1887

Abstract

(PFBV) belongs to the genus (family ) and, as with the remaining members of the group, possesses a monopartite genome of single-stranded, positive-sense RNA that contains five ORFs. The two 5′-proximal ORFs (ORFs 1 and 2) encode two polypeptides of 27 and 86 kDa (p27 and p86), respectively, that show homology with replication proteins. The p27 does not present any motif to explain its presumed involvement in replication, while p86 has the motifs conserved in RNA-dependent RNA polymerases. In this work, we have confirmed the necessity of p27 and p86 for PFBV replication. To gain insights into the function(s) of p27, we have expressed and purified the protein from and tested its ability to bind RNA . The results have shown that p27 is able to bind ssRNA with high affinity and in a cooperative fashion and that it is also capable of binding other types of nucleic acids, though to a lesser extent. Additionally, competition experiments suggest that p27 has a preference for PFBV-derived ssRNAs. Using truncated forms of p27, it can be concluded that several regions of the protein contribute to its RNA-binding properties and that this contribution is additive. This study is the first to show nucleic acid-binding ability of the ORF1 product of a carmovirus and the data obtained suggest that this product plays an essential role in selection and recruitment of viral RNA replication templates.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.023093-0
2010-12-01
2024-12-14
Loading full text...

Full text loading...

/deliver/fulltext/jgv/91/12/3075.html?itemId=/content/journal/jgv/10.1099/vir.0.023093-0&mimeType=html&fmt=ahah

