Tomas Canto,1 Seung Kook Choi2 and Peter Palukaitis1. 1 Virology Department, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK.
Journal of General Virology (2001), 82, 941–945. Printed in Great Britain ..........................................................................................................................................................................................................
A subpopulation of RNA 1 of Cucumber mosaic virus contains 3h termini originating from RNAs 2 or 3 Tomas Canto,1 Seung Kook Choi2 and Peter Palukaitis1 1 2
Virology Department, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK Graduate School of Biotechnology, Korea University, Seoul 136-701, Republic of Korea
Tobacco plants transgenic for RNA 1 of Cucumber mosaic virus and inoculated with transcript of RNAs 2 and 3 regenerated viral RNA 1 from the transgenic mRNA, and the plants became systemically infected by the reconstituted virus. cDNA fragments corresponding to the 3h non-coding region (NCR) of viral RNA 1 were amplified, cloned and sequenced. In some clones the termini of the 3h NCR corresponded to those of viral RNAs 2 or 3. This suggested that in some cases RNA 1 may have been regenerated during replication by a template switching mechanism between the inoculated transcript RNAs and the mRNA. However, encapsidated, recombinant RNA 1 with the 3h NCR ends originating from RNAs 2 or 3 also was found in virus samples that had been passaged exclusively through nontransgenic plants. Thus, these chimeras occur naturally due to recombination between wild-type viral RNAs, and they are found encapsidated in low, but detectable amounts.
Cucumber mosaic virus (CMV) is a tripartite icosahedral virus, with five major messenger-sense RNAs encoding viral proteins. RNAs 1 and 2 encode components of the viral RNAdependent RNA polymerase (RdRp) : the 1a and 2a proteins, with putative helicase and polymerase activities, respectively (Hayes & Buck, 1990). RNA 3 encodes the 3a movement protein and the capsid protein (CP). The latter is expressed from subgenomic RNA 4, which is the fourth encapsidated RNA. Both the 3a protein and the CP are required for the intercellular movement of CMV through plasmodesmata (Canto et al., 1997 ; Blackman et al., 1998). In addition, the small 2b protein is expressed from 3h-proximal sequences of RNA 2, via subgenomic RNA 4A (Ding et al., 1994). This protein is a pathogenicity determinant and plays a role in the longdistance movement of CMV (Brigneti et al., 1998 ; Ding et al., 1995). Author for correspondence : Peter Palukaitis. Fax j44 1382 562426. e-mail ppaluk!scri.sari.ac.uk
0001-7537 # 2001 SGM
The 3h-terminal 270 nucleotides of each CMV RNA are highly conserved, with only 7–10 % sequence differences scattered throughout this non-coding region (NCR) between any two RNAs. These 3h end sequences form tRNA-like structures that provide the initial binding site for the viral RdRp, and promote the synthesis of the (k)-sense RNA (Boccard & Baulcombe, 1993). In the Fny strain of CMV, the four encapsidated RNAs 1–4 do not accumulate in equimolar amounts. There is an inverse correlation between encapsidation level and size, with RNAs 3 and 4 being more abundant than RNAs 1 and 2. In addition to this, the relative levels of RNA 1 are higher at earlier stages than at later stages of infection, corresponding with higher early levels of 1a protein (Gal-On et al., 1995, 2000). The mechanisms that regulate the levels of accumulation of each RNA and the balance between replication and translation\encapsidation are unknown, but could involve the sequences of the 3h NCR (Duggal et al., 1992). If so, one might expect selection of recombinant RNAs generated in the 3h NCR. Recombination in the 3h NCR of CMV RNAs and selection of recombinant RNAs might also occur in transgenic plants expressing such sequences. In tobacco plants transgenic for RNA 1 of Fny-CMV, viral RNA 1 was regenerated from the transgenic mRNA when plants were inoculated with viral RNAs 2 and 3. The plants became systemically infected with reconstituted wild-type virus, inducing typical CMV symptoms (Canto & Palukaitis, 1998). For this to occur, the transgenic mRNA had to lose the non-viral sequences present at both the 5h and the 3h ends (Fig. 1). There are three ways in
Fig. 1. Schematic representation of the transgene harboured by RNA 1transgenic tobacco. NPTII, 35S, T7 and NOS Ter refer to neomycin phosphotransferase II, cauliflower mosaic virus 35S RNA promoter, T7 RNA promoter and nopaline synthase gene transcriptional terminator, respectively. The T7 promoter was included in the transformation vector in order to produce full-length transcript RNA 1 in vitro and confirm that the transgene encodes biologically active 1a protein (Canto & Palukaitis, 1998). The DNA sequence corresponding to the open reading frame of the 1a protein is shown as a black solid rectangle. The 3h end of the 1a protein gene contains a sequence motif that encodes 6ihistidine (Gal-On et al., 2000). The 5h and 3h flanking viral non-coding regions are shown as single lines.
