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1 Laboratory of Plant Physiology and Molecular Biology, University of Turku, FIN-20014 Turku, Finland
2 Nordita, Blegdamsvej 17, DK-2100 Copenhagen, Denmark
Correspondence
Kirsi Lehto
klehto{at}utu.fi
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ABSTRACT |
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A supplementary table showing oligonucleotides used in this study is available in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Kneller et al., 2006; Thivierge et al., 2005). An important role in regulation of viral translation belongs to the mRNA 5' and 3' non-translated regions (NTRs) (Kean, 2003; Kneller et al., 2006). In many cases, the 5' NTRs or other regions of uncapped viral mRNAs contain internal ribosome entry sites (IRESs), allowing the eukaryotic ribosomes to be loaded onto the mRNA substantially downstream from its 5' end (Bedard & Semler, 2004; López-Lastra et al., 2005; Martinez-Salas & Fernandez-Miragall, 2004; Mokrej
et al., 2006; Pisarev et al., 2005; Sarnow et al., 2005). This mode of translation initiation is therefore fundamentally different from the cap-dependent ribosome-scanning mechanism, in which the ribosome has to scan the mRNA from its very 5' end (Jackson, 2000, 2005). Although initially discovered in animal viruses (Jang et al., 1988; Pelletier & Sonenberg, 1988), IRESs were subsequently found in many animal cellular mRNAs (Komar & Hatzoglou, 2005; Merrick, 2004; Pickering & Willis, 2005) and also in plant viruses (Basso et al., 1994; Dorokhov et al., 2006; Ivanov et al., 1997; Jaag et al., 2003; Niepel & Gallie, 1999; Skulachev et al., 1999) and plant cellular mRNAs (Dinkova et al., 2005). However, in other cases, the 5' NTRs of uncapped viral mRNAs utilize mechanisms different from IRESs (Fabian & White, 2004, 2006; Guo et al., 2001; Meulewaeter et al., 1998).
The existence of IRESs in the 5' NTRs of representatives of the family Comoviridae has been under debate (Belsham & Lomonossoff, 1991; Thomas et al., 1991; Verver et al., 1991) and a role of the NTRs in translational regulation of viruses from the genus Nepovirus of this family has been poorly studied. Blackcurrant reversion virus (BRV) is a recently isolated, mite-transmitted virus belonging to subgroup c of the genus Nepovirus in the family Comoviridae (Lemmetty et al., 1997; reviewed by Susi, 2004). It has a genome composed of two positive-sense RNAs, each of which has a poly(A) tail at the 3' end and, most probably, a small viral protein linked covalently to the genome (VPg) at the 5' end, instead of a 5' cap (Fig. 1a). RNA1 is 7711 nt and RNA2 is 6405 nt long (Latvala-Kilby & Lehto, 1999; Pacot-Hiriart et al., 2001). Each of the two RNAs encodes one large polyprotein, cleaved proteolytically by the viral protease into mature proteins. The 3' NTRs of RNA1 and RNA2 are very long: 1360 and 1363 nt for RNA1 and RNA2, respectively. Their sequences are very conserved between RNA1 and RNA2 of the sequenced type isolate (94.8 % identity), as well as between ten field isolates (Lehto et al., 2004). The BRV 3' NTRs are very structured (Karetnikov et al., 2004). However, the 5' NTRs are quite short (66 and 161 nt, respectively) and do not share substantial sequence similarity (Latvala-Kilby & Lehto, 1999; Pacot-Hiriart et al., 2001). The RNA2 5' leader contains little secondary structure, harbouring only one predicted stem–loop at the 5' end (5' SL) (Fig. 1b). We have shown elsewhere that long-distance base pairing between the 5' SL and 3' NTR of RNA2 is essential for maximal cap-independent translational stimulation of a reporter mRNA (Karetnikov et al., 2006).
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METHODS |
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) implemented in the package STAR (http://wwwbio.leidenuniv.nl/
batenburg/STAR.html) at 25 °C.
cDNA constructs.
The construct 2F2-A50 (Fig. 2a) has been described elsewhere (Karetnikov et al., 2006).