References

  1. Ahlquist, P., Noueiry, A. O., Lee, W. M., Kushner, D. B. & Dye, B. T.(2003). Host factors in positive-strand RNA virus genome replication. J Virol 77, 8181–8186.[CrossRef] [Google Scholar]
  2. Bedford, M. T. & Richard, S.(2005). Arginine methylation an emerging regulador of protein function. Mol Cell 18, 263–272.[CrossRef] [Google Scholar]
  3. Burd, C. G. & Dreyfuss, G.(1994). Conserved structures and diversity of functions of RNA-binding proteins. Science 265, 615–621.[CrossRef] [Google Scholar]
  4. Carbonell, A., Martínez de Alba, A. E., Flores, R. & Gago, S.(2008). Double-stranded RNA interferes in a sequence-specific manner with the infection of representative members of the two viroid families. Virology 371, 44–53.[CrossRef] [Google Scholar]
  5. Carey, J.(1991). Gel retardation. Methods Enzymol 208, 103–117. [Google Scholar]
  6. Castaño, A., Ruiz, L. & Hernández, C.(2009). Insights into the translational regulation of biologically active open reading frames of Pelargonium line pattern virus. Virology 386, 417–426.[CrossRef] [Google Scholar]
  7. Citovsky, V., Knorr, D., Schuster, G. & Zambryski, P.(1990). The P30 movement protein of tobacco mosaic virus is a single strand nucleic acid binding protein. Cell 60, 637–647.[CrossRef] [Google Scholar]
  8. Daròs, J. A. & Carrington, J. C.(1997). RNA binding activity of NIa proteinase of tobacco etch potyvirus. Virology 237, 327–336.[CrossRef] [Google Scholar]
  9. Genovés, A., Navarro, J. A. & Pallás, V.(2006). Functional analysis of the five melon necrotic spot virus genome-encoded proteins. J Gen Virol 87, 2371–2380.[CrossRef] [Google Scholar]
  10. Genovés, A., Navarro, J. A. & Pallás, V.(2009). A self-interacting carmovirus movement protein plays a role in binding of viral RNA during the cell-to-cell movement and shows an actin cytoskeleton dependent location in cell periphery. Virology 395, 133–142.[CrossRef] [Google Scholar]
  11. Gross, C. H. & Shuman, S.(1996). The QRxGRxGRxxxG motif of the vaccinia virus DExH box RNA helicase NPH-II is required for ATP hydrolysis and RNA unwinding but not for RNA binding. J Virol 70, 1706–1713. [Google Scholar]
  12. Habili, N. & Symons, R. H.(1989). Evolutionary relationship between luteoviruses and other RNA plant viruses based on sequence motifs in their putative RNA polymerases and nucleic acid helicases. Nucleic Acids Res 17, 9543–9555.[CrossRef] [Google Scholar]
  13. Hacker, D. L., Petty, I. T., Wei, N. & Morris, T. J.(1992). Turnip crinkle virus genes required for RNA replication and virus movement. Virology 186, 1–8.[CrossRef] [Google Scholar]
  14. Herranz, M. C. & Pallás, V.(2004). RNA-binding properties and mapping of the RNA-binding domain from the movement protein of Prunus necrotic ringspot virus. J Gen Virol 85, 761–768.[CrossRef] [Google Scholar]
  15. Hobson, S. D., Rosenblum, E. S., Richards, O. C., Richmond, K., Kirkegaard, K. & Schultz, S. C.(2001). Oligomeric structures of poliovirus polymerase are important for function. EMBO J 20, 1153–1163.[CrossRef] [Google Scholar]
  16. Jones, S., Daley, D. T., Luscombe, N. M., Berman, H. M. & Thornton, J. M.(2001). Protein–RNA interactions: a structural analysis. Nucleic Acids Res 29, 943–954.[CrossRef] [Google Scholar]
  17. Kim, Y. C. & Kao, C. C.(2008). Biochemical analyses of the interactions between viral polymerases and RNAs. Methods Mol Biol 451, 185–200. [Google Scholar]
  18. Koonin, E. V. & Dolja, V. V.(1993). Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Crit Rev Biochem Mol Biol 28, 375–430.[CrossRef] [Google Scholar]
  19. Lewandowski, D. J. & Dawson, W. O.(2000). Functions of the 126- and 183-kDa proteins of tobacco mosaic virus. Virology 271, 90–98.[CrossRef] [Google Scholar]
  20. Li, Q. & Palukaitis, P.(1996). Comparison of the nucleic acid- and NTP-binding properties of the movement protein of cucumber mosaic cucumovirus and tobacco mosaic tobamovirus. Virology 216, 71–79.[CrossRef] [Google Scholar]
  21. Liu, Q. & Dreyfuss, G.(1995).In vivo and in vitro arginine methylation of RNA-binding proteins. Mol Cell Biol 15, 2800–2808. [Google Scholar]
  22. Lohman, T. M., Overman, L. B. & Datta, S.(1986). Salt-dependent changes in the DNA binding cooperativity of Escherichia coli single-strand binding protein. J Mol Biol 187, 603–615.[CrossRef] [Google Scholar]
  23. Lommel, S. A., Martelli, G. P., Rubino, L. & Russo, M.(2005). Family Tombusviridae. In Eighth Report of the International Committee on Taxonomy of Viruses, pp. 907–936. Edited by Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U. & Ball, L. A.. San Diego. : Academic Press. [Google Scholar]
  24. López, C., Navas-Castillo, J., Gowda, S., Moreno, P. & Flores, R.(2000). The 23-kDa protein coded by the 3′-terminal gene of Citrus tristeza virus is an RNA-binding protein. Virology 269, 462–470.[CrossRef] [Google Scholar]
  25. Marcos, J. F., Vilar, M., Pérez-Payá, E. & Pallás, V.(1999).In vivo detection, RNA-binding properties and characterization of the RNA-binding domain of the p7 putative movement protein from carnation mottle carmovirus (CarMV). Virology 255, 354–365.[CrossRef] [Google Scholar]
  26. Martínez-Turiño, S. & Hernández, C.(2009). Inhibition of RNA silencing by the coat protein of Pelargonium flower break virus: distinctions from closely related suppressors. J Gen Virol 90, 519–525.[CrossRef] [Google Scholar]
  27. McCormack, J. C., Yuan, X., Yingling, Y. G., Kasprzak, W., Zamora, R. E., Shapiro, B. A. & Simon, A. E.(2008). Structural domains within the 3′ untranslated region of turnip crinkle virus. J Virol 82, 8706–8720.[CrossRef] [Google Scholar]
  28. Mochizuki, T., Hirai, K., Kanda, A., Ohnishi, J., Ohki, T. & Tsuda, S.(2009). Induction of necrosis via mitochondrial targeting of melon necrotic spot virus replication protein p29 by its second transmembrane domain. Virology 390, 239–249.[CrossRef] [Google Scholar]
  29. Molnár, A., Havelda, Z., Dalmay, T., Szutorisz, H. & Burgyán, J.(1997). Complete nucleotide sequence of tobacco necrosis virus strain DH and genes required for RNA replication and virus movement. J Gen Virol 78, 1235–1239. [Google Scholar]
  30. Na, H. & White, K. A.(2006). Structure and prevalence of replication silencer-3′ terminus RNA interactions in Tombusviridae. Virology 345, 305–316.[CrossRef] [Google Scholar]
