Archives of Virology

, Volume 159, Issue 3, pp 413–423 | Cite as

The N-terminal region containing the zinc finger domain of tobacco streak virus coat protein is essential for the formation of virus-like particles

  • Chhavi Mathur
  • Kalyani Mohan
  • T. R. Usha Rani
  • M. Krishna Reddy
  • Handanahal S. SavithriEmail author
Original Article


Tobacco streak virus (TSV), a member of the genus Ilarvirus (family Bromoviridae), has a tripartite genome and forms quasi-isometric virions. All three viral capsids, encapsidating RNA 1, RNA 2 or RNA 3 and subgenomic RNA 4, are constituted of a single species of coat protein (CP). Formation of virus-like particles (VLPs) could be observed when the TSV CP gene was cloned and the recombinant CP (rCP) was expressed in E. coli. TSV VLPs were found to be stabilized by Zn2+ ions and could be disassembled in the presence of 500 mM CaCl2. Mutational analysis corroborated previous studies that showed that an N-terminal arginine-rich motif was crucial for RNA binding; however, the results presented here demonstrate that the presence of RNA is not a prerequisite for assembly of TSV VLPs. Instead, the N-terminal region containing the zinc finger domain preceding the arginine-rich motif is essential for assembly of these VLPs.


Coat Protein Sucrose Density Gradient Brome Mosaic Virus Tobacco Streak Virus Cowpea Chlorotic Mottle Virus 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We thank Dr. S. S. Indi, Dept. of Microbiology and Cell Biology, IISc, for his assistance in electron microscopy. We thank Department of Biotechnology and Department of Science and Technology (J.C. Bose fellowship to HSS), New Delhi, India, for the financial support. CM thanks CSIR, New Delhi, for Junior and Senior Research fellowships. We thank Prof. M.R.N. Murthy for critical reading of the manuscript.

Supplementary material

705_2013_1822_MOESM1_ESM.tif (1.2 mb)
Supplementary material 1 (TIFF 1223 kb) Supplementary Table S1: List of primers used for cloning and RT-PCR
705_2013_1822_MOESM2_ESM.tif (613 kb)
Supplementary material 2 (TIFF 612 kb) Supplementary Fig. S1 Analysis of TSV CP (a) Mass spectrometric analysis of purified recombinant TSV CP (molecular sizes of monomer and dimer peaks are indicated). (b) Glutaraldehyde crosslinking of TSV CP. TSV coat protein (10 μg) was incubated with 0.01 % glutaraldehyde (Sigma) for 1 h in the dark. The samples were then separated by 10 % SDS-PAGE and stained with CBB R250. Lane 1, TSV CP; lane 2, glutaraldehyde-crosslinked TSV CP
705_2013_1822_MOESM3_ESM.tif (3.5 mb)
Supplementary material 3 (TIFF 3595 kb) Supplementary Fig. S2 Deletion mutants of TSV CP (a) Amino acid sequence of TSV CP indicating the positions where deletions were created. Underlined, zinc-finger domain; bold, RNA-binding N-ARM. (b) SDS-PAGE analysis of TSV CP and its deletion mutants. Lane 1, molecular mass (kDa) markers; lane 2, purified Ndel48 CP; lane 3, purified Ndel26 CP; lane 4, purified Ndel86 CP. (c) Lane 1, uninduced fraction – TSV CP; lane 2, induced fraction – TSV CP; lane 3, uninduced fraction –Cdel38 CP; lane 4, induced fraction – Cdel38 CP; lane 5, uninduced fraction – Cdel55 CP; lane 6, induced fraction – Cdel55 CP; lane 7, uninduced fraction – Cdel74 CP; lane 8, induced fraction – Cdel74 CP; lane 9, molecular mass (kDa) markers


