Biochemistry (Moscow)

, Volume 82, Issue 1, pp 60–66 | Cite as

Subcellular localization and detection of Tobacco mosaic virus ORF6 protein by immunoelectron microscopy

  • T. N. ErokhinaEmail author
  • E. A. Lazareva
  • K. R. Richert-Pöggeler
  • E. V. Sheval
  • A. G. Solovyev
  • S. Y. Morozov


Members of the genus Tobamovirus represent one of the best-characterized groups of plant positive, single stranded RNA viruses. Previous studies have shown that genomes of some tobamoviruses contain not only genes coding for coat protein, movement protein, and the cistron coding for different domains of RNA-polymerase, but also a gene, named ORF6, coding for a poorly conserved small protein. The amino acid sequences of ORF6 proteins encoded by different tobamoviruses are highly divergent. The potential role of ORF6 proteins in replication of tobamoviruses still needs to be elucidated. In this study, using biochemical and immunological methods, we have shown that ORF6 peptide is accumulated after infection in case of two isolates of Tobacco mosaic virus strain U1 (TMV-U1 common and TMV-U1 isolate A15). Unlike virus particles accumulating in the cytoplasm, the product of the ORF6 gene is found mainly in nuclei, which correlates with previously published data about transient expression of ORF6 isolated from TMV-U1. Moreover, we present new data showing the presence of ORF6 genes in genomes of several tobamoviruses. For example, in the genomes of other members of the tobamovirus subgroup 1, including Rehmannia mosaic virus, Paprika mild mottle virus, Tobacco mild green mosaic virus, Tomato mosaic virus, Tomato mottle mosaic virus, and Nigerian tobacco latent virus, sequence comparisons revealed the existence of a similar open reading frame like ORF6 of TMV.


Tobacco mosaic virus ORF6 protein overlapping genes subcellular localization monoclonal antibodies immunoelectron microscopy 



Argonaute (enzyme)


sodium cacodylate buffer


coat protein


monoclonal antibodies


movement protein


open reading frame 6


Tris-buffered saline containing Tween 20


Tobacco mosaic virus


Tomato mosaic virus

VSR (proteins)

