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National Institute of Agrobiological Sciences, Ohwashi, Tsukuba, Ibaraki 305-8634, Japan
Correspondence
Nobuhiko Nakashima
nakaji{at}affrc.go.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Present address: National Institute of Neuroscience, National Center of Neurology and Psychiatry, Ogawahigashi, Kodaira, Tokyo 187-8551, Japan. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). In contrast, viral RNAs, which do not have a 5' 7-methylguanosine cap, usually have a long 5' untranslated region (UTR) with tertiary structure that recruits ribosomes independently of the 5' termini. Such cis-acting RNA elements are known as internal ribosome entry sites (IRES) (Hellen & Sarnow, 2001; Jang et al., 1988; Pelletier & Sönenberg, 1988).
Plautia stali intestine virus (PSIV) is a member of the genus Cripavirus in the family Dicistroviridae. Rhopalosiphum padi virus (RhPV), Triatoma virus (TrV), Cricket paralysis virus (CrPV), Drosophila C virus (DCV) and Taura syndrome virus (TSV) also belong to the family Dicistroviridae (Christian et al., 2005). Dicistroviruses are characterized by a monopartite positive-stranded RNA genome with two non-overlapping open reading frames (ORFs). The 5' and 3' ORFs encode non-structural and structural protein precursors, respectively. The intergenic region (IGR) between the two ORFs contains an IGR-IRES (Czibener et al., 2005; Domier et al., 2000; Sasaki & Nakashima, 1999, 2000; Wilson et al., 2000). Because dicistroviral IGR elements have a common secondary structure with only minor variations, the mechanism for initiation in IGR-IRES-mediated translation is believed to be essentially the same in all dicistroviruses (Kanamori & Nakashima, 2001; Nishiyama et al., 2003; Hatakeyama et al., 2004). Of the IGR-IRES elements that have been tested, all function in numerous eukaryotic lysate systems, including mammal (Domier et al., 2000; Sasaki & Nakashima, 1999), plant (Wilson et al., 2000; Shibuya et al., 2003) and yeast (Thompson et al., 2001).
Translation of a truncated 5' ORF under the control of the 5' UTR of PSIV was previously examined in rabbit reticulocyte lysate (RRL), but no products were detected (Sasaki et al., 1998). Recent reports, however, show that the 5' UTRs of other dicistroviruses, such as RhPV and CrPV, function as an IRES: the 5' IRES of RhPV functions in plant, mammal and insect cell lysate systems (Royall et al., 2004; Woolaway et al., 2001), whereas the 5' IRES of CrPV functions in RRL, but not in wheatgerm extract (WGE) (Wilson et al., 2000). These reports suggest that the 5' UTR of PSIV may function as an IRES, but that its activity is dependent on which cell-free system is used. Data presented here show that the 5' UTR of PSIV contains an IRES that is functional in insect cell lysates, but not in RRL or WGE, suggesting that the requirements for translation initiation mediated by the 5' UTR IRES vary among dicistroviruses.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) was digested with XbaI, treated with Klenow fragment and then cut with EcoRI for ligation with the 2.5 kb PSIV-Fluc fragment to generate pT7Rluc-1IRES736-Fluc. Construction of pT7Rluc-5950IRES6240-Fluc, pFluc and pRluc-Fluc has been described previously (Hatakeyama et al., 2004; Shibuya et al., 2004). Mutants to determine the initiation AUG triplets and the 3' and 5' borders of the 5' IRES were constructed by PCR-based mutagenesis using pT7B5'PSIVFluc as template. The mutated PSIV sequence was recovered by digestion with PstI and NcoI and then ligated into those sites of pT7B5'PSIVFluc to replace wild-type PSIV sequence with mutated sequences. Mutations were confirmed by DNA sequencing. Dicistronic constructs carrying these mutations were prepared according to the methods for pT7Rluc-1IRES736-Fluc described above.
For structure analysis, a partial cDNA of PSIV (nt 225–736) was amplified by RT-PCR with forward and reverse primers that incorporate PstI and NcoI recognition sequences, respectively, into their 5' sequences. The obtained fragment was digested with PstI and NcoI and ligated into those sites of pT7B5'PSIVFluc. The resultant recombinant plasmid was linearized with NcoI prior to in vitro transcription.
In vitro transcription.