  1. 1.
    Anindya R, Savithri HS (2003) Surface-exposed amino- and carboxy-terminal residues are crucial for the initiation of assembly in Pepper vein banding virus: a flexuous rod-shaped virus. Virology 316:325–336PubMedCrossRefGoogle Scholar
  2. 2.
    Ansel-McKinney P, Scott SW, Swanson M, Ge X, Gehrke L (1996) A plant viral coat protein RNA binding consensus sequence contains a crucial arginine. EMBO J 15:5077–5084PubMedCentralPubMedGoogle Scholar
  3. 3.
    Aparicio F, Vilar M, Perez-Paya E, Pallas V (2003) The coat protein of prunus necrotic ringspot virus specifically binds to and regulates the conformation of its genomic RNA. Virology 313:213–223PubMedCrossRefGoogle Scholar
  4. 4.
    Aparicio F, Sanchez-Navarro JA, Pallas V (2006) In vitro and in vivo mapping of the Prunus necrotic ringspot virus coat protein C-terminal dimerization domain by bimolecular fluorescence complementation. J Gen Virol 87:1745–1750PubMedCrossRefGoogle Scholar
  5. 5.
    Bol JF (1999) Alfalfa mosaic virus and ilarviruses: involvement of coat protein in multiple steps of the replication cycle. J Gen Virol 80(Pt 5):1089–1102PubMedGoogle Scholar
  6. 6.
    Bol JF (2005) Replication of alfamo- and ilarviruses: role of the coat protein. Annu Rev Phytopathol 43:39–62PubMedCrossRefGoogle Scholar
  7. 7.
    Chen C, Kao CC, Dragnea B (2008) Self-assembly of brome mosaic virus capsids: insights from shorter time-scale experiments. J Phys Chem A 112:9405–9412PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Clark MF, Lister RM (1971) Preparation and some properties of the nucleic acid of tobacco streak virus. Virology 45:61–74PubMedCrossRefGoogle Scholar
  9. 9.
    Cornelissen BJ, Janssen H, Zuidema D, Bol JF (1984) Complete nucleotide sequence of tobacco streak virus RNA 3. Nucleic Acids Res 12:2427–2437PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Ding SW, Li WX, Symons RH (1995) A novel naturally occurring hybrid gene encoded by a plant RNA virus facilitates long distance virus movement. EMBO J 14:5762–5772PubMedCentralPubMedGoogle Scholar
  11. 11.
    Driedonks RA, Krijgsman PC, Mellema JE (1977) Alfalfa mosaic virus protein polymerization. J Mol Biol 113:123–140PubMedCrossRefGoogle Scholar
  12. 12.
    Edwardson JR, Purcifull DE (1974) Relationship of Datura quercina and Tobacco streak viruses. Phytopathology 64:1322–1324CrossRefGoogle Scholar
  13. 13.
    Fulton RW (1967) Purification and some properties of tobacco streak and Tulare apple mosaic viruses. Virology 32:153–162PubMedCrossRefGoogle Scholar
  14. 14.
    Ge X, Scott SW, Zimmerman MT (1997) The complete sequence of the genomic RNAs of spinach latent virus. Arch Virol 142:1213–1226PubMedCrossRefGoogle Scholar
  15. 15.
    Hema M, Murali A, Ni P, Vaughan RC, Fujisaki K, Tsvetkova I, Dragnea B, Kao CC (2010) Effects of amino-acid substitutions in the Brome mosaic virus capsid protein on RNA encapsidation. Mol Plant Microbe Interact 23:1433–1447PubMedCrossRefGoogle Scholar
  16. 16.
    Johnson JM, Willits DA, Young MJ, Zlotnick A (2004) Interaction with capsid protein alters RNA structure and the pathway for in vitro assembly of cowpea chlorotic mottle virus. J Mol Biol 335:455–464PubMedCrossRefGoogle Scholar
  17. 17.
    Kumar A, Reddy VS, Yusibov V, Chipman PR, Hata Y, Fita I, Fukuyama K, Rossmann MG, Loesch-Fries LS, Baker TS, Johnson JE (1997) The structure of alfalfa mosaic virus capsid protein assembled as a T=1 icosahedral particle at 4.0-A resolution. J Virol 71:7911–7916PubMedCentralPubMedGoogle Scholar
