Virology Department, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK1
Graduate School of Biotechnology, Korea University, Seoul 136-701, Republic of Korea2
Author for correspondence: Peter Palukaitis. Fax +44 1382 562426. e-mail ppaluk{at}scri.sari.ac.uk
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Abstract |
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The 3'-terminal 270 nucleotides of each CMV RNA are highly conserved, with only 710% sequence differences scattered throughout this non-coding region (NCR) between any two RNAs. These 3' end sequences form tRNA-like structures that provide the initial binding site for the viral RdRp, and promote the synthesis of the (-)-sense RNA (Boccard & Baulcombe, 1993 ). In the Fny strain of CMV, the four encapsidated RNAs 14 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 3' NCR (Duggal et al., 1992
). If so, one might expect selection of recombinant RNAs generated in the 3' NCR.
Recombination in the 3' 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 5' and the 3' ends (Fig. 1
). There are three ways in which the 3' non-viral sequences of the transgenic mRNA could be lost: (i) the viral RdRp could initiate replication of the (-)-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 3' end to remove non-viral sequences; (iii) template viral RNA 1 could be generated by the initiation of replication on the 3' 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 3' 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.
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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 3' 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. 2a). The four recombinants were detected in samples from three of the five plants in which reconstitution of viral RNA 1 took place.
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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 3' 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 3' NCRs of RNA 2 and RNA 3, the homologous primers used corresponded to nucleotides 26652676 of RNA 2, and nucleotides 19191932 of RNAs 3/4, respectively. Both primers contained a SpeI site at their 5' ends. Two recombinants were found out of the 18 cDNA clones examined corresponding to the 3' 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. 2c
). No recombinants were found among the 18 different cDNA clones corresponding to the 3' NCR of either RNA 2 or RNAs 3/4 (Fig. 2c
). These results indicate that during replication recombination events in the 3' 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 RTPCR 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 RTPCR, 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 RTPCR amplification of the 3' NCR of RNA 1, as described above. As a control, the same procedure was followed in a parallel sample, but omitting the addition of reverse transcriptase. A DNA fragment was amplified by RTPCR 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 3' NCR (Fig. 2
).
The presence of a subpopulation of RNA 1 with recombinant 3' NCRs after the passage of regenerated virus to non-transformed tobacco (Fig. 2b) 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. 2c
) 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 3' ends of Brome mosaic virus (BMV), in order to restore the functionality of a 3' end-defective RNA 2 (Rao et al., 1990 ; Rao & Hall, 1990
). However, this report provides the first demonstration of homologous recombinations at the 3' 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 3' 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 3' 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 3' 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 3' ends bind the viral RdRp and promote the synthesis of (-)-strand RNA during replication is altered by the nucleotide differences, but 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 5' of the 3' terminus in Fny-CMV RNA 1 could be more compatible with the 3' terminus of the other CMV RNAs than in BMV (Duggal et al., 1992
; Chapman et al., 1998
).
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References |
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Received 10 November 2000;
accepted 21 December 2000.