1 Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Pflanzenvirologie, Mikrobiologie und biologische Sicherheit, Messeweg 11, D-38104 Braunschweig, Germany
2 Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
3 Leiden Institute of Biology, Leiden University, Kaiserstraat 63, 2311 GP Leiden, The Netherlands
Correspondence
R. Koenig
r.koenig{at}bba.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the complete RNA sequence of a Nemesia isolate of Nemesia ring necrosis virus is AY751778; partial RNA sequences of Diascia and Verbena isolates of the virus have accession numbers AY751781 and AY885256, respectively.
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INTRODUCTION |
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METHODS |
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Native turnip yellow mosaic virus (TYMV) RNA was isolated from infected Chinese cabbage according to Dunn & Hitchborn (1965) and native brome mosaic virus (BMV) RNA was purchased from Promega. Tobacco mosaic virus (TMV) particles (U1 strain) were purified from infected Nicotiana tabacum leaves that were frozen in liquid nitrogen prior to grinding in 10 mM EDTA/10 mM NaH2PO4 (adjusted to pH 7·5 with NaOH). Cell debris was removed by centrifugation at 6000 g, 4 °C. The supernatant was filtered and virus particles were precipitated by adding 4 % (w/v) polyethylene glycol 20 000 and 4 % (w/v) NaCl followed by gentle shaking for 2 h at 4 °C. The pellet obtained after 30 min centrifugation at 25 000 g at 4 °C was dissolved in the grinding buffer and the precipitation and centrifugation steps were repeated twice. The final pellet was dissolved in H2O to a concentration of
50 mg ml1. RNA was extracted from the particles by three phenol extractions.
Sequence analysis.
The first genome portions of NeRNV RNA were amplified by PCR using primers derived from sequences that, in alignments, were found to be highly conserved in tymoviral RNAs. The starter sequences thus obtained were bridged up using PCRs with specific primers or were extended by a random primed cDNA approach (Koenig et al., 2004). The sequence of the 5' end of NeRNV RNA was determined using the 5' RACE system of rapid amplification of cDNA ends (Invitrogen) and that of the 3' end using an RNA ligase-mediated RACE method essentially as described by Coutts & Livieratos (2003)
. For cloning into the pGEM-T vector (Promega), PCR products were purified using the Jetsorb Gel Extraction kit (Genomed). Sequencing was done by a commercial company (MWG-Biotech). Sequences were analysed using UWGCG software version 8 (Devereux et al., 1984
). The program STAR was used for prediction of RNA secondary structure, including pseudoknots (Gultyaev et al., 1995
).
Aminoacylation.
Yeast histidyl-tRNA synthetase (HisRS) was partially purified by chromatography on a hydroxyapatite column (Bio-Rad). Fractions with enriched histidylation activity were pooled and dialysed against 50 mM KH2PO4/K2HPO4 (pH 7·2), 50 % glycerol, 0·1 mM EDTA and 10 mM -mercaptoethanol and stored at 20 °C.
All histidylation experiments were performed in a 100 µl reaction mixture containing 55 mM Tris/HCl, pH 7·5, 15 mM MgCl2, 30 mM KCl, 10 mM ATP, 100 µM [3H]His (3·4x1013 Bq mol1; Amersham Biosciences), 100 nM RNA and 5 % (v/v) yeast HisRS extract. Reactions were followed at 30 °C and, at the indicated times, 20 µl samples were precipitated in 1 ml 5 % (w/v) ice-cold trichloroacetic acid and filtered over GF/C filters (Whatman). Radioactivity was measured by liquid scintillation counting.
Translation.
In vitro translation was essentially carried out as described previously (Barends et al., 2003). Briefly, wheat germ extract (Promega) was complemented with 100 mM KCl, 100 µM of each amino acid except methionine, 1 U RNaseOUT µl1 (Invitrogen), 100 nM mRNA and 0·43 µM [35S]Met (MP Biomedicals) and incubated at 25 °C for 90 min. Reactions were stopped by treatment with 2 volumes protein sample buffer (Bio-Rad), prior to separation by SDS-PAGE and analysis by phosphor imaging (Bio-Rad).