  31. Navarro, B., Rubino, L. & Russo, M.(2004). Expression of the Cymbidium ringspot virus 33-kilodalton protein in Saccharomyces cerevisiae and molecular dissection of the peroxisomal targeting signal. J Virol 78, 4744–4752.[CrossRef] [Google Scholar]
  32. Navarro, J. A., Genovés, A., Climent, J., Saurí, A., Martínez-Gil, L., Mingarro, I. & Pallás, V.(2006). RNA-binding properties and membrane insertion of melon necrotic spot virus (MNSV) double gene block movement proteins. Virology 356, 57–67.[CrossRef] [Google Scholar]
  33. Neeleman, L. & Bol, J. F.(1999).Cis-acting functions of alfalfa mosaic virus proteins involved in replication and encapsidation of viral RNA. Virology 254, 324–333.[CrossRef] [Google Scholar]
  34. Oberste, M. S. & Flanegan, J. B.(1988). Measurement of poliovirus RNA polymerase binding to poliovirion and nonviral RNAs using a filter-binding assay. Nucleic Acids Res 16, 10339–10352.[CrossRef] [Google Scholar]
  35. Ohndorf, U. M., Steegborn, C., Knijff, R. & Sondermann, P.(2001). Contributions of the individual domains in human La protein to its RNA 3′-end binding activity. J Biol Chem 276, 27188–27196.[CrossRef] [Google Scholar]
  36. Okamoto, K., Nagano, H., Iwakawa, H., Mizumoto, H., Takeda, A., Kaido, M., Mise, K. & Okuno, T.(2008).Cis-preferential requirement of a −1 frameshift product p88 for the replication of red clover necrotic mosaic virus RNA1. Virology 375, 205–212.[CrossRef] [Google Scholar]
  37. Oster, S. K., Wu, B. & White, K. A.(1998). Uncoupled expression of p33 and p92 permits amplification of tomato bushy stunt virus RNAs. J Virol 72, 5845–5851. [Google Scholar]
  38. Panavas, T., Hawkins, C. M., Panaviene, Z. & Nagy, P. D.(2005). The role of the 33:p33/p92 interaction domain in RNA replication and intracellular localization of p33 and p92 proteins of cucumber necrosis tombusvirus. Virology 338, 81–95.[CrossRef] [Google Scholar]
  39. Pata, J. D., Schultz, S. C. & Kirkegaard, K.(1995). Functional oligomerization of poliovirus RNA-dependent RNA polymerase. RNA 1, 466–477. [Google Scholar]
  40. Pogany, J., Fabian, M. R., White, K. A. & Nagy, P. D.(2003). A replication silencer element in a plus-strand RNA virus. EMBO J 22, 5602–5611.[CrossRef] [Google Scholar]
  41. 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]
  42. 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]
  43. Rajendran, K. S. & Nagy, P. D.(2004). Interaction between the replicase proteins of tomato bushy stunt virus in vitro and in vivo. Virology 326, 250–261.[CrossRef] [Google Scholar]
  44. Richmond, K. E., Chenault, K., Sherwood, J. L. & German, T. L.(1998). Characterization of the nucleic acid binding properties of tomato spotted wilt virus nucleocapsid protein. Virology 248, 6–11.[CrossRef] [Google Scholar]
  45. Rico, P. & Hernández, C.(2004). Complete nucleotide sequence and genome organization of Pelargonium flower break virus. Arch Virol 149, 641–651.[CrossRef] [Google Scholar]
  46. Rico, P. & Hernández, C.(2006). Infectivity of in vitro transcripts from a full length cDNA clone of Pelargonium flower break virus in an experimental and a natural host. J Plant Pathol 88, 103–106. [Google Scholar]
  47. Rico, P. & Hernández, C.(2009). Characterization of the subgenomic RNAs produced by Pelargonium flower break virus: identification of two novel RNA species. Virus Res 142, 100–107.[CrossRef] [Google Scholar]
  48. Rico, P., Ivars, P., Elena, S. F. & Hernández, C.(2006). Insights on the selective pressures restricting Pelargonium flower break virus genome variability: evidence for host adaptation. J Virol 80, 8124–8132.[CrossRef] [Google Scholar]
  49. Siebel, C. W. & Guthrie, C.(1996). The essential yeast RNA binding protein Npl3p is methylated. Proc Natl Acad Sci U S A 93, 13641–13646.[CrossRef] [Google Scholar]
  50. Stork, J., Panaviene, Z. & Nagy, P. D.(2005). Inhibition of in vitro RNA binding and replicase activity by phosphorylation of the p33 replication protein of cumber necrosis tombusvirus. Virology 343, 79–92.[CrossRef] [Google Scholar]
  51. Terribilini, M., Lee, J. H., Yan, C., Jernigan, R. L., Honavar, V. & Dobbs, D.(2006). Prediction of RNA-binding sites in proteins from amino acid sequence. RNA 12, 1450–1462.[CrossRef] [Google Scholar]
  52. Terribilini, M., Sander, J. D., Lee, J.-H., Zaback, P., Jernigan, R. L., Honavar, V. & Dobbs, D.(2007). RNABindR: a server for analyzing and predicting RNA-binding sites proteins. Nucleic Acids Res 35, W578–W584.[CrossRef] [Google Scholar]
  53. Turner, K. A., Sit, T. L., Callaway, A. S., Allen, N. S. & Lommel, S. A.(2004). Red clover necrotic mosaic virus replication proteins accumulate at the endoplasmic reticulum. Virology 320, 276–290.[CrossRef] [Google Scholar]
  54. Wang, R. Y. & Nagy, P. D.(2008). Tomato bushy stunt virus co-opts the RNA-binding function of a host metabolic enzyme for viral genomic RNA synthesis. Cell Host Microbe 3, 178–187.[CrossRef] [Google Scholar]
  55. Wang, H. H. & Wong, S. M.(2004). Significance of the 3′-terminal region in minus strand RNA synthesis of Hibiscus chlorotic ringspot virus. J Gen Virol 85, 1763–1776.[CrossRef] [Google Scholar]
  56. Wang, Q. M., Hockman, M. A., Staschke, K., Johnson, R. B., Case, K. A., Lu, J., Parsons, S., Zhang, F., Rathnachalam, R. & other authors(2002). Oligomerization and cooperative RNA synthesis activity of hepatitis C virus RNA-dependent RNA polymerase. J Virol 76, 3865–3872.[CrossRef] [Google Scholar]
  57. Weber-Lotfi, F., Dietrich, A., Russo, M. & Rubino, L.(2002). Mitocondrial targeting and membrane anchoring of a viral replicase in plant and yeast cells. J Virol 76, 10485–10496.[CrossRef] [Google Scholar]
  58. Weiss, M. A. & Narayana, N.(1998). RNA recognition by arginine-rich peptide motifs. Biopolymers 48, 167–180.[CrossRef] [Google Scholar]
  59. 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]
  60. White, K. A., Skuzeski, J. M., Li, W., Wei, N. & Morris, T. J.(1995). Immunodetection, expression strategy and complementation of turnip crinkle virus p28 and p88 replication components. Virology 211, 525–534.[CrossRef] [Google Scholar]
  61. Wobbe, K. K., Akgoz, M., Dempsey, D. A. & Klessig, D. F.(1998). A single amino acid change in turnip crinkle virus movement protein p8 affects RNA binding and virulence on Arabidopsis thaliana. J Virol 72, 6247–6250. [Google Scholar]
/content/journal/jgv/10.1099/vir.0.023093-0
Loading
/content/journal/jgv/10.1099/vir.0.023093-0
Loading

