The biological and molecular properties of a novel satellite RNA (satRNA L) associated with tomato bushy stunt virus (TBSV) are described. satRNA L consisted of a linear single-stranded RNA of 615 nt, lacked significant open reading frames (ORFs) and had no sequence identity with the helper genome other than in the 5′-proximal 7 nt and in a central region that is also conserved in all tombusvirus genomic, defective interfering and satellite RNAs. Secondary-structure analysis showed the presence of high-order domains similar to those described for other tombusvirus RNAs. Shorter-than-unit-length molecules were shown not to be related to a silencing mechanism. satRNA L did not modify the symptoms induced by TBSV under any of the temperature conditions tested. A full-length cDNA clone was constructed and used in co-inoculations with transcripts of carnation Italian ringspot virus (CIRV) and cymbidium ringspot virus (CymRSV). CIRV, but not CymRSV, supported the replication of satRNA L. Using CIRV–CymRSV hybrid infectious clones, two regions were identified as possible determinants of the different ability to support satRNA L replication. The first region was in the 5′-untranslated region, which folds differently in CymRSV in comparison with CIRV and TBSV; the second region was in the ORF1-encoded protein where a more efficient satRNA L-binding domain is suggested to be present in CIRV.


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  1. Altschul, S. F., Stephen, F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J.(1990). Basic local alignment search tool. J Mol Biol 215, 403–410.[CrossRef] [Google Scholar]
  2. Burgyán, J. & Russo, M.(1998). Tombusvirus isolation and RNA extraction. Methods Mol Biol 81, 225–230. [Google Scholar]
  3. Burgyán, J., Rubino, L. & Russo, M.(1996). The 5′-terminal region of a tombusvirus genome determines the origin of multivesicular bodies. J Gen Virol 77, 1967–1974.[CrossRef] [Google Scholar]
  4. Célix, A., Rodríguez-Cerezo, E. & García-Arenal, F.(1997). New satellite RNAs, but no DI RNAs, are found in natural populations of tomato bushy stunt tombusvirus. Virology 239, 277–284.[CrossRef] [Google Scholar]
  5. Célix, A., Burgyán, J. & Rodríguez-Cerezo, E.(1999). Interactions between tombusviruses and satellite RNAs of tomato bushy stunt virus: a defect in satRNA B1 replication maps to ORF1 of a helper virus. Virology 262, 129–138.[CrossRef] [Google Scholar]
  6. Chernysheva, O. A. & White, K. A.(2005). Modular arrangement of viral cis-acting RNA domains in a tombusvirus satellite RNA. Virology 332, 640–649.[CrossRef] [Google Scholar]
  7. Csorba, T., Pantaleo, V. & Burgyán, J.(2009). RNA silencing: an antiviral mechanism. Adv Virus Res 75, 35–71. [Google Scholar]
  8. Dalmay, T. & Rubino, L.(1994). The nature of multimeric forms of cymbidium ringspot tombusvirus satellite RNA. Arch Virol 138, 161–167.[CrossRef] [Google Scholar]
  9. Dalmay, T., Burgyán, J. & Russo, M.(1993a). Repair in vivo of altered 3′ terminus of cymbidium ringspot tombusvirus RNA. Virology 192, 551–555.[CrossRef] [Google Scholar]
  10. Dalmay, T., Rubino, L., Burgyán, J., Kollár, A. & Russo, M.(1993b). Functional analysis of cymbidium ringspot virus genome. Virology 194, 697–704.[CrossRef] [Google Scholar]
  11. Ding, S. W. & Voinnet, O.(2007). Antiviral immunity directed by small RNAs. Cell 130, 413–426.[CrossRef] [Google Scholar]
  12. Fabian, M. R., Na, H., Ray, D. & White, K. A.(2003). 3′-Terminal RNA secondary structures are important for accumulation of tomato bushy stunt virus DI RNAs. Virology 313, 567–580.[CrossRef] [Google Scholar]
  13. Galetzka, D., Russo, M., Rubino, L. & Krczal, G.(2000). Molecular characterization of a tombusvirus associated with a disease of statice [Goniolimon tataricum (L.) Boiss]. J Plant Pathol 82, 151–155. [Google Scholar]
  14. Gallitelli, D. & Hull, R.(1985). Characterization of satellite RNAs associated with tomato bushy stunt virus and five other definitive tombusviruses. J Gen Virol 66, 1533–1543.[CrossRef] [Google Scholar]