In general, plasmids were linearized with EcoRI. Capped RNAs were transcribed using mMESSAGE mMACHINE (Ambion) and uncapped RNAs were transcribed using T7-RiboMAX Express Large Scale RNA Production system (Promega) according to the manufacturers' recommendations. Transcribed RNAs were extracted with phenol and precipitated with 2-propanol in the presence of ammonium acetate.
In vitro translation and detection of translation products.
In vitro translations using insect Spodoptera frugiperda 21 (Sf21) cell lysate (Transdirect insect cell; Shimadzu), WGE (Promega) and RRL (Promega) were conducted according to the manufacturers' recommendations. To examine the effect of a cap analogue, m7GpppG (Promega) was added to reaction mixtures at a final concentration of 0.5 mM. Rluc and Fluc enzymic activities were measured as described previously (Shibuya et al., 2004). To determine their relative mass, translation products were labelled with 1 µl biotinylated lysyl-tRNA (Transcend tRNA; Promega) per 24 µl reaction mixture containing 3 pmol template RNA. After incubation for 60 min, an aliquot of each reaction mixture was separated by SDS-PAGE (9 % polyacrylamide) and products were detected by chemiluminescence as described previously (Shibuya et al., 2003). To detect translation products by fluorescence, FluoroTect Green Lys-tRNA (Promega) was used instead of biotinylated lysyl-tRNA and translation products were visualized using Typhoon 9410 (Amersham Bioscience).
Chemical and enzymic structure probing analysis.
Modifications of refolded RNAs after heat treatment using dimethyl sulfate (DMS), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMCT) and RNase T1 (Ambion) were carried out as described previously (Shibuya et al., 2003). Primers corresponding to nt 342–327, 428–412, 510–493, 590–573 and 666–650 in the PSIV sequence were used for targeting reverse transcriptase to the template.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) was constructed that expresses Rluc as the first cistron, nt 1–736 of the PSIV sequence and Fluc as the second cistron, all under the control of a T7 promoter. Transcripts from the plasmid were translated in vitro in the presence or absence of cap analogue using lysate prepared from Sf21 insect cells. In a comparison of luciferase activities derived from different translation mixtures, Rluc activity decreased 5-fold in the presence of 0.5 mM cap analogue compared with its activity in the absence of analogue (Fig. 1b). In contrast, Fluc activity did not decrease in the presence of cap analogue, indicating that translation of the second cistron of the RNA was cap-independent.
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). Because Fluc activity originating from the Rluc–Fluc dicistronic RNA that lacked the intervening sequence between the two cistrons was 14-fold lower than activity from Rluc–1IRES736–Fluc RNA, it is concluded that the 5' UTR of PSIV functions as an IRES in the insect cell-free system.
The first in-frame AUG triplet is the initiation site for PSIV ORF1
In sequence databases, the ORF1 start site of dicistroviruses has been assigned in various ways. ORF1 in TrV (GenBank accession no. AF178440
[GenBank]
) opens with the triplet that immediately follows an in-frame stop codon, probably because dicistroviral IGR-IRES do not use an AUG triplet for initiation. In both DCV (AF014388
[GenBank]
) and TSV (AF277675
[GenBank]
), the second in-frame AUG triplet has been assigned as the ORF1 start site because it is present in a suitable context for invertebrate initiation (Cavener & Ray, 1991). In other dicistroviruses, the first in-frame AUG triplet has been assigned as the initiation site. In PSIV, the 5' UTR (nt 1–570) contains nine AUG triplets and the 5' part in the 5' ORF of PSIV also encodes four in-frame AUG triplets (at nt 571–573, 574–576, 628–630 and 697–699) (Fig. 2a). Among these, AUG571–573 and AUG697–699 are most likely to be suitable for translation initiation in invertebrates, but their functionality has not been examined.
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, lane 4) than authentic Fluc (Fig. 2b, lane 3), suggesting that initiation occurred within the upstream PSIV sequence. To compare the relative electrophoretic mobilities of NS–Fluc fusion proteins that could initiate at AUG697–699, AUG628–630, AUG574–576 or AUG571–573 with known standards, monocistronic PSIV-Fluc RNAs that are initiated by ribosome scanning were constructed. The mobilities of products initiated at AUG628–630 and AUG697–699 were faster than those of products of Rluc-1IRES736-Fluc (Fig. 2b, lanes 1, 2 and 4), indicating that the initiation site is likely to be either AUG571–573 or AUG574–576. To distinguish between these two sites, AUG574–576 was changed to AUA by PCR-based mutagenesis and fluorescence was used as a measure of initiation because of the many endogenous biotinylated proteins in insect cell lysates (Fig. 2b). Mutation of AUG574–576 did not affect the amount of product from the second cistron (Fig. 2c, lane 4); however, there was a significant decrease in the amount of product from the second cistron in a mutant in which AUGAUG571–576 was replaced by AUAAUA (Fig. 2c, lane 5). Translation of this mutant resulted in a very faint band that migrated to the same position as NS–Fluc (Fig. 2b, lane 5). It is hypothesized that this was a non-AUG-initiated product from the mutated AUA codon because a UAG stop codon is located five triplets upstream of AUG571–573 (Fig. 2a) and there are no AUG-like triplets between the UAG triplet and AUA571–573 in the mutated RNA. These results indicate that AUG571–573 is the initiation site for viral protein production.