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), the pETBlue-2 vector (Novagen), containing the T7 promoter, was digested with XbaI and NheI and religated, resulting in pET
XbaI-NheI. The HindIII–NotI fluc [Photinus pyralis (firefly) luciferase] fragment and NotI–PvuII fragment of the RNA2 3' NTR were ligated and cloned into pETXbaI-NheI. To add the poly(A)50 tail, two complementary oligonucleotides, 1 and 2 (see Supplementary Table S1, available in JGV Online), were introduced by using the sites PvuII and PmlI.
To obtain 1-82-A50 (Fig. 2a), the SpeI–ClaI fragment of 2F2 (Karetnikov et al., 2006) and ClaI–PacI fragment of R2F30-2 (see below) were ligated and cloned into pETBlue-2, digested with XbaI and PacI. The poly(A)50 tail was inserted as described for CF2-A50.
To create 5'-NheI-XhoI-3', the NheI–XhoI fragment of pETBlue-2 was cloned into 2F2-A50, digested with SpeI and XhoI. The SalI–PstI and PstI–HindIII fragments of 2F2 were ligated and cloned into 5'-NheI-XhoI-3', resulting in 87-161-A50 (Fig. 2a).
To produce 18S1/2-A50, 18S3/4-A50, 18S5/6-A50 and 18S5-A50, the regions of 2F2-A50 upstream and downstream of the corresponding deleted or mutated sequence (nt 23–41, 56–74, 115–138 and 115–124, respectively) were PCR-amplified with two pairs of primers. The upstream forward (primer 3) and downstream reverse (primer 4) primers were common for all four constructs. The other primers, containing the ClaI site, were unique for each clone (Supplementary Table S1, primers 5–11), except for primer 9, common for 18S5/6-A50 and 18S5-A50. The resulting PCR products were inserted into pCR-BluntII-TOPO (Invitrogen) and accumulated in the dam Escherichia coli strain K12 ER2925 (New England Biolabs). The corresponding fragments, digested with AatII/ClaI and ClaI/SphI, respectively, were ligated and introduced into 2F2-A50.
Clones pRL-SV40 and pPVc702, containing Renilla reniformis luciferase and P. pyralis (firefly) luciferase reporter genes (rluc and fluc, respectively), were a kind gift of Matti Karp and Pekka Virtanen (University of Turku, Finland). To obtain R2F0-2, the rluc gene was PCR-amplified with primers 12 and 13, the RNA2 5' NTR with primers 14 and 15, the fluc gene with primers 16 and 17 and the RNA2 3' NTR with primers 18 and 19 (Supplementary Table S1). The corresponding DNA fragments were digested with relevant restriction endonucleases and introduced into pETBlue-2. R2F30-2, encoding a 30 nt fragment of the rluc 3' NTR, was designed by inserting two complementary oligonucleotides, 20 and 21 (Supplementary Table S1), into R2F0-2 by using the site EcoRV. To produce R2F2-A61 (Fig. 3), the poly(A)61-containing SmaI–DraIII fragment of pGLO18A (a kind gift from W. Allen Miller, Iowa State University, IA, USA) was inserted into R2F30-2 digested with PvuII and DraIII.
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; a kind gift from Anthony Griffiths, Harvard Medical School, Boston, MA, USA) into R2F2-A61, using the site XbaI (Fig. 3).
For obtaining R18S5/6F2-A61 and R18S5F2-A61 (Fig. 3), the SpeI–PacI fragment of R2F2-A61 was replaced by the SpeI–PacI fragment of 18S5/6-A50 and 18S5-A50, respectively.
RCF30-2 was produced as described for R2F30-2, except for introducing two complementary oligonucleotides, 22 and 23 (Supplementary Table S1), representing the 27 nt fragment of the 5' leader of Xenopus laevis 
-globin mRNA instead of the RNA2 5' NTR, using the sites XbaI and SmaI. To create RCF2-A61 (Fig. 3), the poly(A)61-containing BamHI–NheI fragment of pGLO18A was inserted into pETBlue-2, leading to pET-A61. The SmaI–XbaI fragment of pET-A61 was cloned into RCF30-2, digested with PvuII and AvrII.