  18. 18.
    Kurstak E (1981) Handbook of plant virus infections: comparative diagnosis. Elsevier/Noth Holland Biomedical Press, AmsterdamGoogle Scholar
  19. 19.
    Larson SB, Lucas RW, McPherson A (2005) Crystallographic structure of the T=1 particle of brome mosaic virus. J Mol Biol 346:815–831PubMedCrossRefGoogle Scholar
  20. 20.
    Lavelle L, Michel JP, Gingery M (2007) The disassembly, reassembly and stability of CCMV protein capsids. J Virol Methods 146:311–316PubMedCrossRefGoogle Scholar
  21. 21.
    Lister RM, Bancroft JB (1970) Alteration of Tobacco streak virus component ratio influenced by host and extraction procedure. Phytopathology 60:689–694CrossRefGoogle Scholar
  22. 22.
    Lister RM, Ghabrial SA, Saksena KN (1972) Evidence that particle size heterogeneity is the cause of centrifugal heterogeneity in tobacco streak virus. Virology 49:290–299PubMedCrossRefGoogle Scholar
  23. 23.
    Lokesh GL, Gowri TD, Satheshkumar PS, Murthy MR, Savithri HS (2002) A molecular switch in the capsid protein controls the particle polymorphism in an icosahedral virus. Virology 292:211–223PubMedCrossRefGoogle Scholar
  24. 24.
    Lucas RW, Larson SB, McPherson A (2002) The crystallographic structure of brome mosaic virus. J Mol Biol 317:95–108PubMedCrossRefGoogle Scholar
  25. 25.
    Pallas V, Sanchez-Navarro JA, Diez J (1999) In vitro evidence for RNA binding properties of the coat protein of prunus necrotic ringspot ilarvirus and their comparison to related and unrelated viruses. Arch Virol 144:797–803PubMedCrossRefGoogle Scholar
  26. 26.
    Phelps JP, Dao P, Jin H, Rasochova L (2007) Expression and self-assembly of cowpea chlorotic mottle virus-like particles in Pseudomonas fluorescens. J Biotechnol 128:290–296PubMedCrossRefGoogle Scholar
  27. 27.
    Prilusky J, Felder CE, Zeev-Ben-Mordehai T, Rydberg EH, Man O, Beckmann JS, Silman I, Sussman JL (2005) FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics 21:3435–3438PubMedCrossRefGoogle Scholar
  28. 28.
    Reddy AS, Prasad Rao RDVJ, Thirumala-Devi K, Reddy SV, Mayo MA, Roberts I, Satyanarayana T, Subramaniam K, Reddy DVR (2002) Occurrence of Tobacco streak virus on Peanut (Arachis hypogaea) in India. Plant Disease 86:173–178CrossRefGoogle Scholar
  29. 29.
    Sastri M, Kekuda R, Gopinath K, Kumar CT, Jagath JR, Savithri HS (1997) Assembly of physalis mottle virus capsid protein in Escherichia coli and the role of amino and carboxy termini in the formation of the icosahedral particles. J Mol Biol 272:541–552PubMedCrossRefGoogle Scholar
  30. 30.
    Satheshkumar PS, Lokesh GL, Sangita V, Saravanan V, Vijay CS, Murthy MR, Savithri HS (2004) Role of metal ion-mediated interactions in the assembly and stability of Sesbania mosaic virus T=3 and T=1 capsids. J Mol Biol 342:1001–1014PubMedCrossRefGoogle Scholar
  31. 31.
    Scott SW, Zimmerman MT, Ge X (1998) The sequence of RNA 1 and RNA 2 of tobacco streak virus: additional evidence for the inclusion of alfalfa mosaic virus in the genus Ilarvirus. Arch Virol 143:1187–1198PubMedCrossRefGoogle Scholar
  32. 32.
    Scott SW (2001) AAB Description of Plant Viruses. 381Google Scholar
  33. 33.
    Sdoodee R, Teakle DS (1993) Studies on the mechanism of transmission of pollen-associated tobacco streak ilarvirus virus by Thrips tabaci. Plant Pathol 42:88–92CrossRefGoogle Scholar
