–derived tubules in developing phloem cells. J Virol 71:3726–3733

    CAS  Article  Google Scholar 

  38. Maggioni C, Braakman I (2005) Synthesis and quality control of viral membrane proteins. Curr Top Microbiol Immunol 285:175–198

    CAS  PubMed  Google Scholar 

  39. Boutin JA (1997) Myristoylation. Cell Signal 9:15–35

    CAS  Article  Google Scholar 

  40. Mei Y, Wang Y, Hu T, Yang X, Lozano-Duran R, Sunter G, Zhou X (2018) Nucleocytoplasmic shuttling of geminivirus C4 protein mediated by phosphorylation and myristoylation is critical for viral pathogenicity. Mol Plant 11:1466–1481

    CAS  Article  Google Scholar 

  41. Kopito RR (2000) Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol 10:524–530

    CAS  Article  Google Scholar 

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Acknowledgements

The facilities provided at University of Stuttgart by Prof. Holger Jeske for part of this work are gratefully acknowledged. The study was funded by EU INCO-DEV Grant ICA-CT-2000-30001. BLP acknowledges Research Fellowship from Council for Scientific and Industrial Research, New Delhi, the DAAD Scholarship and the financial support of Vater-ünd-Sohn-Eiselen-Stiftung. Help in photography from Frau Sigrid Kober is gratefully acknowledged.

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ID and BLP conceived and designed the study, BLP performed the experiments and drafted the manuscript, ID supervised the study and corrected the manuscript.

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Correspondence to Indranil Dasgupta.

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This research did not involve any animal and/or human participants.

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Patil, B.L., Dasgupta, I. Characterization of the functional domains of nuclear shuttle protein (NSP) of Indian cassava mosaic virus using green fluorescent protein as reporter. Virus Genes 58, 308–318 (2022). https://doi.org/10.1007/s11262-022-01909-5

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  • DOI: https://doi.org/10.1007/s11262-022-01909-5

Keywords

  • Nuclear shuttle protein (NSP)
  • Nuclear localization signal
  • Geminivirus
  • Indian cassava mosaic virus (ICMV)