To obtain as-fluc, a 600 nt HindIII–EcoRI fluc fragment was cloned in the antisense (as) orientation into pETXbaI-NheI.
In vitro transcription.
DNA templates were linearized by PscI (2F2-A50 and its derivatives), PmlI (CF2-A50 and 1-82-A50), NheI (dicistronic constructs) and HindIII (as-fluc). RNAs were synthesized by using a RiboMAX kit (Promega) as described elsewhere (Karetnikov et al., 2006). To produce the digoxigenin (DIG)-labelled RNA probe as-fluc, transcription was performed with DIG RNA labelling mix (Roche) according to the manufacturer's instructions.
In vivo expression and protein analysis.
Protoplasts from Nicotiana benthamiana plants were isolated, electroporated with RNA and incubated after electroporation as described elsewhere (Karetnikov et al., 2006). A Gene Pulser II (Bio-Rad) for electroporation was kindly provided by Ari Hinkkanen, Åbo Akademi University, Turku, Finland. Each experiment was repeated three times, with each electroporation performed in triplicate.
The cells were lysed by shaking for 15 min at room temperature in 100 µl passive lysis buffer (Promega) followed by two freeze–thaw cycles. Aliquots (50 µl) of cell lysate were taken for FLUC assays, performed in duplicate in 200 µl FLUC assay buffer (Gallie, 2002), using a BioOrbit-1250 luminometer (kindly provided by Matti Karp). For RLUC assays, 20 µl cell lysate was mixed with 200 µl RLUC assay buffer (Gaur et al., 2004). Total protein concentration was measured according to Bradford (1976). Translational efficiency was estimated as the rate of protein synthesis during a transient steady-state phase of translation, and corresponded to the maximal slope of the curve describing the kinetics of FLUC production. mRNA functional half-life was determined as the time required, following mRNA delivery, to reach 50 % of the final FLUC level produced from a given mRNA (Gallie, 2002; Krab et al., 2005).
Northern blotting.
After 6 h incubation of electroporated protoplasts, total RNA was extracted with TRIzol (Invitrogen), separated by electrophoresis in 0.8 % agarose gel with 2 % formaldehyde and transferred onto a Hybond-N+ membrane (Amersham Biosciences). The membrane was hybridized with the 600 nt DIG-labelled antisense RNA probe as-fluc. Samples were visualized by using DIG detection antibody (Roche), the chemiluminescent substrate CSPD (Roche) and membrane exposure to X-ray film.
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RESULTS |
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) contained the fluc gene flanked by the authentic RNA2 5' leader and RNA2 3' NTR, followed by the poly(A)50 tail, which is the native 3' element of BRV RNAs. The fluc RNAs were electroporated into protoplasts of N. benthamiana, shown previously to be a BRV laboratory host species (Lemmetty et al., 1997).
The RNA2 5' leader provided in vivo FLUC expression from uncapped 2F2-A50 that was 333-fold higher than that with uncapped CF2-A50 mRNA, containing a vector-derived 5' NTR (Fig. 2b). At the same time, the expression level from uncapped 2F2-A50 was as high as that from m7G-capped CF2-A50 (100 versus 98 %, respectively; Fig. 2b). Replacement of either the 5' (nt 1–82) or 3' (nt 87–161) half of the RNA2 leader by vector-derived sequences, using the site SpeI (see Methods; Figs 1b, 2a), decreased the FLUC level to 0.5 and 1.5 %, respectively, when weighed against the wild type, 2F2-A50 (Fig. 2b, mutants 1-82-A50 and 87-161-A50). To test whether the observed differences in expression resulted from different translational efficiencies (rates of protein synthesis) or, alternatively, distinct mRNA stabilities, we performed a time-course analysis of FLUC accumulation for all four uncapped RNAs. We carried out analysis of mRNA functional stability as a method superior to Northern blotting when detecting functionally important nucleotide losses. This approach can distinguish between translationally active and inert mRNA pools and has been used successfully in translation studies (Chiu et al., 2005; Gallie, 2002; Krab et al., 2005; Matsuda et al., 2004). Functional half-lives (functional stabilities) and translational efficiencies of mRNAs were determined as described in Methods. Time-course analysis showed that mRNAs CF2-A50, 1-82-A50 and 87-161-A50 had translational efficiencies of 0.4, 0.9 and 3.2 %, respectively, relative to that of the wild type, with mRNA functional stabilities altered to a much lower extent (Fig. 2c, d). Therefore, the RNA2 5' NTR can stimulate efficient translation from the fluc reporter mRNA and each half of the RNA2 5' leader is essential for its functioning.
