Expression of tombusvirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, RNA

Luisa Rubino, Vitantonio Pantaleo, Beatriz Navarro and Marcello Russo

Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi and Istituto di Virologia Vegetale del CNR, Sezione di Bari, Via Amendola 165/A, 70126 Bari, Italy

Correspondence
Luisa Rubino
l.rubino{at}area.ba.cnr.it


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Yeast cells co-expressing the replication proteins p36 and p95 of Carnation Italian ringspot virus (CIRV) support the RNA-dependent replication of several defective interfering (DI) RNAs derived from either the genome of CIRV or the related Cymbidium ringspot virus (CymRSV), but not the replication of a satellite RNA (sat RNA) originally associated with CymRSV. DI, but not sat RNA, was replicated in yeast cells co-expressing both DI and sat RNA. Using transgenic Nicotiana benthamiana plants constitutively expressing CymRSV replicase proteins (p33 and p92), or transiently expressing either these proteins or CIRV p36 and p95, it was shown that expression of replicase proteins alone was also not sufficient for the replication of sat RNA in plant cells. However, it was also shown that replicating CIRV genomic RNA deletion mutants encoding only replicase proteins could sustain replication of sat RNA in plant cells. These results suggest that sat RNA has a replication strategy differing from that of genomic and DI RNAs, for it requires the presence of a cis-replicating genome acting as a trans-replication enhancer.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Tombusviruses are small isometric plant viruses belonging to the genus Tombusvirus, family Tombusviridae. Their genome is a monopartite, single-stranded, positive-sense RNA of 4800 nt containing five ORFs. ORF1 and -2 encode the replicase proteins of 33 or 36 kDa (p33 or p36), depending on the species, and the corresponding readthrough products, p92 and p95. ORF3 encodes the coat protein (CP), and two nested ORFs (ORF4 and -5) encode two proteins involved in virus movement and pathogenesis (Russo et al., 1994; Silhavy et al., 2002). Tombusvirus infections are often associated with small parasitic RNAs, which can be either defective interfering (DI) or satellite (sat) RNAs. DI RNAs are shortened forms of genomic RNA deprived of all viral genes required for replication, encapsidation and movement of the viral genome. They are generated by errors of the viral replicase, which bypasses local intramolecular base-paired regions of genomic RNA, thus leading to the synthesis of deletion mutants. All described tombusvirus DI RNA molecules contain segments of viral RNA, derived from the 5' leader sequence, the replicase and movement protein genes and the 3' non-coding sequence. Tombusviruses can support the replication of homologous or heterologous DI RNA in plant (Havelda et al., 1998) or yeast cells (Pantaleo et al., 2003, 2004; Panavas & Nagy, 2003). Tombusvirus DI RNAs have been widely used for the analysis of cis-acting factors directing the replication of viral RNA both in vivo (Havelda & Burgyan, 1995; Havelda et al., 1995; Chang et al., 1995; Ray & White, 1999, 2003; White & Morris, 1994; Wu & White, 1998; Wu et al., 2001) and in vitro (Nagy & Pogany, 2000; Panavas et al., 2002a, b; Panavas & Nagy, 2003).

The molecular biology of tombusvirus sat RNAs has been much less studied. So far, the best characterized sat RNA is the 621 nt sat RNA of Cymbidium ringspot virus (CymRSV) whose cis-acting sequence requirements essential for replication and the significance and nature of multimeric forms found in infected tissues have been studied (Rubino et al., 1990; Dalmay & Rubino, 1994, 1995). Two additional satellites, associated with natural infections by Tomato bushy stunt virus (TBSV) (Celix et al., 1997), have been studied in relation to helper virus replication and symptom modulation in infected plants (Celix et al., 1999). Sat RNAs contain little sequence homology with the viral genome other than a region of approximately 50 nt present both in the 5' non-coding region of the corresponding helper genomes and in all DI RNAs of tombusviruses (Rubino et al., 1990; Russo et al., 1994; Celix et al., 1997). The replication of sat RNA originally associated with CymRSV infection can also be supported in plants by the related species Artichoke mottled crinkle virus and Carnation Italian ringspot virus (CIRV) (M. Russo, unpublished information).