The 5' IRES of PSIV is composed of approximately 350 nt
To examine the 5' boundary of the 5' IRES of PSIV, deletion mutants were constructed from pT7Rluc-1IRES736-Fluc (Fig. 3a). When uncapped transcripts from the mutants were translated, luciferase activity from 5'-
150 and 5'-225 did not decrease (Fig. 3b). This indicates that the 5'-terminal 225 nt of PSIV are not necessary for IRES-mediated translation. However, the luciferase activity of p5'-300 and p5'-375 transcripts was two-thirds and one-third of that of the wild-type, respectively. Although these results do not define a clear 5' boundary for the IRES, they suggest that a 5' UTR that includes a sequence downstream of position 225 would be necessary for a fully functioning IRES.
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). These results are in contrast to similar assays of RhPV; the RhPV 5' IRES does not require any viral coding sequence (Woolaway et al., 2001). The G+C content of the PSIV 5' UTR is unusually high (43 %) relative to RhPV (34 %). Based on previous experiments with the IGR-IRES of PSIV, in which a G+C-rich introduced nucleotide sequence inhibited Fluc (Shibuya et al., 2003), it is predicted that the high G+C content downstream of the 5' IRES might also be inhibitory to translation. The requirement for the viral coding sequence, on the other hand, was conditional, and there was a positive correlation between high IGR-IRES activity and low G+C content within the 5' regions of coding sequences (Shibuya et al., 2003).
To test whether the same correlation existed within the 5' IRES, silent mutations that lowered the G+C content of Fluc at positions 570-Fluc and 573-Fluc were constructed as described previously (Shibuya et al., 2004). Luciferase activity from 570-Fluc(mut) and 573-Fluc(mut) templates was 4- and 11-fold higher, respectively, than from the wild-type (Fig. 3b), suggesting that the high G+C content of wild-type Fluc decreases translation efficiency. The 573-Fluc(mut) showed 5-fold higher translation activity compared with 570-Fluc(mut). The difference in nucleotide sequences in the two constructs is the presence of an additional AUG triplet after the initiator AUG in the 573-Fluc(mut). Because other constructs were not examined, the reason for this difference in translation activity is difficult to explain; however, efficiency of translation initiation is sometimes affected by nucleotide sequences downstream of the initiation codon. Although 570-Fluc(mut) showed lower translation activity of the second cistron compared with 573-Fluc(mut), detected Fluc activity from 570-Fluc(mut) was apparently higher than 570-Fluc containing the wild-type Fluc sequences (Fig. 3b). These results suggest that sequences downstream of the initiation codon affect translation efficiency, but the viral coding sequence is not an absolute requirement for 5' IRES-mediated translation of PSIV.
Structure of the 5' IRES elements in PSIV and RhPV is distinct
Chemical modifications that probe the structure of 5' IRES have been utilized with RhPV, but not other dicistroviruses. Among the compounds tested, DMS modifies unpaired adenine and cytosine residues, CMCT modifies unpaired uridines and guanines and RNase T1 cleaves unpaired guanines. Subsequent reverse transcription of modified RNA templates with 33P-labelled primers produces bands at position –1 relative to any modified nucleotide.