The RNA2 5' leader contains an IRES
To test the possibility that the stimulatory effect of the RNA2 5' leader on translation would be exerted through an IRES mechanism, we generated a series of dicistronic constructs containing the rluc and fluc genes as upstream and downstream cistron, respectively, as well as the RNA2 3' NTR and poly(A)61 tail (Fig. 3). As an intergenic region, these constructs harboured the RNA2 5' leader (R2F2-A61) or control sequence, composed of a vector part and a fragment of the cap-dependent -globin 5' leader (RCF2-A61). Both mRNAs were capped and, hence, the RLUC expression should be cap-dependent, whilst possible FLUC expression would depend on IRES activity of the intercistronic region.
Both dicistronic mRNAs were electroporated into N. benthamiana protoplasts and the expression ratio of FLUC : RLUC was determined. The construct R2F2-A61 was characterized by an FLUC : RLUC ratio 17-fold higher than that of RCF2-A61 (Fig. 3). Such a difference stemmed from very distinct FLUC expression levels between the two mRNAs, with the RLUC amount being relatively constant (Fig. 3).
Inserting a stable SL into R2F2-A61 immediately upstream of the rluc gene diminished its expression to 2.9 % relative to that of R2F2-A61, without significant change in FLUC level (Fig. 3, construct SR2F2-A61).
To test the possibility that FLUC would be expressed from a monocistronic degradation product of R2F2-A61 RNA instead of the full-length dicistronic R2F2-A61, we performed a Northern blot analysis of total RNA isolated from protoplasts electroporated with either R2F2-A61 or 2F2-A50. For R2F2-A61, we did not observe any outstanding degradation products having a length comparable with that of monocistronic 2F2-A50, implying that FLUC was produced from the full-length R2F2-A61 (Fig. 4). These data provide evidence that the RNA2 5' leader contains the IRES.
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). These complementary stretches were located throughout the 5' NTR length and included 18S1 (nt 23–31), 18S2 (nt 34–41), 18S3 (nt 56–64), 18S4 (nt 65–74), 18S5 (nt 115–124) and 18S6 (nt 130–138) (Fig. 1b). To test the possible importance of these regions for translation, we constructed monocistronic mutants based on 2F2-A50 by using deletions and site-directed mutagenesis. Each of the three deletion mutants was lacking two stretches complementary to 18S rRNA. It should be noted that the mutant 18S1/2-A50 was designed in such a way that the 5' SL and 5' NTR–3' NTR base pairing would not be disrupted (Karetnikov et al., 2006). After RNA electroporation into N. benthamiana protoplasts, all of the mutants exhibited dramatically decreased FLUC expression. The FLUC level observed for deletion mutants was 18 % of that of the wild type for 18S1/2-A50, 13 % for 18S3/4-A50 and 6 % for 18S5/6-A50 (Fig. 5a). For the segment 18S5, we performed site-directed mutagenesis, which would compromise its base-pairing potential with 18S rRNA, by replacement of the corresponding BRV complementary sequence with AUCGAU, representing the ClaI restriction site (Fig. 6a). The resulting mutant 18S5-A50 had FLUC expression reduced to 14 % of that of the wild type, 2F2-A50 (Fig. 6b). Time-course expression analysis demonstrated that the observed effect of mutations was primarily a result of decreasing translational efficiencies (to 9–24 % of that of 2F2-A50), with mRNA functional stabilities being changed only slightly (to 56–75 %) (Figs 5b–d, 6c–d).