  15. Havelda, Z. & Burgyán, J.(1995). 3′ Terminal putative stem–loop structure required for the accumulation of cymbidium ringspot viral RNA. Virology 214, 269–272.[CrossRef] [Google Scholar]
  16. Hearne, P. Q., Knorr, D. A., Hillman, B. I. & Morris, T. J.(1990). The complete genome structure and synthesis of infectious RNA from clones of tomato bushy stunt virus. Virology 177, 141–151.[CrossRef] [Google Scholar]
  17. Hwang, Y. T., McCartney, A. W., Gidda, S. K. & Mullen, R. T.(2008). Localization of the carnation Italian ringspot virus replication protein p36 to the mitochondrial outer membrane is mediated by an internal targeting signal and the TOM complex. BMC Cell Biol 9, 54[CrossRef] [Google Scholar]
  18. Jones, R. W., Jackson, A. O. & Morris, T. J.(1990). Defective-interfering RNAs and elevated temperatures inhibit replication of tomato bushy stunt virus in inoculated protoplasts. Virology 176, 539–545.[CrossRef] [Google Scholar]
  19. Kollar, A. & Burgyán, J.(1994). Evidence that ORF 1 and 2 are the only virus-encoded replicase genes of cymbidium ringspot tombusvirus. Virology 201, 169–172.[CrossRef] [Google Scholar]
  20. Marck, C.(1988). ‘DNA Strider’: a ‘C’ program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res 16, 1829–1836.[CrossRef] [Google Scholar]
  21. Martelli, G. P. & Russo, M.(1984). Use of thin sectioning for visualization and identification of plant viruses. J Virol Methods 8, 143–224. [Google Scholar]
  22. Martelli, G. P., Gallitelli, D. & Russo, M.(1988). Tombusviruses. In The Plant Viruses. Polyhedral Virions with Monopartite RNA Genomes, pp. 13–72. Edited by R. Koenig. New York: Plenum Press.
  23. Mathews, D. H., Sabina, J., Zuker, M. & Turner, D. H.(1999). Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol 288, 911–940.[CrossRef] [Google Scholar]
  24. Militão, V., Moreno, I., Rodríguez-Cerezo, E. & García-Arenal, F.(1998). Differential interactions among isolates of peanut stunt cucumovirus and its satellite RNA. J Gen Virol 79, 177–184. [Google Scholar]
  25. Panavas, T., Pogany, J. & Nagy, P. D.(2002). Analysis of minimal promoter sequences for plus-strand synthesis by the Cucumber necrosis virus RNA-dependent RNA polymerase. Virology 296, 263–274.[CrossRef] [Google Scholar]
  26. Pantaleo, V. & Burgyán, J.(2008). Cymbidium ringspot virus harnesses RNA silencing to control the accumulation of virus parasite satellite RNA. J Virol 82, 11851–11858.[CrossRef] [Google Scholar]
  27. Pantaleo, V., Rubino, L. & Russo, M.(2003). Replication of Carnation Italian ringspot virus defective interfering RNA in Saccharomyces cerevisiae. J Virol 77, 2116–2123.[CrossRef] [Google Scholar]
  28. Pogany, J., White, K. A. & Nagy, P. D.(2005). Specific binding of tombusvirus replication protein p33 to an internal replication element in the viral RNA is essential for replication. J Virol 79, 4859–4869.[CrossRef] [Google Scholar]
  29. Rajendran, K. S. & Nagy, P. D.(2003). Characterization of the RNA-binding domains in the replicase proteins of tomato bushy stunt virus. J Virol 77, 9244–9258.[CrossRef] [Google Scholar]
  30. Ray, D., Wu, B. & White, P. D.(2003). A second functional RNA domain in the 5′ UTR of the Tomato bushy stunt virus genome intra- and interdomain interactions mediate viral RNA replication. RNA 9, 1232–1245.[CrossRef] [Google Scholar]
  31. Rice, P., Longden, I. & Bleasby, A.(2000).emboss: the European molecular biology open software suite. Trends Genet 16, 276–277.[CrossRef] [Google Scholar]
  32. Roossinck, M. J., Sleat, D. & Palukaitis, P.(1992). Satellite RNAs of plant viruses: structures and biological effects. Microbiol Rev 56, 265–279. [Google Scholar]
  33. Rubino, L. & Russo, M.(1995). Characterization of resistance to cymbidium ringspot virus in transgenic plants expressing a full-length viral replicase gene. Virology 212, 240–243.[CrossRef] [Google Scholar]