The availability of a yeast-based system to analyse the replication of tombusviruses prompted us to reinvestigate the flexibility of the system using several CIRV or CymRSV DI RNA species and CymRSV sat RNA. It was found that the yeast system allowing the replication of homologous and heterologous DI RNAs did not support the replication of sat RNA.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Yeast strain, transformation, cell growth and plasmids.
The yeast strain used and its growth were as described by Pantaleo et al. (2003). CIRV p36 and p95 were tagged at their N termini with the epitopes c-Myc and haemagglutinin (HA), respectively, and expressed from plasmids pAc-Myc36K and YEHA95K using the alcohol dehydrogenase gene (ADH1) promoter (Pantaleo et al., 2004). The replication templates were amplified from the appropriate clones in pUC18 by PCR with primers designed to represent the exact 5' and 3' ends and cloned into plasmid pB3MI3S, digested with SnaBI and SmaI (Ishikawa et al., 1997; Pantaleo et al., 2003). The resulting plasmids (indicated throughout as pB*, where * stands for the intended sequence) had a transcription cassette comprising the galactose-inducible (GAL1) promoter, the viral sequence, the Tobacco ringspot virus ribozyme, the ADH1 terminator and one of the selectable markers TRP1 or URA3. The replication templates originated from: (i) CIRV DI-7. This molecule was the same as that used previously (Rubino et al., 1995; Pantaleo et al., 2003), except that the pBDI-7 clone used in the present work contained the natural DI RNA 5' end of AGAAA instead of GGAAA; (ii) CymRSV DI-13 and DI-3. These templates (679 and 481 nt, respectively) were derived from CymRSV genomic RNA (Burgyan et al., 1991, 1992); (iii) CymRSV sat RNA. Prior to cloning, the 5'-terminal nucleotide of CymRSV satellite RNA, which had not been determined by previous sequence analysis (Rubino et al., 1990), was identified as an A by RT-PCR, as previously described (Rubino et al., 1995). The cDNA was cloned either in vector pBMI3S carrying TRP1 as a selectable marker or in the same vector after substituting URA3 for the TRP1 marker. To do this, two BamHI sites were introduced by site-directed mutagenesis (QuickChange; Stratagene) at the borders of the TRP1 gene, which was substituted with the URA3 gene excised from the plasmid YDp-U containing the URA3 gene bordered by BamHI sites (Berben et al., 1991); (iv) CIRV {Delta}B/H, an artificial deletion mutant of the CIRV genome obtained by digestion of the full-length infectious clone with BamHI (at nt 2653, 20 nt downstream of the termination codon of ORF2) and HpaI (at nt 4154, inside the nested ORF4 and -5), followed by filling in and religation.

RNA and protein analysis.
Total yeast RNA was extracted and analysed as described previously (Pantaleo et al., 2003). Riboprobes for positive- and negative-strand DI or sat RNAs were obtained by cloning approximately 300 nt of the 3'-terminal regions of DI or sat RNAs in the vector pTL7SN (Oh & Carrington, 1989) in the appropriate orientation.

Protein extraction, electrophoresis and Western blot analysis were done as previously described (Rubino et al., 2000), except that the replicase proteins were detected using anti-c-Myc or anti-HA antibodies (Santa Cruz Biotechnology) (Pantaleo et al., 2004).

Plant growth, agroinfiltration and protoplasts.
Transgenic Nicotiana benthamiana plant line 92KA11, expressing the full-length CymRSV replicase gene (Rubino et al., 1993), and non-transgenic plants were grown in growth chambers at 25 °C with a 14 h photoperiod. For agroinfiltration, sequences encoding the proteins p92 and p95 were cloned into the expression vector pRTL2 (Carrington et al., 1990) and DI-3 and sat RNA sequences were cloned into the vector pRT101 (Töpfer et al., 1987). Expression cassettes were excised from the corresponding constructs and inserted into the binary vector pGA482. The resulting plasmids were electroporated into Agrobacterium tumefaciens strain C58C1. Recombinant clones were cultured and bacterial suspensions of each clone were infiltrated alone or in combination into N. benthamiana leaves as described previously (Rubino et al., 2001). For co-infiltration, equal volumes of the designated cultures were mixed before infiltration, i.e. p92 and DI-3 or sat RNA, p95 and DI-3 or sat RNA. Several small tissue pieces showing clear signs of infiltration were excised after 24–48 h and nucleic acids were extracted as described previously (Rubino et al., 2001). Preparation of protoplasts from fully expanded leaves, transfection and RNA analysis were done as previously described (Dalmay et al., 1993a; Rubino et al., 2001). Protoplasts were transfected with full-length CIRV genomic RNA (Burgyan et al., 1996) or deletion mutants {Delta}B/H (see above) or {Delta}B/N (obtained by restriction of the CIRV full-length clone with BamHI and NcoI at nt 3870, at the beginning of ORF4), together with DI (Burgyan et al., 1992) or sat (Rubino et al., 1990) RNA in vitro transcripts.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
DI, but not satellite, RNAs replicate in yeast cells
To investigate whether the yeast system could replicate subviral molecules other than DI RNAs, pBsat-transformed yeast cells expressing the CIRV replicase proteins (p36+p95+; Fig. 1, lower panel, lanes 3 and 4) or not (p36p95; Fig. 1, lower panel, lanes 1 and 2) were cultured for 24 h in the presence of galactose at 26 °C, to induce GAL1-driven transcription. A small aliquot (0·25 OD) was then inoculated on to a GAL1-repressive medium, containing glucose, where yeasts were cultured for an additional 24 h at 26 °C. Total RNA was extracted from induced and repressed cultures and analysed by Northern blotting using a radioactive riboprobe. As shown in Fig. 1, analysis of RNA extracted from p36p95 (lane 1) or p36+p95+ (lane 3) cells expressing the CymRSV sat RNA grown in the presence of galactose showed the presence of two sat RNA-related bands, regardless of the presence of the viral proteins. By analogy with previous analysis of DI RNA expression (Pantaleo et al., 2003), the fastest band, co-migrating approximately with sat RNA extracted from CIRV plus sat RNA-infected plants (not shown), was composed of transcripts cleaved by the ribozyme, and the slowest, with an approximate size of 1·2 kb, of uncleaved molecules of sat RNA plus the vector terminator sequence and the poly(A) tail. When the same transformants were grown in the GAL1-repressive medium (containing 0·1 % glucose), these bands were no longer present (Fig. 1, lanes 2 and 4), suggesting that no CIRV replicase-dependent replication of sat RNA took place.