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, mutants R18S5F2-A61 and R18S5/6F2-A61). This resulted in a decrease in FLUC production to 35 and 27 % relative to that of R2F2-A61, for R18S5F2-A61 and R18S5/6F2-A61, respectively, without substantial effect on RLUC expression (Fig. 3). These observations indicate the importance of the regions containing the 18S rRNA-complementary sequences for the RNA2 5' NTR function in translational stimulation of both monocistronic and dicistronic fluc mRNAs.
Regions complementary to 18S rRNA in the RNA2 5' leaders of other nepoviruses
Nucleotide sequences of the RNA2 5' NTRs of different members of the genus Nepovirus do not have any consensus elements that would be common for all of the species (Mayo & Robinson, 1996). However, we found that, similar to BRV, the RNA2 5' leaders of all other analysed nepoviruses contained several (up to 11) 7–11 nt segments complementary to parts of the same region of plant 18S rRNA, nt 1110–1123 (Table 1). These complementary stretches resided in various locations inside the 5' NTRs.
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; Guo et al., 2001) and their 5' leaders seem to use 5' end-dependent ribosomal scanning instead of an IRES mechanism (Fabian & White, 2006; Guo et al., 2001). Yet, the 5' NTRs of many other uncapped viral mRNAs contain IRESs (Dreher & Miller, 2006; Jackson, 2005; Kneller et al., 2006; López-Lastra et al., 2005; Sarnow et al., 2005). In this work, we studied a role of the BRV RNA2 5' leader in translation in vivo using the reporter fluc and rluc mRNAs. We found that the RNA2 5' NTR was able to confer very efficient translation of the uncapped mRNA, the level of which was much higher than that of the uncapped control leader (Fig. 2b). Adding a 5' cap to the control mRNA stimulated its expression by 256-fold, which corresponded to values obtained earlier for cellular mRNAs (Gallie, 1991; Gallie et al., 1995; Meulewaeter et al., 1998), and this level was very similar to that of uncapped 2F2-A50 (Fig. 2b), in agreement with previously reported data for Tobacco etch virus (TEV) (Carrington & Freed, 1990; Gallie et al., 1995). Thus, the 5' leader of uncapped BRV RNA2 was expected to contain some determinants responsible for conferring efficient cap-independent translation. Each half of the BRV leader was required for this stimulation (Fig. 2). The observed translation enhancement was exerted, at least in part, through the IRES mechanism, as shown in experiments with the dicistronic mRNAs in which the BRV 5' NTR, placed between two reporter genes, mediated expression of the downstream cistron much more efficiently than did the control leader (Fig. 3). The IRES-driven translation mechanism was confirmed in the SL test, when a stable SL introduced upstream of the first cistron blocked only cap-dependent translation from this gene, but not translation from the BRV 5' NTR-driven cistron (Fig. 3). As it has been shown that maximal cap-independent translational stimulation of BRV RNA2 requires base pairing between the 5' SL and 3' NTR (Karetnikov et al., 2006), BRV can be considered as an example of a virus using both an IRES mechanism and long-distance RNA–RNA interactions for efficient translation, unlike the tombusviruses and luteoviruses mentioned above. As to other representatives of the family Comoviridae, IRES activity has been demonstrated previously in vitro for Cowpea mosaic virus (Thomas et al., 1991; Verver et al., 1991), but could not be reproduced in animal cells (Belsham & Lomonossoff, 1991). However, this activity has not been tested in plant cells.
Although the general IRES mechanism of translation is common between BRV and many other viruses, some dramatic differences exist. Unlike picornaviral IRESs (Jang et al., 1988; Pelletier & Sonenberg, 1988) and 5' IRESs of representatives of the family Dicistroviridae (Czibener et al., 2005; Terenin et al., 2005), the BRV RNA2 5' leader is relatively short (161 nt). Nevertheless, its length is quite close to those of the 143–144 nt leaders of Turnip mosaic virus (Basso et al., 1994) and TEV (Niepel & Gallie, 1999), both containing IRESs. Still, some viral IRES-containing 5' leaders may be even shorter, such as a 75 nt 5' NTR of tobamoviral uncapped subgenomic I2 RNA (Skulachev et al., 1999).