T. Canto, S. K. Choi and P. Palukaitis
Fig. 2. Schematic representation of the cloned sequences corresponding to the viral 3h non-coding regions amplified from : RNA 1 of CMV regenerated in RNA 1-transgenic plants that had been inoculated with transcript RNAs 2 and 3 (a) ; RNA 1 of CMV regenerated in a RNA 1-transgenic plant, after its passage to and propagation in a non-transgenic plant (b) ; encapsidated RNAs 1, 2 and 3/4 of CMV obtained from non-transgenic plants that had been inoculated with transcript RNAs 1, 2 and 3 (c). The frequency with which wild-type or recombinant sequences were encountered over the total of clones sequenced is shown to the right. RNA 1 and recombinant sequences from RNAs 2 and 3 are indicated by black or hatched boxes, respectively. The
3h termini of CMV RNA 1 subpopulation
which the 3h non-viral sequences of the transgenic mRNA could be lost : (i) the viral RdRp could initiate replication of the (k)-strand at the correct viral RNA initiation site internally on the intact transgenic mRNA ; (ii) initiation could occur only on templates of transgenic mRNA partially degraded at the 3h end to remove non-viral sequences ; (iii) template viral RNA 1 could be generated by the initiation of replication on the 3h end of viral RNAs 2, 3 or 4, followed by early template-switching to the transgenic mRNA. In the latter case, the regenerated viral RNA 1 would be recombinant, with the terminus of the 3h NCR originating from one of the other viral RNAs. Therefore, an examination of the terminal sequences of individual RNA 1 molecules should allow a determination as to whether recombination played any role in the regeneration of RNA 1. Transgenic plants were inoculated with CMV transcript RNAs 2 and 3 and 2 weeks after the inoculation the plants were monitored for presence of symptoms of infection in systemic tissue. This is an indicator of regeneration of viral RNA 1 (Canto & Palukaitis, 1998). Five out of nine inoculated plants became systemically infected. Total nucleic acids were extracted from systemic leaves as described previously (Kaplan et al., 1995). RNAs were precipitated selectively with lithium chloride, and the precipitated RNA was treated with RQ1 DNase in order to eliminate any contaminating transgene DNA from the RNA preparation. The 3h NCR sequences of the viral RNA 1 were transcribed into cDNA by reverse transcription, using a primer complementary to the last 11 nucleotides of all CMV RNAs (nucleotides 3346– 3357 in the sequence of Fny-CMV RNA 1) and containing a SacI site at its 3h end. This cDNA was amplified by PCR, also using this same complementary primer plus a primer homologous to nucleotides 3074–3087 of Fny-CMV RNA 1 (Rizzo & Palukaitis, 1989). The latter primer also had a SpeI site at its 5h end. The cDNA fragments thus obtained were digested with SacI and SpeI, and were cloned into plasmid pUC18, linearized with XbaI and SacI. All RT–PCR, enzyme digestions and cloning manipulations were done as described by Sambrook et al. (1989). In total 81 different clones isolated from individual E. coli colonies were analysed by automated DNA sequencing (Perkin Elmer), using primers homologous to sequences flanking the polylinker of pUC18. Of the 81 clones of the 3h NCR of CMV RNA 1, 77 contained the wild-type sequence of Fny-CMV RNA 1, while four contained sequences indicating that recombination had occurred between RNA 1 and RNA 2 (three clones), or between RNA 1 and RNA 3 (or 4) (one clone) (Fig. 2 a). The four recombinants were detected in samples from three of the five plants in which reconstitution of viral RNA 1 took place. Virus reconstituted in transgenic plants was mechanically
inoculated to non-transformed tobacco in order to ascertain whether these subpopulations of RNA 1 with recombinant 3h NCRs could still be found after the passage to a non-transgenic host. The 3h NCRs of the viral RNA 1 isolated from systemic leaves were analysed in a way similar to the analysis performed in transgenic plants, omitting the lithium chloride precipitation and RQ1 DNase treatment (due to the absence of transgenic DNA sequences). From 24 different clones obtained from individual E. coli colonies, 20 contained the 3h NCR of wildtype CMV RNA 1, while four were recombinants : one between RNA 1 and RNA 2, and three between RNA 1 and RNA 3 (or 4) (Fig. 2 b). These 24 clones were obtained from five different plants, and the four recombinants were detected in samples from three of the five plants analysed. To determine whether recombinants between wild-type viral RNAs also occurred during replication, Fny-CMV transcript RNAs 1, 2 and 3 (Rizzo & Palukaitis, 1990) were inoculated to non-transgenic plants, the encapsidated viral RNAs were purified from systemic tissue as described by Kaplan et al. (1995), and the 3h NCRs were cloned and sequenced. The cloning procedure for RNA 1 sequences was as described above. For the amplification and cloning of cDNAs corresponding to the 3h NCRs of RNA 2 and RNA 3, the homologous primers used corresponded to nucleotides 2665– 2676 of RNA 2, and nucleotides 1919–1932 of RNAs 3\4, respectively. Both primers contained a SpeI site at their 5h ends. Two recombinants were found out of the 18 cDNA clones examined