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RESULTS AND DISCUSSION |
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The 3' end
In view of the many above-described similarities of the NeRNV RNA with those of previously analysed tymoviruses, it was surprising to find that the predicted secondary structure of the 3' end of NeRNV RNA greatly differs from those of other tymoviral RNAs. For the majority of tymoviral RNAs, 3'-terminal valine-specific tRNA-like structures (TLS) are predicted that are usually preceded upstream by various stemloops or one or more pseudoknots (Hellendoorn et al., 1996a; Koenig et al., 2005
). The 3' end of NeRNV RNA, however, strongly resembles that of tobamoviral RNAs (Rietveld et al., 1984
; van Belkum et al., 1985
) in that it can be folded into a so-called UPD and a TLS with an anticodon for histidine (Fig. 4
). This unusual 3' end is obviously not a specific feature of the Nemesia isolate of NeRNV. Short stretches of 441 and 675 nt, respectively, of Verbena and Diascia isolates of NeRNV were also amplified. The RNA sequence of the Diascia isolate contained the entire coat protein gene and the entire UPD. The sequence of the Verbena isolate contained the 3'-terminal part of the coat protein gene, the entire UPD and, in addition, a 5' portion of the TLS including most of the stemloop with the anticodon for histidine. Sequences of the Diascia and Verbena isolates of NeRNV showed 99·4 and 99·1 % identity with the corresponding RNA parts of the Nemesia isolate suggesting that the three isolates are essentially identical.
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The 3' TLS
The TLS of NeRNV RNA (Fig. 4) has the typical 11 bp acceptor domain, including the pseudoknot structure (PKa) and the strongly conserved seven-membered T-like loop (Mans et al., 1991
). Furthermore, five out of the six base pairs that form pseudoknot PKb are identical in both TMV and NeRNV RNA and the D-like loop in NeRNV RNA also contains the conserved sequence UGGA. The two G residues in this sequence are supposed to interact with the T-like loop in a way analogous to elongator tRNAs (Mans et al., 1991
; Felden et al., 1996
). NeRNV RNA, like TMV RNA, was effectively charged with histidine (Fig. 6
), thus proving the functionality of the anticodon arm in its TLS. Neither NeRNV nor TMV RNA were charged with valine (not shown).
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Concluding remarks
In summary, NeRNV RNA, which is tymovirus-like in most of its parts, has a 3' end with almost the same organization as tobamovirus RNAs, including a TLS that can be specifically aminoacylated with histidine. The tymovirus WCMV was shown previously to also have a UPD domain, but this domain is followed by the valylatable TLS typical for tymoviral RNAs (Hellendoorn et al., 1996a). In fact, NeRNV is more or less the counterpart of Sunn-hemp mosaic virus (SHMV or, previously, CcTMV) (Meshi et al., 1981
; Rietveld et al., 1982
), the only tobamovirus known to have a valine-specific TLS downstream of a UPD domain. Interestingly, both SHMV and NeRNV have a hairpin structure between the UPD domain and the TLS (van Belkum et al., 1985
). In both cases, this may represent a remnant of the recombination event, although it should be kept in mind that SHMV RNA could have received its 3'-UTR from a pomo- or furoviral RNA as an alternative to a tymoviral RNA. Another possibility is that this hairpin is needed to ensure proper mutual orientation of the UPD and TLS. The same phenomenon is observed in the 3'-UTR of tyrosylatable hordeivirus RNAs, which also have a UPD domain (Pleij et al., 1987
). Interestingly, a chimeric TYMV RNA harbouring the TLS of TMV RNA was not infectious to plants (Skuzeski et al., 1996
) and it was only after introducing TYMV sequences into the amino acid acceptor arm and anticodon loop (including the valine anticodon) and passaging in plants, which produced further mutations, that the resulting viruses, TYMC-XX and TYMC-YY, became highly infectious to plants (Goodwin et al., 1997
; Filichkin et al., 2000
).
Fig. 7 summarizes the various compositions of the RNA 3' ends of tobamo- and tymoviruses and compares them with those of pomo- and furoviruses. It illustrates the high flexibility in the make-up of 3' ends among these virus genera, which is presumably due to recombination events in doubly infected plant hosts. This suggestion is supported by the occurrence of frequent duplications of UPDs and parts of the TLS in several tobamoviruses (Gultyaev et al., 1994
; Bodaghi et al., 2000
; Ruiz del Pino et al., 2003
). In view of the observations summarized in Figs 5 and 7
, it might not be surprising if some day a pomo- or furovirus would be detected with an RNA that has a histidylatable TLS on its 3' end rather than the common valylatable TLS.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Barends, S., Rudinger-Thirion, J., Florentz, C., Giegé, R., Pleij, C. W. A. & Kraal, B. (2004). tRNA-like structure regulates translation of Brome mosaic virus RNA. J Virol 78, 40034010.
Bink, H. H. J., Hellendoorn, K., van der Meulen, J. & Pleij, C. W. A. (2002). Protonation of non-WatsonCrick base pairs and encapsidation of turnip yellow mosaic virus RNA. Proc Natl Acad Sci U S A 99, 1346513470.
Bodaghi, S., Ngon A Yassi, M. & Dodds, J. A. (2000). Heterogeneity in the 3'-terminal untranslated region of tobacco mild green mosaic tobamoviruses from Nicotiana glauca resulting in variants with three or six pseudoknots. J Gen Virol 81, 577586.