Data & Media loading...

Supplements

Control EMSA with distinct amounts of BSA. The P-labelled 3′-PFBV ssRNA probe (at 33 pM) was incubated with no protein (lane 1) or with increasing concentrations (50, 75 and 100 nM) of BSA (lanes 2–4, respectively). The unbound, free RNA probe is marked on the right; no shifted (bound) RNA complexes were detected.

IMAGE

Illustrative EMSA with distinct amounts of recombinant proteins. The P-labelled 3′-PFBV ssRNA probe (at 33 pM) was incubated with no protein (lane 1) or with two concentrations (100 nM in lanes 2, 4, 6, 8, 10, 12 and 14 and 0.1 µM in lanes 3, 5, 7, 9, 11, 13 and 15) of His-tagged p27 and derivatives. The unbound, free RNA probe and the shifted (bound) RNA complexes are marked on the right.

IMAGE

RNA-binding predictions for PFBV p27 and related carmoviral proteins using the program RNABindR. Residues predicted to bind RNA (in 'optimal prediction mode') are indicated by grey boxes. Numbers denote positions of the amino acid residues in the corresponding full-length proteins. Sequences used for comparison were retrieved from the sequence databases and correspond to the following viruses: PFBV ( flower break virus; GenBank accession no. AJ514833), SCV (saguaro cactus virus; NC_001780), CbMV ( mottle virus; GQ244431), NLVCV ( vein-clearing virus; EF207438), CarMV (carnation mottle virus; X02986), AnFBV ( flower break virus; DQ219415), TCV (turnip crinkle virus; M22445), CCFV (cardamine chlorotic fleck virus; L16015), HCRSV (hibiscus chlorotic ringspot virus; X86448), MNSV (melon necrotic spot virus; M29671), PSNV (pea stem necrosis virus; NC_004995), JINRV (Japanese iris necrotic ring virus; NC_002187), CPMoV (cowpea mottle virus; U20976) and GaMV ( mosaic virus; Y13463).

IMAGE
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error