1 Centrum Grüne Gentechnik, Dienstleistungszentrum Ländlicher Raum Rhein-Pfalz, Breitenweg 71, D-67435 Neustadt, Germany
2 Klinikum der Johannes Gutenberg Universität Mainz, Institut für Humangenetik, Langenbeckstr. 1, D-55101 Mainz, Germany
Correspondence
K. Boonrod
kjohn.boonrod{at}dlr.rlp.de
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ABSTRACT |
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MAIN TEXT |
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Tomato bushy stunt virus (TBSV), the prototypical tombusvirus, encodes the two overlapping replicase proteins, p33 and p92 (Russo et al., 1994), which are essential for TBSV replication (Oster et al., 1998
; Scholthof et al., 1995
). Recently, Rajendran & Nagy (2003)
characterized RNA-binding domains, one of them being an arginine/proline-rich motif termed RPR, which has the sequence RPRRRP. This motif is highly conserved among tombusviruses and related carmoviruses and lies in the overlapping domain of p33 and p92. It is similar to the arginine-rich motif present in the Tat transactivator protein of human immunodeficiency virus type 1 (Bayer et al., 1995
; Calnan et al., 1991
).
Wang & Gillam (2001) investigated the functional role of the GDD motif of Rubella virus (RUBV). RUBV is a small positive-strand RNA virus belonging to the genus Rubivirus of the family Togaviridae (Weaver et al., 2000
). BHK cells were infected with RUBV RNA carrying mutations in the GDD motif and, by characterizing revertants isolated from infected BHK cells, Wang & Gillam (2001)
could determine the essential amino acid residues of the GDD motif. To establish a similar test system for a plant virus, we first generated a TBSV RdRp GDD mutant. The glycine residue in the GDD motif of the TBSV RdRp in position 620 was substituted by alanine (G620A) by using the QuikChange Site-Directed Mutagenesis kit (Stratagene) and primers 5'-GCAAACTGTGCAGATGACTGTG-3' and 5'-CACAGTCATCTGCACAGTTTGC-3' (nucleotide position 20352075, mutated codons are underlined). The resulting viral mutant was tested for infectivity on mechanically inoculated Nicotiana benthamiana plants. In contrast to N. benthamiana plants that were inoculated with infectious transcripts of non-mutated TBSV, these plants did not show any symptoms and no systemic infection 5 days post-inoculation (p.i.). However, about half of these plants established systemic infection 14 days p.i., suggesting reversion of the mutation. Viral RNA was extracted from these plants and the mutated region was amplified by RT-PCR (RT-PCR kit; Gibco-BRL). PCR products were cloned into a T-tailed pUC19 vector and three individual clones were sequenced (Big dye reaction mix; MWG). In all three clones the substituted alanine residue had reverted to the original glycine (G620). The G620A exchange was a double base change (GGG
GCA), in which only one base (C) reverted back (G), resulting in GGA. The non-reverted base (A) functions as a marker in our system, which helped us to exclude the possibility that our preparation of infectious transcripts or of viral RNA from infected plants was contaminated with (low amounts of) wild-type (wt) TBSV.
Our data are in accordance with previous studies on viral RdRps suggesting that the glycine residue of the GDD motif is somewhat flexible and that replacement of this residue does not completely abolish the functionality of RdRps (Hong & Hunt, 1996; Jablonski et al., 1991
; Lohmann et al., 1997
). Similarly, the G620 of the TBSV RdRp appeared to not completely abolish virus replication, allowing the mutation to revert. Until now, for tombusviruses, only the strict requirement of the first aspartate residue of the GDD motif has been demonstrated (Molinari et al., 1998
), confirming that any changes at this position are not tolerated for in vivo virus replication and/or in vitro RNA synthesis (Lohmann et al., 1997
; Longstaff et al., 1993
; Inokuchi & Hirashima, 1987
). Moreover, we were able to demonstrate that N. benthamiana is a suitable plant system to detect revertants of tombusviruses.