Bozarth, C. S., Weiland, J. J. & Dreher, T. W. (1992). Expression of ORF-69 of turnip yellow mosaic virus is necessary for viral spread in plants. Virology 187, 124130.[CrossRef][Medline]
Bransom, K. L. & Dreher, T. W. (1994). Identification of the essential cysteine and histidine residues of the turnip yellow mosaic virus protease. Virology 198, 148154.[CrossRef][Medline]
Bransom, K. L., Wallace, S. E. & Dreher, T. W. (1996). Identification of the cleavage site recognized by the turnip yellow mosaic virus protease. Virology 217, 404406.[CrossRef][Medline]
Chen, J., Li, W. X., Xie, D., Peng, J. R. & Ding, S. W. (2004). Viral virulence protein suppresses RNA silencing-mediated defense but upregulates the role of microRNA in host gene expression. Plant Cell 16, 13021313.
Coutts, R. H. A. & Livieratos, I. C. (2003). A rapid method for sequencing the 5'- and 3'-termini of dsRNA viral templates using RLM-Race. J Phytopathol 151, 525527.[CrossRef]
Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12, 387395.[Abstract]
Dunn, D. B. & Hitchborn, J. H. (1965). The use of bentonite in the purification of plant viruses. Virology 25, 171192.[CrossRef][Medline]
Felden, B., Florentz, C., Giegé, R. & Westhof, E. (1996). A central pseudoknotted three-way junction imposes tRNA-like mimicry and the orientation of three 5' upstream pseudoknots in the 3' terminus of tobacco mosaic virus RNA. RNA 2, 201212.[Abstract]
Filichkin, S. A., Bransom, K. L., Goodwin, J. B. & Dreher, T. W. (2000). The infectivities of turnip yellow mosaic virus genomes with altered tRNA mimicry are not dependent on compensating mutations in the viral replication protein. J Virol 74, 83688375.
Goodwin, J. B., Skuzeski, J. M. & Dreher, T. W. (1997). Characterization of chimeric turnip yellow mosaic virus genomes that are infectious in the absence of aminoacylation. Virology 230, 113124.[CrossRef][Medline]
Gultyaev, A. P., van Batenburg, E. & Pleij, C. W. A. (1994). Similarities between the secondary structure of satellite tobacco mosaic virus and tobamovirus RNAs. J Gen Virol 75, 28512856.[Abstract]
Gultyaev, A. P., van Batenburg, F. H. D. & Pleij, C. W. A. (1995). The computer simulation of RNA folding pathways using a genetic algorithm. J Mol Biol 250, 3751.[CrossRef][Medline]
Hellendoorn, K., Mat, A. W., Gultyaev, A. P. & Pleij, C. W. A. (1996a). Secondary structure model of the coat protein gene of turnip yellow mosaic virus RNA: long, C-rich, single-stranded regions. Virology 224, 4354.[CrossRef][Medline]
Hellendoorn, K., Michiels, P. J., Buitenhuis, R. & Pleij, C. W. A. (1996b). Protonatable hairpins are conserved in the 5'-untranslated region of tymovirus RNAs. Nucleic Acids Res 24, 49104917.
Hellendoorn, K., Verlaan, P. W. & Pleij, C. W. A. (1997). A functional role for the conserved protonatable hairpins in the 5' untranslated region of turnip yellow mosaic virus RNA. J Virol 71, 87748779.[Abstract]
Kadare, G., Rozanov, M. & Haenni, A.-L. (1995). Expression of the turnip yellow mosaic virus proteinase in Escherichia coli and determination of the cleavage site within the 206 kDa protein. J Gen Virol 76, 28532857.[Abstract]
Kashiwazaki, S., Scott, K. P., Reavy, B. & Harrison, B. D. (1995). Sequence analysis and gene content of potato mop top virus RNA 3: further evidence of heterogeneity in the genome organization of furoviruses. Virology 206, 701706.[CrossRef][Medline]
Koenig, R. & Lesemann, D.-E. (2000). Ein Tymovirus aus den Zierpflanzen Diascia und Nemesia - wie zuverlässig ist die Serologie bei der Identifizierung von Pflanzenviren? Phytomedizin 30, 1617.