corresponding to the 3h NCR of RNA 1. In one of them, recombination took place between RNA 1 and RNA 2 and in the other one, between RNA 1 and RNA 3 (or 4) (Fig. 2 c). No recombinants were found among the 18 different cDNA clones corresponding to the 3h NCR of either RNA 2 or RNAs 3\4 (Fig. 2 c). These results indicate that during replication recombination events in the 3h NCR occurred between wild-type viral RNA 1 and the remaining viral RNAs. The possibility that the recombinants detected were artefactual chimeras generated by the analysis procedure is unlikely. The precise nature of all the recombinants found, containing neither additions nor deletions of viral sequences, would not be expected if the sequences obtained were artefacts of the copying activity of the reverse transcriptase or the Taq polymerase during the RT–PCR process. Rather, the lack of sequence alterations suggests that the sequences have been selected to be functional. Nevertheless, to test the possibility that the recombinants were an artefact of the RT–PCR, a mixture of Fny-CMV transcript RNAs 1, 2 and 3 was treated with RQ1 DNase to remove the original plasmid templates, and the remaining RNA was precipitated with lithium chloride. This RNA sample was used as template for the RT–PCR amplification of the 3h NCR of RNA 1, as described above. As
nucleotide domains within which recombination took place are indicated in white. The nucleotide numbers refer to the Fny-CMV RNA 1 sequence. The numbers in (a) and (b) were obtained by pooling the clones obtained from five different plants, in both cases.
T. Canto, S. K. Choi and P. Palukaitis
a control, the same procedure was followed in a parallel sample, but omitting the addition of reverse transcriptase. A DNA fragment was amplified by RT–PCR only from the sample that contained reverse transcriptase, indicating that there was no contaminating DNA left in the transcript RNA sample (data not shown). This PCR fragment was cloned, and the 18 cDNA clones were sequenced. No recombinants were found (data not shown). Due to the very high level of sequence identity of the region, the exact site at which recombination took place could not be determined for any recombinant (Fig. 2). However, the recombination process was precise with neither loss nor addition of viral or non-viral sequences, and the recombination events did take place along most of the 3h NCR (Fig. 2). The presence of a subpopulation of RNA 1 with recombinant 3h NCRs after the passage of regenerated virus to nontransformed tobacco (Fig. 2 b) suggested that these recombinants either were not lost during the mechanical passage or were being generated continuously during replication by recombination between wild-type RNAs. The detection of recombinants in virus samples that had no contact with transgenic plants (Fig. 2 c) indicated that the latter was the case. The analysis of the sequencing data also suggests that the rescue of viral RNA 1 in transgenic plants inoculated with transcript RNAs 2 and 3 occurred only to a low extent, if at all, by template switching between the transgenic mRNA and the inoculated viral RNAs 2 and 3. Initiation by the RdRp at the appropriate terminal sequence in the transgenic mRNA, yielding wild-type viral RNA 1, is more likely. In any event, the latter is the ultimate source of most of the RNA 1 that accumulated systemically after the regeneration of virus in transgenic plants. Chimeric RNAs have been reported to occur in vivo through recombination at the 3h ends of Brome mosaic virus (BMV), in order to restore the functionality of a 3h enddefective RNA 2 (Rao et al., 1990 ; Rao & Hall, 1990). However, this report provides the first demonstration of homologous recombinations at the 3h termini of a member of the Bromoviridae, CMV, which took place in vivo among functional, wild-type RNAs, between RNA 1 and the other CMV RNAs. Furthermore, it provides evidence for the presence of a subpopulation of RNA 1 with heterologous 3h NCR ends. The driving force for recombination may have been the high degree of secondary structure in this region. However, recombination took place along most of the 3h NCR (Fig. 2). Does the existence of these recombinant viral RNA 1 molecules have any biological purpose? It is known that in BMV, another member of the Bromoviridae, the nucleotide differences at the 3h end of the different viral RNAs, within the tRNA-like structures, have a modulatory effect on the levels of replication and accumulation of each viral RNA (Duggal et al., 1992). The effectiveness with which these 3h ends bind the viral RdRp and promote the synthesis of (k)-strand RNA during replication is altered by the nucleotide differences, but JEE
the context upstream of these sequences is also important (Duggal et al., 1992 ; Chapman et al., 1998). The fact that our recombinants can be detected with relative facility indicates that they are successfully replicated by the viral RdRp. This suggests that the sequences 5h of the 3h terminus in Fny-CMV RNA 1 could be more compatible with the 3h terminus of the other CMV RNAs than in BMV (Duggal et al., 1992 ; Chapman et al., 1998). This work was supported by grant-in-aid from the Scottish Executive Rural Affairs Department.