  34. Rubino, L., Burgyán, J., Grieco, F. & Russo, M.(1990). Sequence analysis of cymbidium ringspot virus satellite and defective interfering RNAs. J Gen Virol 71, 1655–1660.[CrossRef] [Google Scholar]
  35. Rubino, L., Carrington, J. C. & Russo, M.(1992). Biologically active cymbidium ringspot virus satellite RNA in transgenic plants suppresses accumulation of DI RNAs. Virology 188, 429–437.[CrossRef] [Google Scholar]
  36. Rubino, L., Burgyán, J. & Russo, M.(1995). Molecular cloning and complete nucleotide sequence of carnation Italian ringspot tombusvirus genomic and defective interfering RNAs. Arch Virol 140, 2027–2039.[CrossRef] [Google Scholar]
  37. Rubino, L., Weber-Lotfi, F., Dietrich, A., Stussi-Garaud, C. & Russo, M.(2001). The open reading frame 1-encoded (‘36K’) protein of Carnation Italian ringspot virus localizes to mitochondria. J Gen Virol 82, 29–34. [Google Scholar]
  38. Rubino, L., Pantaleo, V., Navarro, B. & Russo, M.(2004). Expression of tombusvirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, RNA. J Gen Virol 85, 3115–3122.[CrossRef] [Google Scholar]
  39. Ruiz-Ferrer, V. & Voinnet, O.(2009). Roles of plant small RNAs in biotic stress responses. Annu Rev Plant Biol 60, 485–510.[CrossRef] [Google Scholar]
  40. Russo, M., Burgyán, J. & Martelli, G. P.(1994). Molecular biology of Tombusviridae. Adv Virus Res 44, 381–428. [Google Scholar]
  41. Shen, B. & Goodman, H. M.(2004). Uridine addition after microRNA-directed cleavage. Science 306, 997[CrossRef] [Google Scholar]
  42. Silhavy, D., Molnar, D., Lucioli, A., Szittya, G., Horniyik, C. & Burgyán, J.(2002). A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs. EMBO J 21, 3070–3080.[CrossRef] [Google Scholar]
  43. Simon, A. E., Roossinck, M. J. & Havelda, Z.(2004). Plant virus satellite and defective interfering RNAs: new paradigm. Annu Rev Phytopathol 42, 415–437.[CrossRef] [Google Scholar]
  44. Szittya, G., Molnar, A., Silhavy, D., Hornyik, C. & Burgyán, J.(2002). Short defective interfering RNAs of tombusviruses are not targeted but trigger post-transcriptional gene silencing against their helper virus. Plant Cell 14, 359–372.[CrossRef] [Google Scholar]
  45. Szittya, G., Silhavy, D., Molnár, A., Havelda, Z., Lovas, A., Lakatos, L., Bánfalvi, Z. & Burgyán, J.(2003). Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation. EMBO J 22, 633–640.[CrossRef] [Google Scholar]
  46. White, J. L. & Kaper, J. M.(1989). A simple method for detection of viral satellite RNAs in small tissue samples. J Virol Methods 23, 83–94.[CrossRef] [Google Scholar]
  47. White, K. A. & Nagy, P. D.(2004). Advances in the molecular biology of tombusviruses: gene expression, genome replication, and recombination. Prog Nucleic Acid Res Mol Biol 78, 187–226. [Google Scholar]
  48. Wu, B., Vanti, W. B. & White, K. A.(2001). An RNA domain within the 5′ untranslated region of the tomato bushy stunt virus genome modulates viral RNA replication. J Mol Biol 305, 741–756.[CrossRef] [Google Scholar]
  49. Zuker, M.(2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31, 3406–3415.[CrossRef] [Google Scholar]

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vol. , part 9, pp. 2393–2401

Systemic necrosis and death of an plant inoculated with TBSV L plus satRNA L.

Northern blot analysis of satRNA L using a probe complementary to the 3′-terminal region or to the complete negative strand.

Positions of the 5′ or 3′ termini in the smaller-than-unit-length satRNA L molecules extracted from plants co-inoculated with TBSV L and grown at 24 or 15 °C.

Northern blot analysis of RNA extracts from protoplasts co-inoculated with satRNA L and selected CymRSV–CIRV hybrid clones with decreasing amounts of the 5′ portion of the CymRSV genome fused to the CIRV genome, CymRSV or CIRV.

Northern blot analysis of RNA extracts from plants co-inoculated with satRNA L and CIRV C80 or wild-type CIRV.

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