In contrast to highly structured picornaviral IRESs, the BRV 5' NTR contains only one predicted SL (Fig. 1b). Also, we could not predict for BRV any pseudoknots, which are typical for the moderately structured TEV leader and involved in its IRES activity (Zeenko & Gallie, 2005). However, the lack of extensive RNA secondary structure is a common feature of the BRV leader and 5' IRES of Rhopalosiphum padi virus (RhPV) (Terenin et al., 2005), both having a low G+C content and long, polypyrimidine-rich tracts. Binding of eukaryotic translation-initiation factors (eIFs) to a long (380 nt), unstructured region upstream of the RhPV initiation codon has been proposed to mediate its IRES activity (Terenin et al., 2005). Yet, it should be noted that secondary structure of the BRV leader has so far been predicted only by computer analysis and it needs to be verified by enzymic and chemical RNA-structural probing.
We found six 8–10 nt segments in the RNA2 5' NTR (18S1–18S6) complementary to nt 1113–1123 of plant 18S rRNA and located throughout the length of the BRV leader (Fig. 1b). Deletion of these RNA2 stretches or site-directed mutagenesis disrupting a putative 5' NTR–18S rRNA base pairing reduced FLUC expression severely, mainly at the level of translational efficiency and altering mRNA stability much less (Figs 5, 6). The effect of mutations was observed for both monocistronic constructs, where the BRV IRES resided as a part of the 5' leader (Figs 5, 6), and dicistronic mRNAs, with the BRV IRES in the intergenic region (Fig. 3). The corresponding 18S rRNA sequence is a part of the larger region, nt 1105–1124, demonstrated previously to be conserved among eukaryotes, to be exposed and accessible for intermolecular mRNA–18S rRNA base pairing and to stimulate translation when its base-pairing mRNA counterpart is present in either the leader or intercistronic region of mRNA (Akbergenov et al., 2004). A similar complementary interaction between the 7 nt part of the same 18S rRNA region, nt 1117–1123, and the corresponding stretch in the TEV 5' NTR has been shown to contribute to the TEV IRES activity (Zeenko & Gallie, 2005). All of these observations appear to be in agreement with the ‘ribosome-filter’ hypothesis (Mauro & Edelman, 2002), postulating that direct mRNA–18S rRNA base pairing is an important mechanism of translational control in eukaryotes by which the 40S ribosomal subunit could be recruited efficiently to mRNA, with some (although limited) analogy to prokaryotic translation initiation. Indeed, complementary interactions between different mRNAs and various accessible, conserved 18S rRNA regions have been proposed to mediate high translation levels in a number of unrelated eukaryotic systems (Akbergenov et al., 2004; Dresios et al., 2006; Hu et al., 1999; Tranque et al., 1998; Vanderhaeghen et al., 2006; Zeenko & Gallie, 2005), including IRES elements. Evidence to support this hypothesis has recently been demonstrated for the IRES of the mouse Gtx homeodomain mRNA (Dresios et al., 2006). Although direct interactions between the BRV leader and plant 18S rRNA remain to be demonstrated, our results suggest the importance of the segments complementary to 18S rRNA for BRV IRES function. In addition, the existence of multiple stretches complementary to the same 18S rRNA region in the 5' leaders of other nepoviruses (Table 1) should trigger further experiments to test the relevance of possible base pairing for translational stimulation in these viruses.
It is worth mentioning that all of the six regions of the 5' NTR of BRV RNA2 complementary to 18S rRNA were in the polypyrimidine-rich tracts (Fig. 1b). Such tracts have been shown to stimulate translation of various IRES-containing viral and cellular mRNAs by interacting with the polypyrimidine tract-binding proteins (PTBs) (Florez et al., 2005; Hunt & Jackson, 1999; Mitchell et al., 2005; Song et al., 2005; Spriggs et al., 2005). Although the regulatory role of these elements has so far been investigated only in mammalian systems, proteins with similarity to mammalian PTBs are predicted to exist in plants (Marín & Boronat, 1998). Thus, alternatively or in addition to the mRNA–18S rRNA base-pairing mechanism, at least some of the 18S rRNA-complementary segments might exert their effect on BRV translation through interactions of the respective polypyrimidine-rich tracts with plant PTBs. Involvement of some of the 18S rRNA-complementary stretches of the RNA2 leader (or BRV sequences overlapping with the 18S1–18S6 regions) in translational stimulation through their binding to PTBs or other protein factors essential for BRV IRES function could also explain a strong effect on translation upon modification or deletion of just one or two of the six stretches (Figs 5, 6). In this case, different complementary regions would function through dissimilar mechanisms, resulting in the absence of functional redundancy. On the other hand, the 18S rRNA-complementary stretches might constitute separate parts of an integral complex, and destroying such a functional ensemble would compromise translation. In support of the second suggestion, each half of the RNA2 5' leader was required for translation (Fig. 2). The third alternative, which could explain the absence of functional redundancy, would be unpredicted changes in RNA secondary structure inactivating at least some 18S rRNA-complementary stretches upon deletion of one or two of them.