  34. 34.
    Sehnke PC, Mason AM, Hood SJ, Lister RM, Johnson JE (1989) A “zinc-finger”-type binding domain in tobacco streak virus coat protein. Virology 168:48–56PubMedCrossRefGoogle Scholar
  35. 35.
    Sehnke PC, Johnson JE (1993) Crystallization and preliminary X-ray characterization of tobacco streak virus and a proteolytically modified form of the capsid protein. Virology 196:328–331PubMedCrossRefGoogle Scholar
  36. 36.
    Sehnke PC, Johnson JE (1994) A chromatographic analysis of capsid protein isolated from alfalfa mosaic virus: zinc binding and proteolysis cause distinct charge heterogeneity. Virology 204:843–846PubMedCrossRefGoogle Scholar
  37. 37.
    Speir JA, Munshi S, Wang G, Baker TS, Johnson JE (1995) Structures of the native and swollen forms of cowpea chlorotic mottle virus determined by X-ray crystallography and cryo-electron microscopy. Structure 3:63–78PubMedCrossRefGoogle Scholar
  38. 38.
    Swanson MM, Ansel-McKinney P, Houser-Scott F, Yusibov V, Loesch-Fries LS, Gehrke L (1998) Viral coat protein peptides with limited sequence homology bind similar domains of alfalfa mosaic virus and tobacco streak virus RNAs. J Virol 72:3227–3234PubMedCentralPubMedGoogle Scholar
  39. 39.
    Tama F, Brooks CL 3rd (2002) The mechanism and pathway of pH induced swelling in cowpea chlorotic mottle virus. J Mol Biol 318:733–747PubMedCrossRefGoogle Scholar
  40. 40.
    Tang J, Johnson JM, Dryden KA, Young MJ, Zlotnick A, Johnson JE (2006) The role of subunit hinges and molecular “switches” in the control of viral capsid polymorphism. J Struct Biol 154:59–67PubMedCrossRefGoogle Scholar
  41. 41.
    Terribilini M, Sander JD, Lee JH, Zaback P, Jernigan RL, Honavar V, Dobbs D (2007) RNABindR: a server for analyzing and predicting RNA-binding sites in proteins. Nucleic Acids Res 35:W578–W584PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    van Vloten-doting L (1975) Coat protein is required for infectivity of tobacco streak virus: Biological equivalence of the coat proteins of tobacco streak and alfalfa mosaic viruses. Virology 65:215–225PubMedCrossRefGoogle Scholar
  43. 43.
    Xin HW, Ji LH, Scott SW, Symons RH, Ding SW (1998) Ilarviruses encode a Cucumovirus-like 2b gene that is absent in other genera within the Bromoviridae. J Virol 72:6956–6959PubMedCentralPubMedGoogle Scholar
  44. 44.
    Yusibov V, Kumar A, North A, Johnson JE, Loesch-Fries LS (1996) Purification, characterization, assembly and crystallization of assembled alfalfa mosaic virus coat protein expressed in Escherichia coli. J Gen Virol 77(Pt 4):567–573PubMedCrossRefGoogle Scholar
  45. 45.
    Zlotnick A, Aldrich R, Johnson JM, Ceres P, Young MJ (2000) Mechanism of capsid assembly for an icosahedral plant virus. Virology 277:450–456PubMedCrossRefGoogle Scholar
  46. 46.
    Zlotnick A (2003) Are weak protein-protein interactions the general rule in capsid assembly? Virology 315:269–274PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2013

Authors and Affiliations

  • Chhavi Mathur
    • 1
  • Kalyani Mohan
    • 1
  • T. R. Usha Rani
    • 2
  • M. Krishna Reddy
    • 2
  • Handanahal S. Savithri
    • 1
    Email author
  1. 1.Department of BiochemistryIndian Institute of ScienceBangaloreIndia
  2. 2.Division of Plant PathologyIndian Institute of HorticultureBangaloreIndia

Personalised recommendations