Koenig, R., Pleij, C. W. A., Beier, C. & Commandeur, U. (1998). Genome properties of beet virus Q, a new furo-like virus from sugarbeet, determined from unpurified virus. J Gen Virol 79, 20272036.[Abstract]
Koenig, R., Pleij, C. W. A. & Huth, W. (1999). Molecular characterization of a new furovirus mainly infecting rye. Arch Virol 144, 21252140.[CrossRef][Medline]
Koenig, R., Pleij, C. W. A. & Büttner, G. (2000). Structure and variability of the 3' end of RNA 3 of beet soil-borne pomovirus a virus with uncertain pathogenic effects. Arch Virol 145, 11731181.[CrossRef][Medline]
Koenig, R., Pleij, C. W. A., Loss, S., Burgermeister, W., Aust, H. & Schiemann, J. (2004). Molecular characterisation of potexviruses isolated from three different genera in the family Cactaceae. Arch Virol 149, 903914.[CrossRef][Medline]
Koenig, R., Pleij, C. W. A., Lesemann, D.-E., Loss, S. & Vetten, H. J. (2005). Molecular characterization of Anagyris vein yellowing virus, Plantago mottle virus and Scrophularia mottle virus comparison of various approaches for tymovirus classification. Arch Virol (in press).
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, 375430.[Abstract]
Mans, R. M., Pleij, C. W. A. & Bosch, L. (1991). tRNA-like structures. Structure, function and evolutionary significance. Eur J Biochem 201, 303324.[CrossRef][Medline]
Meshi, T., Ohno, T., Iba, H. & Okada, Y. (1981). Nucleotide sequence of a cloned cDNA copy of TMV (cowpea strain) RNA, including the assembly origin, the coat protein cistron, and the 3' non-coding region. Mol Gen Genet 184, 2025.[CrossRef][Medline]
Nameki, N., Asahara, H., Shimizu, M., Okada, N. & Himeno, H. (1995). Identity elements of Saccharomyces cerevisiae tRNAHis. Nucleic Acids Res 23, 389394.[Abstract]
Pleij, C. W. A., Abrahams, J. P., van Belkum, A., Rietveld, K. & Bosch, L. (1987). The spatial folding of the 3' noncoding region of aminoacylatable plant viral RNAs. In Positive Strand RNA Viruses, UCLA Symposia on Molecular and Cellular Biology, New Series, vol. 54, pp. 299316. Edited by M. A. Brinton & R. Rueckert. New York: Alan Riss.
Rietveld, K., van Poelgeest, R., Pleij, C. W. A., van Boom, J. H. & Bosch, L. (1982). The tRNA-like structure at the 3' terminus of turnip yellow mosaic virus RNA. Differences and similarities with canonical tRNA. Nucleic Acids Res 10, 19291946.[Abstract]
Rietveld, K., Linschooten, K., Pleij, C. W. A. & Bosch, L. (1984). The three-dimensional folding of the tRNA-like structure of tobacco mosaic virus RNA. A new building principle applied twice. EMBO J 3, 26132619.
Rudinger, J., Florentz, C. & Giegé, R. (1994). Histidylation by yeast HisRS of tRNA or tRNA-like structure relies on residues 1 and 73 but is dependent on the RNA context. Nucleic Acids Res 22, 50315037.[Abstract]
Ruiz del Pino, M., Moreno, A., Garcia de Lacoba, M., Castillo-Lluva, S., Gilardi, P., Serra, M. T. & Garcia-Luque, I. (2003). Biological and molecular characterization of P101 isolate, a tobamoviral pepper strain from Bulgaria. Arch Virol 148, 21152135.[CrossRef][Medline]
Seigner, L. (2003). Schäden an Zierpflanzen durch INSV und Nemesiavirus. Das Magazin für Zierpflanzenbau 19, 4749.
Shimamoto, I., Sonoda, S., Vazquez, P., Minaka, N. & Nishiguchi, M. (1998). Nucleotide sequence analysis of the 3' terminal region of a wasabi strain of crucifer tobamovirus genomic RNA: subgrouping of crucifer tobamoviruses. Arch Virol 143, 18011813.[CrossRef][Medline]
Shirako, Y. & Wilson, T. M. A. (1993). Complete nucleotide sequence and organization of the bipartite RNA genome of soil-borne wheat mosaic virus. Virology 195, 1632.[CrossRef][Medline]
Skelton, A. L., Jarvis, B., Koenig, R., Lesemann, D.-E. & Mumford, R. A. (2004). The isolation and identification of a tymovirus from Nemesia in the UK. Plant Pathol 53, 798.[CrossRef]
Skuzeski, J. M., Bozarth, C. S. & Dreher, T. W. (1996). The turnip yellow mosaic virus tRNA-like structure cannot be replaced by generic tRNA-like elements or by heterologous 3' untranslated regions known to enhance mRNA expression and stability. J Virol 70, 21072115.[Abstract]
van Belkum, A., Abrahams, J. P., Pleij, C. W. A. & Bosch, L. (1985). Five pseudoknots are present at the 204 nucleotides long 3' noncoding region of tobacco mosaic virus RNA. Nucleic Acids Res 13, 76737686.[Abstract]
Received 24 January 2005;
accepted 8 March 2005.
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