viral suppressors of silencing


yellow fluorescent protein


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Pumplin, N., and Voinnet, O. (2013) RNA silencing suppression by plant pathogens: defense, counter-defense and counter-counter-defense, Nat. Rev. Microbiol., 11, 745–760.CrossRefPubMedGoogle Scholar
  2. 2.
    Mandadi, K. K., and Scholthof, K. B. (2013) Plant immune responses against viruses: how does a virus cause disease? Plant Cell, 25, 1489–1505.Google Scholar
  3. 3.
    Kachroo, A., and Robin, G. P. (2013) Systemic signaling during plant defense, Curr. Opin. Plant Biol., 16, 527–533.CrossRefPubMedGoogle Scholar
  4. 4.
    Gao, Q.-M., Zhu, S., Kachroo, P., and Kachroo, A. (2015) Signal regulators of systemic acquired resistance, Front. Plant Sci., 6, doi: 10.3389/fpls.2015.00228.Google Scholar
  5. 5.
    Callis, J. (2014) The ubiquitination machinery of the ubiquitin system, Arabidopsis Book, 6, doi: 10.1199/tab. 0174.Google Scholar
  6. 6.
    Ziebel, H. (2016) Plant defense and viral interference, in Plant Virus Interactions (Kleinow, T., ed.) Springer Cham Heidelberg, New YorkDordrecht-London.Google Scholar
  7. 7.
    Pooggin, M. M. (2016) Role of small RNAs in virus–host interaction, in Plant Virus Interactions (Kleinow, T., ed.) Springer Cham Heidelberg, New York-Dordrecht-London.Google Scholar
  8. 8.
    Incarbone, M., and Dunoyer, P. (2013) RNA silencing and its suppression: novel insights from in planta analyses, Trends Plant Sci., 18, 382–392.CrossRefPubMedGoogle Scholar
  9. 9.
    Csorba, T., Kontra, L., and Burgyan, J. (2015) Viral silencing suppressors: tools forged to fine-tune host–pathogen coexistence, Virology, 479/480, 85–103.CrossRefGoogle Scholar
  10. 10.
    Sanfacon, H. (2015) Plant translation factors and virus resistance, Viruses, 7, 867–878.CrossRefGoogle Scholar
  11. 11.
    Gibbs, A. (1999) Evolution and origins of tobamoviruses, Philos. Trans. R Soc. Lond. B Biol. Sci., 354, 593–602.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Stobbe, A. H., Melcher, U., Palmer, M. W., Roossinck, M. J., and Shen, G. (2012) Co-divergence and host-switching in the evolution of tobamoviruses, J. Gen. Virol., 93, 408–418.CrossRefPubMedGoogle Scholar
  13. 13.
    Morozov, S. Yu., Denisenko, O. N., Zelenina, D. A., Fedorkin, O. N., Solovyev, A. G., Maiss, E., Casper, R., and Atabekov, J. G. (1993) A novel open reading frame in tobacco mosaic virus genome coding for a putative small, positively charged protein, Biochimie, 75, 659–665.PubMedGoogle Scholar
  14. 14.
    Canto, T., MacFarlane, S. A., and Palukaitis, P. (2004) ORF6 of tobacco mosaic virus is a determinant of viral pathogenicity in Nicotiana benthamiana, J. Gen. Virol., 85, 3123–3133.CrossRefPubMedGoogle Scholar
  15. 15.
    Gushchin, V. A., Lukhovitskaya, N. I., Andreev, D. E., Wright, K. M., Taliansky, M. E., Solovyev, A. G., Morozov, S. Y., and MacFarlane, S. A. (2013) Dynamic localization of two tobamovirus ORF6 proteins involves distinct organellar compartments, J. Gen. Virol., 94, 230–240.Google Scholar
  16. 16.
    Tatineni, S., Robertson, C. J., Garnsey, S. M., and Dawson, W. O. (2011) A plant virus evolved by acquiring multiple nonconserved genes to extend its host range, Proc. Natl. Acad. Sci. USA, 108, 17366–17371.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Roossinck, M. J., Martin, D. P., and Roumagnac, P. (2015) Plant virus metagenomics: advances in virus discovery, Phytopathology, 105, 716–727.CrossRefPubMedGoogle Scholar
  18. 18.
    Yelina, N. E., Erokhina, T. N., Lukhovitskaya, N. I., Minina, E. A., Schepetilnikov, M. V., Lesemann, D.-E., Schiemann, J., Solovyev, A. G., and Morozov, S. Yu. (2005) Localization of Poa semilatent virus cysteine-rich protein in peroxisomes is dispensable for its ability to suppress RNA silencing, J. Gen. Virol., 86, 479–489.CrossRefPubMedGoogle Scholar
  19. 19.
    Erokhina, T. N., Vitushkina, M. V., Zinovkin, R. A., Lesemann, D.-E., Jelkmann, W., Koonin, E. V., and Agranovsky, A. A. (2001) Ultrastructural localization and epitope mapping of the methyltransferase-like and helicase-like proteins of beet yellows virus, J. Gen. Virol., 82, 1983–1994.CrossRefPubMedGoogle Scholar
  20. 20.
    Firth, A. E. (2014) Mapping overlapping functional elements embedded within the protein-coding regions of RNA viruses, Nucleic Acids Res., 42, 12425–12439.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Heinze, C., Lesemann, D.-E., Ilmberger, N., Willingmann, P., and Adam, G. (2006) The phylogenetic structure of the cluster of tobamovirus species serologically related to ribgrass mosaic virus (RMV) and the sequence of Streptocarpus flower break virus (SFBV), Arch. Virol., 151, 763–774.CrossRefPubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2017

Authors and Affiliations

  • T. N. Erokhina
    • 1
    Email author
  • E. A. Lazareva
    • 2
  • K. R. Richert-Pöggeler
    • 3
  • E. V. Sheval
    • 2
  • A. G. Solovyev
    • 2
  • S. Y. Morozov
    • 2
  1. 1.Shemyakin–Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussia
  2. 2.Belozersky Institute of Physico-Chemical BiologyLomonosov Moscow State UniversityMoscowRussia
  3. 3.Julius-Kühn-InstitutInstitute for Epidemiology and Pathogen DiagnosticsBraunschweigGermany

Personalised recommendations