In this study, renatured RNA molecules encoding nt 225–736 of PSIV were probed with DMS, CMCT and RNase T1 (Fig. 4). The resulting data were analysed with the mathematical predictions that operate in the program MFOLD (Zuker, 2003) to construct a secondary structure model of the 5' IRES (Fig. 5). The resulting models suggest that the 5' IRES of PSIV forms several stem–loop structures. In addition, a pseudoknot formation was suggested between nt 323–326 (gccc) and 344–341 (cggg) (Fig. 5), because these nucleotides were not modified by probing reagents (Fig. 4). To examine the effect of these base-pair interactions on 5' IRES-mediated translation, nt 323–326 (gccc) in the Rluc-1IRES736-Fluc construct were changed to cggg. Translation efficiency of the second cistron in this construct was estimated to be 54 % of that of the authentic Rluc-1IRES736-Fluc by measuring Fluc activity of the reaction mixture of the Sf21 insect cell lysate. Additional mutations restoring base-pair interaction between 323–326 (cggg) and 344–341 (gccc) recovered translation efficiency of the second cistron to 84 % of that of the authentic Rluc-1IRES736-Fluc construct. Because disruption of base-pair interactions between nt 322–326 and nt 345–341 moderately inhibited Fluc translation, formation of the pseudoknot may not be an absolute requirement for 5' IRES activity in vitro. However, the recovery of luciferase activity in the construct containing the compensatory mutation, 323–326 (cggg) and 344–341 (gccc), indicates that base-pair interactions in this region would affect IRES-mediated initiation of translation.
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). In contrast, only about 50 % of the 200 PSIV nt proximal to the initiation codon were accessible to DMS, CMCT and RNase T1 (Fig. 5). These data demonstrate that the PSIV sequence upstream of the initiation site has a different structure from that of RhPV and that the 5' IRES elements of PSIV and RhPV are distinct from one another.
The 5' IRES of PSIV does not function in plant or mammalian cell-free translation systems
When Fluc activity generated by PSIV 5' IRES-mediated translation was measured in WGE, the extremely low RLU values of the experimental template and the negative control (with no inserted sequence between Rluc and Fluc coding sequences) were nearly identical (Fig. 6a). In contrast, the activity mediated by the PSIV IGR-IRES measured 2.09x106 RLU, which is 275-fold higher than that of the negative control. These data indicate that the 5' IRES of PSIV does not function in WGE. The activity measured for in vitro translation of a PSIV 5' IRES template using RRL was 1.82x104 RLU (Fig. 6b), which was only one-twentieth of the activity of the IGR-IRES template, but was 2-fold higher than that of a negative control template with no inserted sequence (Fig. 6b).
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). Extrapolation of the magnesium acetate concentration curve (Fig. 6d) intersects the vertical axis at a value less than 1.5, suggesting that the concentration of magnesium acetate in RRL translation also is nearly optimal for the PSIV 5' IRES. Despite these optimal conditions, the PSIV 5' IRES template yielded very low luciferase activity, similar to previous experiments in which there were no translation products detected from PSIV 5' UTR-mediated translation in RRL (Sasaki et al., 1998). These results indicate that the 5' IRES of PSIV does not function in an RRL translation system.
5' IRES elements have distinct structures and functions in dicistroviruses
There are two IRES elements within the monopartite positive-stranded RNA genome of dicistroviruses. Whereas 5' IRES elements control translation of non-structural protein precursors, IGR-IRES elements control translation of capsid protein precursors.
Characterization of dicistroviral IGR-IRES elements indicates that the structure and function of these elements are conserved among dicistroviruses. In contrast, the 5' IRES of dicistroviruses do not appear to be conserved. To date, three 5' IRES elements have been reported in dicistroviruses: in CrPV (Wilson et al., 2000), RhPV (Woolaway et al., 2001) and TrV (Czibener et al., 2005). The 5' IRES of RhPV can function in RRL, WGE and insect cell lysate systems (Royall et al., 2004; Woolaway et al., 2001). Similarly, the 5' IRES of TrV functions in Xenopus oocytes, baby hamster kidney cells and insect cells, suggesting a simplified mode of internal ribosome entry, since host-specific trans-acting factors are unlikely to be required (Czibener et al., 2005). These data indicate that the 5' IRES elements in RhPV and TrV work independently of the translational specificity of the host apparatus.
In contrast, the 5' IRES of CrPV functions in RRL, but not in WGE (Wilson et al., 2000), and that of PSIV functions only in insect cell lysate. These differences demonstrate that individual dicistroviral 5' IRES elements have distinct requirements for mediating internal initiation and that, unlike IGR-IRES elements, initiation mechanisms mediated by 5' IRES elements vary among dicistroviruses. Furthermore, the stem–loop structures suggested by the structure probing analysis of PSIV 5' IRES do not appear to be conserved in the 5' UTR of other dicistroviruses. Contrary to our expectations, these facts imply that translation strategies for the non-structural protein precursor in dicistroviruses are diverse.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 10 May 2006;
accepted 21 August 2006.
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