References Blackman, L. M., Boevinck, P., Santa Cruz, S., Palukaitis, P. & Oparka, K. J. (1998). The movement protein of cucumber mosaic virus traffics
into sieve elements in minor veins of Nicotiana clevelandii. Plant Cell 10, 525–537. Boccard, F. & Baulcombe, D. (1993). Mutational analysis of cis-acting sequences and gene function in RNA 3 of cucumber mosaic virus. Virology 193, 563–578. Brigneti, G., Voinnet, O., Li, W.-X., Ding, S.-W. & Baulcombe, D. (1998). Viral pathogenicity determinants are suppressors of transgene
silencing in Nicotiana benthamiana. EMBO Journal 22, 6739–6746. Canto, T. & Palukaitis, P. (1998). Transgenically expressed cucumber mosaic virus RNA 1 simultaneously complements replication of cucumber mosaic virus RNAs 2 and 3 and confers resistance to systemic infection. Virology 250, 325–336. Canto, T., Prior, D. A. M., Hellwald, K-H., Oparka, K. J. & Palukaitis, P. (1997). Characterization of cucumber mosaic virus. IV. Movement
protein and coat protein are both essential for cell-to-cell movement of cucumber mosaic virus. Virology 237, 237–248. Chapman, M. R., Rao, A. L. N. & Kao, C. C. (1998). Sequences 5h of the conserved tRNA-like promoter modulate the initiation of minus-strand synthesis by brome mosaic virus RNA-dependent RNA polymerase. Virology 252, 458–467. Ding, S.-W., Anderson, B. J., Haase, H. R. & Symons, R. H. (1994).
New overlapping gene encoded by the cucumber mosaic virus genome. Virology 198, 593–601. Ding, S.-W., Li, W.-X. & Symons, R. H. (1995). A novel naturally occurring hybrid gene encoded by a plant RNA virus facilitates long distance virus movement. EMBO Journal 14, 5762–5772. Duggal, R., Rao, A. L. N. & Hall, T. C. (1992). Unique nucleotide differences in the conserved 3h termini of brome mosaic virus RNAs are maintained through their optimization of genome replication. Virology 187, 261–270. Gal-On, A., Kaplan, I. & Palukaitis, P. (1995). Differential effects of satellite RNA on the accumulation of cucumber mosaic virus RNAs and their encoded proteins in tobacco vs zucchini squash with two strains of CMV helper virus. Virology 208, 58–66. Gal-On, A., Canto, T. & Palukaitis, P. (2000). Characterisation of genetically modified cucumber mosaic virus expressing histidine-tagged 1a and 2a proteins. Archives of Virology 145, 37–50. Hayes, R. J. & Buck, K. W. (1990). Complete replication of a eukaryotic virus RNA in vitro by a purified RNA-dependent RNA polymerase. Cell 63, 363–368. Kaplan, I. B., Shintaku, M. H., Li, Q., Zhang, L., Marsh, L. & Palukaitis, P. (1995). Complementation of virus movement in transgenic tobacco
expressing the cucumber mosaic virus 3a gene. Virology 209, 188–199.
3h termini of CMV RNA 1 subpopulation Rao, A. L. N. & Hall, T. C. (1990). Requirement of a viral trans-acting factor encoded by brome mosaic virus RNA-2 provides strong selection in vivo for functional recombinants. Journal of Virology 64, 2437–2441. Rao, A. L. N., Sullivan, B. P. & Hall, T. C. (1990). Use of Chenopodium hybridum facilitates isolation of brome mosaic virus RNA recombinants. Journal of General Virology 71, 1403–1407. Rizzo, T. M. & Palukaitis, P. (1989). Nucleotide sequence and evolutionary relationships of cucumber mosaic virus (CMV) strains : CMV RNA 1. Journal of General Virology 70, 1–11.
Rizzo, T. M. & Palukaitis, P. (1990). Construction of full-length cDNA clones of cucumber mosaic virus RNAs 1, 2, and 3 : generation of infectious RNA transcripts. Molecular and General Genetics 222, 249–256. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning : A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY : Cold Spring Harbor Laboratory.
Received 10 November 2000 ; Accepted 21 December 2000