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Abstract

ORF6 is a small gene that overlaps the movement and coat protein genes of subgroup 1a tobamoviruses. The ORF6 protein of tomato mosaic virus (ToMV) strain L (L-ORF6), interacts with eukaryotic elongation factor 1α, and mutation of the ORF6 gene of tobacco mosaic virus (TMV) strain U1 (U1-ORF6) reduces the pathogenicity of TMV, whereas expression of this gene from two other viruses, tobacco rattle virus (TRV) and potato virus X (PVX), increases their pathogenicity. In this work, the properties of the L-ORF6 and U1-ORF6 proteins were compared to identify sequences that direct the proteins to different subcellular locations and also influence virus pathogenicity. Site-specific mutations in the ORF6 protein were made, hybrid ORF6 proteins were created in which the N-terminal and C-terminal parts were derived from the two proteins, and different subregions of the protein were examined, using expression either from a recombinant TRV vector or as a yellow fluorescent protein fusion from a binary plasmid in . L-ORF6 caused mild necrotic symptoms in when expressed from TRV, whereas U1-ORF6 caused severe symptoms including death of the plant apex. The difference in symptoms was associated with the C-terminal region of L-ORF6, which directed the protein to the endoplasmic reticulum (ER), whereas U1-ORF6 was directed initially to the nucleolus and later to the mitochondria. Positively charged residues at the N terminus allowed nucleolar entry of both U1-ORF6 and L-ORF6, but hydrophobic residues at the C terminus of L-ORF6 directed this protein to the ER.

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2013-01-01
2024-10-12
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References