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Fig. 1. Northern (upper panel) and Western (lower panel) blot analysis of RNA and protein extracts from yeast cells transformed with plasmid pBsat and expressing p36 and p95 (lanes 3 and 4) or not (lanes 1 and 2) grown in the presence of galactose (Gal) or glucose (Glu). The positions of sat RNA molecules cleaved or not by the ribozyme (Rz) are shown.

 
As a control of the system composed of CIRV-derived replicase/CymRSV-derived template, yeast cells were transformed with the same plasmids expressing the CIRV replicase and pBDI-3 or pBDI-13, both containing CymRSV-derived DI RNA sequences, as well as with pBDI-7, containing the CIRV-derived DI-7 RNA sequence (Pantaleo et al., 2003). RNA extracted from p36p95 (Fig. 2a, lanes 1–3) and p36+p95+ (Fig. 2a, lanes 4–6) cells expressing the different DI RNAs and cultured in the GAL1-inductive medium, contained two bands, the fastest with the expected size of monomeric DI RNAs and the slowest composed of transcripts not cleaved by the ribozyme. One major band corresponding to monomeric DI RNAs was detected in the extracts from p36+p95+ transformants grown in the GAL1-repressive medium, occasionally accompanied by a faint slower band corresponding in size to dimers (Fig. 2a, lanes 10–12). No DI RNA-related signal was detected in the cell extracts of p36p95 transformants grown in the GAL1-repressive medium (Fig. 2a, lanes 7–9).



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Fig. 2. Northern (a, upper panel; b) and Western (a, lower panel) blot analysis of RNA and protein extracts from yeast cells transformed with plasmids pBDI-3 (lanes 1, 4, 7 and 10), pBDI-13 (lanes 2, 5, 8 and 11) or pBDI-7 (lanes 3, 6, 9 and 12) and expressing p36 and p95 (lanes 4–6 and 10–12) or not (lanes 1–3 and 7–9). Cells were grown in the presence of galactose (Gal) or glucose (Glu). Northern blot analysis showed DI RNA-derived positive-strand (a) and negative-strand (b) RNAs. The positions of the diverse DI RNA molecules cleaved or not by the ribozyme (Rz) are shown at the left of (a) and those resulting from replication (monomers and dimers) are shown at the right of (a) and (b). Positive-strand dimers of DI-13 and DI-7 RNAs were generally very faint and barely detectable.

 
When RNA extracts were analysed for the presence of negative-sense RNA, two bands were detected in extracts of p36+p95+ yeasts expressing the different DI RNAs grown either in galactose (Fig. 2b, lanes 4–6) or glucose (Fig. 2b, lanes 10–12). The fastest bands corresponded to monomers and the slowest to dimers. The amount of monomeric and dimeric DI RNAs was approximately equivalent, in contrast to positive-sense RNA, where dimers were scarcely present (Fig. 2a). No negative-strand RNA bands were detected in the extracts of p36p95 cells expressing DI RNAs (Fig. 2b, lanes 1–3 and 7–9) or p36+p95+ or p36p95 cells expressing sat RNA (not shown).

The above results indicated that the p36+p95+ transformants were indeed fully competent for the replication of subviral RNAs, regardless of whether these were originally associated with CIRV (DI-7 RNA) or CymRSV (DI-3 and DI-13 RNAs).

Since previous analysis has shown that the replication of sat RNA in plants is inhibited by temperatures above 25 °C (Burgyan & Russo, 1988), p36+p95+ yeast cells expressing either DI-3 or sat RNA were cultured at 26 and 20 °C. At the mid-exponential phase, cells were extracted and examined by Northern blotting. While DI RNA replication still took place in cells grown at 20 °C although yielding fewer progeny than at 26 °C, no sat RNA was synthesized at either temperature (not shown).