In a second experiment we generated a TBSV clone carrying mutations within the RPR motif (primers 5'-TCAACAGGAGGCTCTGAGGGAAGTCCCTACGCGG-3' and 5'-CCGCGTAGGGACTTCCTCTAGAGCCTCCTGTTGA-3', nucleotide position 795829, mutated codons are underlined). In this clone the RNA-binding domain was mutated by changing R213G, P214S, R216G and R217S (Table 1). After verification of the sequence transcripts of the mutated (TBSV KB1) and non-mutated TBSV clones, the clones were inoculated on N. benthamiana and Chenopodium quinoa (local lesion host of TBSV) plants, respectively. In contrast to the TBSV-infected plants that developed typical symptoms 4 days p.i., plants infected with transcripts of TBSV KB1 did not display symptoms during the whole period of the experiment (4 weeks) (Fig. 1
). RT-PCR with TBSV-specific primers using total RNA extracted from upper leaves of these plants also failed to amplify any product, demonstrating that TBSV KB1 was not able to establish (systemic) infection. In summary, these data clearly indicate that the RPR motif is essential for TBSV RdRp function and does not tolerate the above sequence alteration.
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Several revertants were isolated, and the genetic changes in their RdRps were analysed by RT-PCR amplification and sequencing of the corresponding products. In all three clones tested only the G216 had reverted to the original arginine residue while all other substitutions remained unchanged (Table 1). To confirm that the reversion of the G216 to arginine is sufficient to restore infectivity, we created a second mutated clone (TBSV KB2), displaying the same sequence as the infectious revertant of TBSV KB1. Transcripts of TBSV KB2 were inoculated on non-transgenic N. benthamiana plants. By 4 days p.i. all inoculated plants developed typical symptoms as plants inoculated with the wt virus (Fig. 1
). This clearly demonstrates that this single reversion was sufficient to restore infectivity.
In order to verify that the inability of the RPR mutant to infect plants systemically was caused by a defect in replication and not by e.g. a block in long-distance movement, we did a protoplast assay. Protoplasts were prepared from N. benthamiana leaves as described by Panaviene et al. (2003). Purified in vitro RNA transcripts of wt TBSV, TBSV KB1 and KB2 (1 µg) were electroporated into 5x105 protoplasts. After electroporation, the samples were incubated in 2 ml protoplast culture medium in the dark for 48 h at 22 °C. Total RNA was extracted from protoplasts (Nagy et al., 2001
) and analysed.
Aliquots of the total RNA were electrophoresed through a 1 % formaldehyde denaturing agarose gel, and viral RNAs were detected by transfer to a Nylon membrane (Amersham) followed by a Northern blot analysis using a digoxigenin probe complementary to the 3' end of genomic TBSV RNA. The result shows that the infectious TBSV clone and TBSV KB2 replicated in the protoplasts, whereas in the protoplasts infected with TBSV KB1 viral RNA could no longer be detected (Fig. 2).
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Panaviene et al. (2003) demonstrated that mutations within the RNA-binding domains of the CNV replicase proteins affected the frequency of recombination by delaying the formation of recombinants. Because the RNA-binding domain was also affected in the TBSV KB1, we assume that the recombination frequency was also hampered in the complementation system. Thus, to restore a functional RPR motif, reversion was more likely to occur than recombination. Alternatively or in addition, low expression of the RdRp construct, as was observed in all of our transgenic N. benthamiana plants, may explain the failure to detect any recombination of the TBSV KB1 with the transgene mRNA. However, it cannot be excluded that recombinants can also be isolated when the replicase function is provided in trans. Only three of the infectious TBSV isolates were analysed in detail. Characterization of a large number of infectious clones may result in detection of recombinants. However, in this study we would like to focus on revertants. In contrast to recombinants, revertants highlight single amino acid residues that are essential for the functionality of the motifs to be analysed.
Analysis of revertants was also successfully used to elucidate viral RNA and/or gene function of MS2 and Q phages (Klovins & van Duin, 1999
; Licis et al., 2000
; Olsthoorn et al., 1994
). In contrast to our work, however, viable phages were mainly restored by insertions or deletions and not by single base changes as we showed with our system.