  1. Brigneti G., Voinnet O., Li W.-X., Ji L.-H., Ding S.-W., Baulcombe D. C. 1998; Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana . EMBO J 17:6739–6746 [View Article][PubMed]
    [Google Scholar]
  2. Canto T., MacFarlane S. A., Palukaitis P. 2004; ORF6 of Tobacco mosaic virus is a determinant of viral pathogenicity in Nicotiana benthamiana . J Gen Virol 85:3123–3133 [View Article][PubMed]
    [Google Scholar]
  3. Chen H., Wurm T., Britton P., Brooks G., Hiscox J. A. 2002; Interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell. J Virol 76:5233–5250 [View Article][PubMed]
    [Google Scholar]
  4. Csorba T., Pantaleo V., Burgyán J. 2009; RNA silencing: an antiviral mechanism. Adv Virus Res 75:35–71 [View Article][PubMed]
    [Google Scholar]
  5. Duan C.-G., Fang Y.-Y., Zhou B.-J., Zhao J.-H., Hou W.-N., Zhu H., Ding S.-W., Guo H.-S. 2012; Suppression of Arabidopsis ARGONAUTE1-mediated slicing, transgene-induced RNA silencing, and DNA methylation by distinct domains of the Cucumber mosaic virus 2b protein. Plant Cell 24:259–274 [View Article][PubMed]
    [Google Scholar]
  6. Fedorkin O. N., Denisenko O. N., Sitikov A. S., Zelenina D. A., Lukasheva L. I., Morozov S. Iu., Atabekov I. G. 1995; [A protein product of the tobamovirus open translation frame forms a stable complex with translation elongation factor eEF-1α]. Dokl Akad Nauk 343:703–704 (in Russian) [PubMed]
    [Google Scholar]
  7. González I., Martínez L., Rakitina D. V., Lewsey M. G., Atencio F. A., Llave C., Kalinina N. O., Carr J. P., Palukaitis P., Canto T. 2010; Cucumber mosaic virus 2b protein subcellular targets and interactions: their significance to RNA silencing suppressor activity. Mol Plant Microbe Interact 23:294–303 [View Article][PubMed]
    [Google Scholar]
  8. Goodin M. M., Chakrabarty R., Banerjee R., Yelton S., Debolt S. 2007; New gateways to discovery. Plant Physiol 145:1100–1109 [View Article][PubMed]
    [Google Scholar]
  9. Hammani K., Gobert A., Hleibieh K., Choulier L., Small I., Giegé P. 2011; An Arabidopsis dual-localized pentatricopeptide repeat protein interacts with nuclear proteins involved in gene expression regulation. Plant Cell 23:730–740 [View Article][PubMed]
    [Google Scholar]
  10. Han B., Izumi H., Yasuniwa Y., Akiyama M., Yamaguchi T., Fujimoto N., Matsumoto T., Wu B., Tanimoto A. other authors 2011; Human mitochondrial transcription factor A functions in both nuclei and mitochondria and regulates cancer cell growth. Biochem Biophys Res Commun 408:45–51 [View Article][PubMed]
    [Google Scholar]
  11. Hiscox J. A. 2007; RNA viruses: hijacking the dynamic nucleolus. Nat Rev Microbiol 5:119–127 [View Article][PubMed]
    [Google Scholar]
  12. Ikeda R., Watanabe E., Watanabe Y., Okada Y. 1993; Nucleotide sequence of tobamovirus Ob which can spread systemically in N gene tobacco. J Gen Virol 74:1939–1944 [View Article][PubMed]
    [Google Scholar]
  13. Kubota K., Tsuda S., Tamai A., Meshi T. 2003; Tomato mosaic virus replication protein suppresses virus-targeted posttranscriptional gene silencing. J Virol 77:11016–11026 [View Article][PubMed]
    [Google Scholar]
  14. Kyte J., Doolittle R. F. 1982; A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132 [View Article][PubMed]
    [Google Scholar]
  15. Liu Y., Schiff M., Marathe R., Dinesh-Kumar S. P. 2002; Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J 30:415–429 [View Article][PubMed]
    [Google Scholar]
  16. Lukhovitskaya N. I., Yelina N. E., Zamyatnin A. A. Jr, Schepetilnikov M. V., Solovyev A. G., Sandgren M., Morozov S. Y., Valkonen J. P. T., Savenkov E. I. 2005a; Expression, localization and effects on virulence of the cysteine-rich 8 kDa protein of Potato mop-top virus . J Gen Virol 86:2879–2889 [View Article][PubMed]
    [Google Scholar]
  17. Lukhovitskaya N., Solovyev A., Koshkina T., Zavriev S., Morozov S. 2005b; Interaction of the carlavirus cysteine-rich protein with the plant defense system. Mol Biol (Mosk) 39:785–791 [View Article]
    [Google Scholar]
  18. Lukhovitskaya N. I., Ignatovich I. V., Savenkov E. I., Schiemann J., Morozov S. Y., Solovyev A. G. 2009; Role of the zinc-finger and basic motifs of chrysanthemum virus B p12 protein in nucleic acid binding, protein localization and induction of a hypersensitive response upon expression from a viral vector. J Gen Virol 90:723–733 [View Article][PubMed]
    [Google Scholar]
  19. Martin K., Kopperud K., Chakrabarty R., Banerjee R., Brooks R., Goodin M. M. 2009; Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced definition of protein localization, movement and interactions in planta . Plant J 59:150–162 [View Article][PubMed]
    [Google Scholar]
  20. MacFarlane S. A., Popovich A. H. 2000; Efficient expression of foreign proteins in roots from tobravirus vectors. Virology 267:29–35 [CrossRef]
    [Google Scholar]
  21. Morozov S. Y., Denisenko O. N., Zelenina D. A., Fedorkin O. N., Solovyev A. G., Maiss E., Casper R., 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 [View Article][PubMed]
    [Google Scholar]
  22. Mueller A. M., Mooney A. L., MacFarlane S. A. 1997; Replication of in vitro tobravirus recombinants shows that the specificity of template recognition is determined by 5′ non-coding but not 3′ non-coding sequences. J Gen Virol 78:2085–2088[PubMed]
    [Google Scholar]
  23. Musinova Y. R., Lisitsyna O. M., Golyshev S. A., Tuzhikov A. I., Polyakov V. Y., Sheval E. V. 2011; Nucleolar localization/retention signal is responsible for transient accumulation of histone H2B in the nucleolus through electrostatic interactions. Biochim Biophys Acta 1813:27–38 [View Article][PubMed]
    [Google Scholar]
  24. Paape M., Solovyev A. G., Erokhina T. N., Minina E. A., Schepetilnikov M. V., Lesemann D.-E., Schiemann J., Morozov S. Y., Kellmann J.-W. 2006; At-4/1, an interactor of the Tomato spotted wilt virus movement protein, belongs to a new family of plant proteins capable of directed intra- and intercellular trafficking. Mol Plant Microbe Interact 19:874–883 [View Article][PubMed]
    [Google Scholar]
  25. Rajamäki M.-L., Valkonen J. P. T. 2009; Control of nuclear and nucleolar localization of nuclear inclusion protein a of picorna-like Potato virus A in Nicotiana species. Plant Cell 21:2485–2502 [View Article][PubMed]
    [Google Scholar]
  26. Raska I. 2003; Oldies but goldies: searching for Christmas trees within the nucleolar architecture. Trends Cell Biol 13:517–525 [View Article][PubMed]
    [Google Scholar]
  27. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  28. Shaw P., Brown J. 2012; Nucleoli: composition, function, and dynamics. Plant Physiol 158:44–51 [View Article][PubMed]
    [Google Scholar]
  29. Taliansky M. E., Brown J. W. S., Rajamäki M. L., Valkonen J. P. T., Kalinina N. O. 2010; Involvement of the plant nucleolus in virus and viroid infections: parallels with animal pathosystems. Adv Virus Res 77:119–158 [View Article][PubMed]
    [Google Scholar]
  30. Tatineni S., Robertson C. J., Garnsey S. M., Dawson W. O. 2011; A plant virus evolved by acquiring multiple nonconserved genes to extend its host range. Proc Natl Acad Sci U S A 108:17366–17371 [View Article][PubMed]
    [Google Scholar]
  31. Verwoerd T. C., Dekker B. M., Hoekema A. 1989; A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res 17:2362 [View Article][PubMed]
    [Google Scholar]
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