The replication of DI RNA does not transactivate the replication of sat RNA
Knowing that both DI and sat RNA can replicate in the same plant cell (Rubino et al., 1992), yeast cells were co-transformed with plasmids expressing CIRV replicase proteins and plasmids pBsat and pBDI-3 to see whether sat RNA transcripts could replicate in yeast cells in which an active replication complex was formed by another template RNA, such as DI RNA. However, since these plasmids must contain different nutritional markers to permit simultaneous selection, pBsat was first engineered to have the marker URA3 in place of TRP1. Cell growth and GAL1 induction and repression were performed as before, and Northern blot analysis was carried out in duplicate using DI RNA- and sat RNA-specific probes. Robust DI RNA replication took place either in galactose-containing (Fig. 3a, lanes 1 and 2) or glucose-containing (Fig. 3a, lanes 4 and 5) medium, regardless of whether yeast cells expressed sat RNA (Fig. 3a, lanes 1 and 4) or not (Fig. 3a, lanes 2 and 5). By contrast, there was no evidence of sat RNA replication, regardless of the presence (Fig. 3b, lanes 1 and 4) or absence (Fig. 3b, lanes 3 and 6) of DI RNA, notwithstanding the active plasmid DNA-dependent transcription (Fig. 3b, lanes 1 and 3), which was not affected by the concomitant transcription of DI RNA (Fig. 3b, lane 1). These results suggested that a replication complex was correctly formed in transformed yeast cells, but that it was unsuitable for the replication of sat RNA, even if already activated by the replicating DI RNA.



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Fig. 3. Northern blot analysis of RNA extracts from yeast cells expressing p36 and p95 replicase proteins and both DI-3 and sat RNA (lanes 1 and 4) or either DI-3 (lanes 2 and 5) or sat RNA (lanes 3 and 6) alone. Blots were hybridized with DI (a) or sat (b) RNA-specific probes.

 
The replication of CymRSV sat RNA with defective helper genomes
The question arose as to whether the inability to obtain replication of sat RNA expressing only the viral replicase proteins was a characteristic of recombinant yeasts or whether the same phenomenon occurred in plant cells. It was previously shown that uninfected transgenic N. benthamiana plant cells expressing the native ORF2 sequence of CymRSV (i.e. containing the leaky amber stop codon of p33), could synthesize both p33 and p92 proteins (Lupo et al., 1994). Moreover, protoplasts from these plants transfected with CymRSV DI RNA in vitro transcripts were shown to support the replication of this molecule (Rubino & Russo, 1995). A similar approach was therefore followed to test whether expression of p33 and p92 alone was sufficient to promote the replication of sat RNA in planta.

Protoplasts were extracted from fully expanded leaves of the transgenic N. benthamiana line 92KA11, which expresses the active CymRSV replicase under the control of the cauliflower mosaic virus 35S promoter and terminator (Rubino & Russo, 1995). Approximately 106 cells were transfected with 50 ng of in vitro-synthesized DI or sat RNA together or not with the helper genome (200 ng) or with water, as described previously (Dalmay et al., 1993a). Protoplasts were incubated for 24 h at room temperature (22–25 °C) under continuous fluorescent light, then disrupted and extracted with phenol. RNA extracts were analysed by Northern blotting. Whereas transgenic protoplasts supported the replication of DI RNA in the absence or presence of the helper (Fig. 4a, lanes 1 and 3, respectively), sat RNA replication was not detected when it was transfected alone, but occurred when there was co-transfection with genomic RNA (Fig. 4b, lane 1 and 3, respectively). No signal was detected in extracts of mock-inoculated protoplasts (Fig. 4a and b, lane 2).



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Fig. 4. (a, b) Northern blot analysis of extracts from protoplasts obtained from transgenic N. benthamiana plants expressing CymRSV p92 (a, b) transfected with DI RNA alone, water, or DI and CIRV genomic RNAs (a, lanes 1, 2 and 3, respectively), or sat RNA alone, water, or satellite and CIRV genomic RNAs (b, lanes 1, 2 and 3, respectively). (c) Northern blot analysis of extracts from non-transgenic N. benthamiana plants agroinfiltrated with bacteria expressing DI RNA together with CymRSV p92 (lane 1) or CIRV p95 (lane 2), or alone (lane 3). Blots were hybridized with DI (a, c) or sat (b) RNA-specific probes.