Reversion of the TBSV KB1 mutation as reported here may illustrate the power of selection as a means of eliminating genetic drifts in viral genomes. A practical aspect of this observation concerns the identification of essential amino acids or amino acid motifs in viral proteins. Analysis of a population of viruses carrying random mutations in one or more essential viral genes and characterization of evolving revertants may help to identify essential virus functions.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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![]() ![]() ![]() ![]() |
---|
Calnan, B. J., Tidor, B., Biancalana, S., Hudson, D. & Frankel, A. D. (1991). Arginine-mediated RNA recognition: the arginine fork. Science 252, 11671171.[Medline]
Carrington, J. C. & Freed, D. D. (1990). Cap-independent enhancement of translation by a plant potyvirus 5' nontranslated region. J Virol 64, 15901597.[Medline]
Hong, Y. & Hunt, A. G. (1996). RNA polymerase activity catalyzed by a potyvirus-encoded RNA-dependent RNA polymerase. Virology 226, 146151.[CrossRef][Medline]
Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Rogers, S. G. & Fraley, R. T. (1985). A simple and general method for transferring genes into plants. Science 227, 12291231.
Inokuchi, Y. & Hirashima, A. (1987). Interference with viral infection by defective RNA replicase. J Virol 61, 39463949.[Medline]
Jablonski, S. A., Luo, M. & Morrow, C. D. (1991). Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif. J Virol 65, 45654572.[Medline]
Kamer, G. & Argos, P. (1984). Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses. Nucleic Acids Res 12, 72697282.[Abstract]
Klovins, J. & van Duin, J. (1999). A long-range pseudoknot in Q RNA is essential for replication. J Mol Biol 294, 875884.[CrossRef][Medline]
Koonin, E. V. (1991). The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses. J Gen Virol 72, 21972206.[Abstract]
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]
Licis, N., Balklava, Z. & Van Duin, J. (2000). Forced retroevolution of an RNA bacteriophage. Virology 271, 298306.[CrossRef][Medline]
Lohmann, V., Korner, F., Herian, U. & Bartenschlager, R. (1997). Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity. J Virol 71, 84168428.[Abstract]
Longstaff, M., Brigneti, G., Boccard, F., Chapman, S. & Baulcombe, D. (1993). Extreme resistance to potato virus X infection in plants expressing a modified component of the putative viral replicase. EMBO J 12, 379386.[Abstract]
Molinari, P., Marusic, C., Lucioli, A., Tavazza, R. & Tavazza, M. (1998). Identification of artichoke mottled crinkle virus (AMCV) proteins required for virus replication: complementation of AMCV p33 and p92 replication-defective mutants. J Gen Virol 79, 639647.[Abstract]
Nagy, P. D., Pogany, J. & Simon, A. E. (2001). In vivo and in vitro characterization of an RNA replication enhancer in a satellite RNA associated with Turnip crinkle virus. Virology 288, 315324.[CrossRef][Medline]
Olsthoorn, R. C., Licis, N. & van Duin, J. (1994). Leeway and constraints in the forced evolution of a regulatory RNA helix. EMBO J 13, 26602668.[Abstract]
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, 58455851.
Panaviene, Z., Baker, J. M. & Nagy, P. D. (2003). The overlapping RNA-binding domains of p33 and p92 replicase proteins are essential for tombusvirus replication. Virology 308, 191205.[CrossRef][Medline]
Poch, O., Sauvaget, I., Delarue, M. & Tordo, N. (1989). Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J 8, 38673874.[Abstract]
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, 92449258.
Rubino, L. & Russo, M. (1995). Characterization of resistance to cymbidium ringspot virus in transgenic plants expressing a full-length viral replicase gene. Virology 212, 240243.[CrossRef][Medline]
Russo, M., Burgyan, J. & Martelli, G. P. (1994). Molecular biology of tombusviridae. Adv Virus Res 44, 381428.[Medline]
Scholthof, K. B., Scholthof, H. B. & Jackson, A. O. (1995). The tomato bushy stunt virus replicase proteins are coordinately expressed and membrane associated. Virology 208, 365369.[CrossRef][Medline]
Wang, X. & Gillam, S. (2001). Mutations in the GDD motif of rubella virus putative RNA-dependent RNA polymerase affect virus replication. Virology 285, 322331.[CrossRef][Medline]
Weaver, S. C., Dalgarno, L., Frey, T. K., Huang, H. V., Kinney, R. M., Rice, C. M., Roehrig, J. T., Shope, R. E. & Strauss, E. G. (2000). Togaviridae. In Virus Taxonomy. Classification and Nomenclature of Viruses. Seventh Report of the International Committee on Taxonomy of Viruses, pp. 879889. Edited by M. H. V. van Regenmortel, C. M. Fauquet & D. H. L Bishop. San Diego: Academic Press.
Received 21 September 2004;
accepted 18 November 2004.
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