The observed functional importance of the 18S1–18S6 segments does not imply that these regions would be the only RNA2 5' leader elements critical for translational enhancement, and putative roles of other 5' NTR parts should be tested in future experiments. Indeed, the important role of one such element, the 5' SL, has been determined and is reported elsewhere (Karetnikov et al., 2006). Furthermore, we found that another RNA2 5' leader region, nt 140–146, is complementary to nt 1765–1771 of plant 18S rRNA (Fig. 1b), located in a single-stranded part of its 3' end, next to the segment shown previously to be accessible for interaction with mRNA of plant ribosomal protein S18 and essential for its cap-independent translation (Vanderhaeghen et al., 2006), although we did not test putative significance of this complementarity.
In addition, the 5' termini of BRV genomic RNAs are supposed to be bound covalently to VPg by analogy with other members of the family Comoviridae (Mayo & Robinson, 1996). The role of VPg in translatability of virus RNAs has been under debate. Although potyvirus, nepovirus and calicivirus VPg has been shown to bind certain eIFs, the significance, if any, of these interactions for translation in vivo is not known (reviewed by Dreher & Miller, 2006), although calicivirus VPg does contribute to efficiency of in vitro translation (Goodfellow et al., 2005). Moreover, potyvirus VPg has been proposed to participate in the infection-associated host translation shutoff, instead of affecting viral mRNA translation directly (Cotton et al., 2006). Furthermore, comovirus and picornavirus VPg is not required for either infectivity or translation (Hewlett et al., 1976; Nomoto et al., 1976, 1977; Stanley et al., 1978). Nepovirus VPg is dispensable for in vitro translation (Chu et al., 1981; Hellen & Cooper, 1987; Koenig & Fritsch, 1982). In our in vivo experiments, the reporter fluc mRNA with the BRV NTRs was translated efficiently without VPg, in agreement with earlier data for potyviruses (Basso et al., 1994; Niepel & Gallie, 1999). More direct studies are needed to shed light on a putative role of VPg in translation in vivo.
In summary, we found that the 5' leader of BRV RNA2 mediates efficient in vivo translation through an IRES mechanism. According to our knowledge, this is the first demonstration of the IRES activity for a virus species from the family Comoviridae in plant cells. Our results suggest that multiple 5' NTR regions complementary to plant 18S rRNA play an important role in translational stimulation by the BRV 5' leader.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 19 June 2006;
accepted 28 August 2006.
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, putative VPg. BRV polyprotein 1 is predicted to be cleaved into: CoPro, protease co-factor; NTB, nucleotide-binding protein; VPg, viral protein genome-linked (grey box); CysPro, cysteine protease; RdRp, RNA-dependent RNA polymerase. BRV polyprotein 2 is predicted to be cleaved into: X, protein X; MP, movement protein; CP, coat protein. (b) Nucleotide sequence of the BRV RNA2 5' leader. The regions complementary to nt 1113–1123 of plant 18S rRNA (18S1–18S6) are depicted by white letters on a grey background. The corresponding 18S rRNA sequence [from Akbergenov et al. (2004)
. Mutants 1-82-A50 and 87-161-A50 are named according to the deleted regions. 2F2-A50, wild-type construct; CF2-A50, construct with the control 5' leader. Other designations are as described for Fig. 1