 
As reported by Rubino et al. (2001), no transgenic plants expressing the CIRV replicase could be obtained. A transient-expression approach was therefore adopted to test the capability of the CIRV replicase (p36/p95) to replicate sat RNA. The same approach was also followed for the CymRSV replicase (p33/p92) to confirm the results obtained with transgenic plants. Northern blot analysis of tissues co-infiltrated with cultures expressing p92 or p95 and DI RNA sequences clearly showed the occurrence of an RNA species corresponding to DI-3 (Fig. 4c, lanes 1 and 2, respectively), which was absent in control samples infiltrated with bacteria expressing only DI RNA (Fig. 4c, lane 3). By contrast, no signal corresponding to sat RNA was found in extracts of tissue pieces infiltrated with sat RNA alone or co-infiltrated with CIRV or CymRSV replicase sequences (not shown), thus confirming the inability of the tombusvirus replicase alone to support the replication of sat RNA.

It was next decided to test whether the competence of the helper genome to sustain the replication of sat RNA was due to expression of genes other than the viral replicase. To do this, two deletion mutants of the full-length infectious clone of CIRV (Burgyan et al., 1996) were constructed (Fig. 5a). The first mutant clone ({Delta}B/N) contained a deletion from nt 20 of the CP gene to the start codon of ORF5, while the second mutant clone ({Delta}B/H) had a longer deletion as it lacked not only the entire CP gene, but also most of the ORF4 and -5 sequences. Protoplasts were obtained from non-transgenic N. benthamiana plants and co-transfected with in vitro-synthesized wild-type genomic or deletion mutant {Delta}B/N or {Delta}B/H RNAs and DI or sat RNA. Both DI (Fig. 5b, upper panel, lanes 2, 4 and 6) and sat RNA (Fig. 5b, lower panel, lanes 1, 3 and 5) replication was supported by the different helpers. It was concluded that sat RNA can be replicated only in the presence of a cis-replicating helper molecule.



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Fig. 5. Analysis of DI and sat RNA replication using deletion mutants of the helper genome. (a) Schematic representation of the full-length genome (CIRV) and deletion mutants {Delta}B/N and {Delta}B/H. The positions of relevant restriction sites are indicated. (b) Northern blot analysis of RNA extracts from N. benthamiana protoplasts transfected with full-length (CIRV) or defective viral genomes {Delta}B/N or {Delta}B/H together with sat (lanes 1, 3 and 5) or DI-3 RNA (lanes 2, 4 and 6). Blots were hybridized with DI (upper panel) or sat (lower panel) RNA-specific probes. sg1, sg2, Subgenomic viral RNAs.

 
The capability of genomic deletion mutant {Delta}B/H RNA to sustain the replication of sat RNA in plant cells prompted us to test whether this could be the same in yeast cells. To do this, the clone pB{Delta}B/H was produced and transformed into yeast cells, either alone or with plasmids YEHA95K and pAc-Myc36K. The replication of {Delta}B/H RNA was detected only in cells expressing the replicase proteins, thus indicating that this RNA can replicate in yeasts only in trans (not shown).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
As previously suggested by experiments with CymRSV DI-3/sat RNA hybrid molecules (Burgyan et al., 1992), the results of this work confirm that a significant difference exists in the replication machinery activated by two different subviral RNAs supported by the same tombusvirus helper genome. Whereas expression of ORF1 and -2 in yeast and plant cells is sufficient to sustain the replication of DI RNA molecules (Kollar & Burgyan, 1994; Rubino & Russo, 1995; Pantaleo et al., 2003), this is not enough for the replication of CymRSV sat RNA. Analysis of primary and secondary structures of DI and sat RNA reveals major differences, which may have relevance in explaining the different replication strategies of these molecules. For instance, all tombusvirus DI RNAs (as well as the genomic RNAs from which they are derived) terminate at the 3' end with -GCAGCCC, whereas the CymRSV sat RNA terminates with -CAACCC (Russo et al., 1994). Mutational studies have shown that genomic and DI RNAs tolerate only deletion of the terminal sequence -CCC, whereas deletion of the G residue at position –4, or even substitution with an A residue, which does not alter the secondary structure of the 3' region, abolishes infectivity (Dalmay et al., 1993a; Havelda & Burgyan, 1995; Pantaleo et al., 2003). In fact, it was suggested that minus-strand synthesis may initiate at the –4 position (Dalmay et al., 1993b). Conversely, deletion of up to 7 nt from the 3' terminus of sat RNA does not abolish infectivity (Dalmay & Rubino, 1995). However, introduction by site-directed mutagenesis of a G residue upstream of the 3'-terminal triplet CCC of sat RNA was not sufficient to make this molecule replicable in yeast (result not shown). Another difference found between genomic RNA, DI and sat RNA results from the observation of the predicted secondary structure of the 3' region. Genomic and DI RNAs contain a conserved sequence with three stem–loop structures, which is absent from the 3' end of sat RNA (Fabian et al., 2003). Taken together, these observations suggest that the mechanism of minus-strand synthesis may be different in the two systems. Further support to this conclusion is given by the analysis of molecules approximately twice the size of DI and sat RNA found in infected tissues. In the case of DI RNA, these molecules are head-to-tail dimers of unit length (Dalmay et al., 1995; Finnen & Rochon, 1995; Pantaleo et al., 2003), whereas in the case of sat RNA they consist exclusively of double-stranded monomers (Dalmay & Rubino, 1994). It has been suggested that DI RNA oligomers may be intermediates in the replication of DI RNA (Finnen & Rochon, 1995). A similar role was attributed to dimers found in infections with the bipartite genome animal virus Flock house virus (FHV) (Albariño et al., 2001). If so, this would constitute a major difference in the replication strategies of CymRSV DI and sat RNAs, which could explain the different requirements for the replication of these two subviral tombusvirus RNAs.

In particular, the replication of sat RNA requires the replication of the helper genome, thus suggesting an interaction with the self-replicating helper molecule. The nature of this interaction is unknown. It could be hypothesized that the replicating genome, besides supplying the replicase, acts as a trans-replication enhancer. Long-distance RNA–RNA interactions have been identified for several viruses for the regulation of subgenomic RNAs, as in the case of Red clover necrotic mosaic virus (Sit et al., 1998) and TBSV (Zhang et al., 1999). With FHV, the replication of subgenomic RNA3, which is synthesized by transcription from FHV RNA1, is necessary for the replication of RNA2 (Eckerle & Ball, 2002; Eckerle et al., 2003). We speculate that a similar interaction regulates the replication of CymRSV sat RNA.

In yeast cells, genomic {Delta}B/H RNA could only be replicated in trans, although it is fully competent for autonomous replication in plant cells. Contrary to our results, Brome mosaic virus (BMV) RNA2 was shown to act both as mRNA for the synthesis of the polymerase protein 2a and as replication template in protein 1a-expressing yeast cells (Chen et al., 2001). A major difference between BMV, a tripartite-genome virus, and CIRV, a monopartite-genome virus, lies in the strategy of expression of replication-associated proteins. In particular, because the presence of an amber stop codon in CIRV may be a stumbling block to the efficiency of our replication system in yeast, overcoming this impairment is currently being addressed.


   ACKNOWLEDGEMENTS
 
The authors wish to thank Professor G. P. Martelli and Dr L. Stavolone for advice and critical reading of the manuscript and Mrs A. Antonacci for the valuable technical help. This research was partially supported by MIUR, Project Cluster CO3, Legge 488/92, ‘Studi di geni di interesse biomedico e agroalimentare’. B. N. was recipient of a Marie Curie Fellowship of the European Commission Programme Human Potential, under contract number HPMF-CT-2001-01109.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Albariño, C. G., Price, B. D., Eckerle, L. D. & Ball, L. A. (2001). Characterization and template properties of RNA dimers generated during Flock house virus RNA replication. Virology 289, 269–282.[CrossRef][Medline]

Berben, G., Dumont, J., Gilliquet, V., Bolle, P. A. & Hilger, F. (1991). The YDp plasmids: a uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae. Yeast 7, 475–477.[Medline]

Burgyan, J. & Russo, M. (1988). Studies on the replication of a satellite RNA associated with cymbidium ringspot virus. J Gen Virol 69, 3089–3092.

Burgyan, J., Rubino, L. & Russo, M. (1991). De novo generation of cymbidium ringspot virus defective interfering RNA. J Gen Virol 72, 505–509.[Abstract]

Burgyan, P., Dalmay, T., Rubino, L. & Russo, M. (1992). The replication of cymbidium ringspot tombusvirus defective interfering-satellite RNA hybrid molecules. Virology 190, 579–586.[CrossRef][Medline]

Burgyan, J., Rubino, L. & Russo, M. (1996). The 5'-terminal region of a tombusvirus genome determines the origin of multivesicular bodies. J Gen Virol 77, 1967–1974.[Abstract]

Carrington, J. C., Freed, D. D. & Oh, C. S. (1990). Expression of potyviral polyproteins in transgenic plants reveals three proteolytic activities required for complete processing. EMBO J 9, 1347–1353.[Abstract]

Celix, A., Rodriguez-Cerezo, E. & Garcia-Arenal, F. (1997). New satellite RNAs, but no DI RNAs, are found in natural populations of tomato bushy stunt tombusvirus. Virology 239, 277–284.[CrossRef][Medline]

Celix, A., Burgyan, J. & Rodriguez-Cerezo, E. (1999). Interactions between tombusviruses and satellite RNAs of tomato bushy stunt virus: a defect in sat RNA B1 replication maps to ORF1 of a helper virus. Virology 262, 129–138.[CrossRef][Medline]

Chang, Y. C., Borja, M., Scholthof, H. B., Jackson, A. O. & Morris, T. J. (1995). Host effects and sequences essential for accumulation of defective interfering RNAs of cucumber necrosis and tomato bushy stunt tombusviruses. Virology 210, 41–53.[CrossRef][Medline]

Chen, J., Noueiry, A. & Ahlquist, P. (2001). Brome mosaic virus protein 1a recruits viral RNA2 to RNA replication through a 5' proximal RNA2 signal. J Virol 75, 3207–3219.[Abstract/Free Full Text]

Dalmay, T. & Rubino, L. (1994). The nature of multimeric forms of cymbidium ringspot tombusvirus satellite RNA. Arch Virol 138, 161–167.[Medline]

Dalmay, T. & Rubino, L. (1995). Replication of cymbidium ringspot virus satellite RNA mutants. Virology 206, 1092–1098.[CrossRef][Medline]

Dalmay, T., Rubino, L., Burgyan, J., Kollar, A. & Russo, M. (1993a). Functional analysis of cymbidium ringspot virus genome. Virology 194, 697–704.[CrossRef][Medline]

Dalmay, T., Russo, M. & Burgyan, J. (1993b). Repair in vivo of altered 3' terminus of cymbidium ringspot tombusvirus RNA. Virology 192, 551–555.[CrossRef][Medline]

Dalmay, T., Szittya, G. & Burgyan, J. (1995). Generation of defective interfering RNA dimers of cymbidium ringspot tombusvirus. Virology 207, 510–517.[CrossRef][Medline]

Eckerle, L. D. & Ball, L. A. (2002). Replication of the RNA segments of a bipartite viral genome is coordinated by a transactivating subgenomic RNA. Virology 296, 165–176.[CrossRef][Medline]

Eckerle, L. D., Albarino, C. G. & Ball, L. A. (2003). Flock house virus subgenomic RNA3 is replicated and its replication correlates with transactivation of RNA2. Virology 317, 95–108.[CrossRef][Medline]

Fabian, M. R., Na, H., Ray, D. & White, K. A. (2003). 3'-Terminal RNA secondary structures are important for accumulation of tomato bushy stunt virus DI RNAs. Virology 313, 567–580.[CrossRef][Medline]

Finnen, R. L. & Rochon, D. M. (1995). Characterization and biological activity of DI RNA dimers formed during cucumber necrosis virus coinfections. Virology 207, 282–286.[CrossRef][Medline]

Havelda, Z. & Burgyan, J. (1995). 3' Terminal putative stem–loop structure required for the accumulation of cymbidium ringspot viral RNA. Virology 214, 269–272.[CrossRef][Medline]

Havelda, Z., Dalmay, T. & Burgyan, J. (1995). Localization of cis-acting sequences essential for cymbidium ringspot tombusvirus defective interfering RNA replication. J Gen Virol 76, 2311–2316.[Abstract]

Havelda, Z., Szittya, G. & Burgyan, J. (1998). Characterization of the molecular mechanism of defective interfering RNA-mediated symptom attenuation in tombusvirus-infected plants. J Virol 72, 6251–6256.[Abstract/Free Full Text]

Ishikawa, M., Janda, M., Krol, M. A. & Ahlquist, P. (1997). In vivo DNA expression of functional brome mosaic virus RNA replicons in Saccharomyces cerevisiae. J Virol 71, 7781–7790.[Abstract]

Kollar, A. & Burgyan, J. (1994). Evidence that ORF 1 and 2 are the only virus-encoded replicase genes of cymbidium ringspot tombusvirus. Virology 201, 169–172.[CrossRef][Medline]

Lupo, R., Rubino, L. & Russo, M. (1994). Immunodetection of the 33K/92K polymerase proteins in cymbidium ringspot-infected and in transgenic plant tissue extracts. Arch Virol 138, 135–142.[Medline]

Nagy, P. D. & Pogany, J. (2000). Partial purification and characterization of Cucumber necrosis virus and Tomato bushy stunt virus RNA-dependent RNA polymerases: similarities and differences in template usage between tombusvirus and carmovirus RNA-dependent RNA polymerases. Virology 276, 279–288.[CrossRef][Medline]

Oh, C. S. & Carrington, J. C. (1989). Identification of essential residues in potyvirus proteinase HC-Pro by site-directed mutagenesis. Virology 173, 692–699.[CrossRef][Medline]

Panavas, T. & Nagy, P. D. (2003). Yeast as a model host to study replication and recombination of defective interfering RNA of Tomato bushy stunt virus. Virology 314, 315–325.[CrossRef][Medline]

Panavas, T., Pogany, J. & Nagy, P. D. (2002a). Analysis of minimal promoter sequences for plus-strand synthesis by the Cucumber necrosis virus RNA-dependent RNA polymerase. Virology 296, 263–274.[CrossRef][Medline]

Panavas, T., Pogany, J. & Nagy, P. D. (2002b). Internal initiation by the Cucumber necrosis virus RNA-dependent RNA polymerase is facilitated by promoter-like sequences. Virology 296, 275–287.[CrossRef][Medline]

Pantaleo, V., Rubino, L. & Russo, M. (2003). Replication of Carnation Italian ringspot virus defective interfering RNA in Saccharomyces cerevisiae. J Virol 77, 2116–2123.[Abstract/Free Full Text]

Pantaleo, V., Rubino, L. & Russo, M. (2004). The p36 and p95 replicase proteins of Carnation Italian ringspot virus cooperate in stabilizing defective interfering RNA. J Gen Virol 85, 2429–2433.[Abstract/Free Full Text]

Ray, D. & White, K. A. (1999). Enhancer-like properties of an RNA element that modulates Tombusvirus RNA accumulation. Virology 256, 162–171.[CrossRef][Medline]

Ray, D. & White, K. A. (2003). An internally located RNA hairpin enhances replication of tomato bushy stunt virus RNAs. J Virol 77, 245–257.[CrossRef][Medline]

Rubino, L. & Russo, M. (1995). Characterization of resistance to cymbidium ringspot virus in transgenic plants expressing a full-length viral replicase gene. Virology 212, 240–243.[CrossRef][Medline]

Rubino, L., Burgyan, J., Grieco, F. & Russo, M. (1990). Sequence analysis of cymbidium ringspot virus satellite and defective interfering RNAs. J Gen Virol 71, 1655–1660.[Abstract]

Rubino, L., Carrington, J. C. & Russo, M. (1992). Biologically active cymbidium ringspot virus satellite RNA in transgenic plants suppresses accumulation of DI RNAs. Virology 188, 429–437.[CrossRef][Medline]

Rubino, L., Lupo, R. & Russo, M. (1993). Resistance to cymbidium ringspot tombusvirus infection in transgenic Nicotiana benthamiana plants expressing a full-length viral replicase gene. Mol Plant Microbe Interact 6, 729–734.

Rubino, L., Burgyan, J. & Russo, M. (1995). Molecular cloning and complete nucleotide sequence of carnation Italian ringspot tombusvirus genomic and defective interfering RNAs. Arch Virol 140, 2027–2039.[Medline]

Rubino, L., Di Franco, A. & Russo, M. (2000). Expression of a plant virus non-structural protein in Saccharomyces cerevisiae causes membrane proliferation and altered mitochondrial morphology. J Gen Virol 81, 279–286.[Abstract/Free Full Text]

Rubino, L., Weber-Lotfi, F., Dietrich, A., Stussi-Garaud, C. & Russo, M. (2001). The open reading frame 1-encoded (‘36K’) protein of Carnation Italian ringspot virus localizes to mitochondria. J Gen Virol 82, 29–34.[Abstract/Free Full Text]

Russo, M., Burgyan, J. & Martelli, G. P. (1994). Molecular biology of Tombusviridae. Adv Virus Res 44, 381–428.[Medline]

Silhavy, D., Molnar, A., Lucioli, A., Szittya, G., Hornyik, C., Tavazza, M. & Burgyan, J. (2002). A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs. EMBO J 21, 3070–3080.[Abstract/Free Full Text]

Sit, T. L., Vaewhongs, A. A. & Lommel, S. A. (1998). RNA-mediated transactivation of transcription from a viral RNA. Science 281, 829–832.[Abstract/Free Full Text]

Töpfer, R., Matzeit, V., Gronenborn, B., Schell, J. & Steinbiss, H. H. (1987). A set of plant expression vectors for transcriptional and translational fusions. Nucleic Acids Res 15, 5890.[Medline]

White, K. A. & Morris, T. J. (1994). Recombination between defective tombusvirus RNAs generates functional hybrid genomes. Proc Natl Acad Sci U S A 91, 3642–3646.[Abstract]

Wu, B. & White, K. A. (1998). Formation and amplification of a novel tombusvirus defective RNA which lacks the 5' nontranslated region of the viral genome. J Virol 72, 9897–9905.[Abstract/Free Full Text]

Wu, B., Vanti, W. B. & White, K. A. (2001). An RNA domain within the 5' untranslated region of the tomato bushy stunt virus genome modulates viral RNA replication. J Mol Biol 305, 741–756.[CrossRef][Medline]

Zhang, G., Slowinski, V. & White, K. A. (1999). Subgenomic mRNA regulation by a distal RNA element in a (+)-strand RNA virus. RNA 5, 550–561.[Abstract/Free Full Text]

Received 20 May 2004; accepted 23 June 2004.



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