1 Fundación Instituto Leloir, Avenida Patricias Argentinas 435, Buenos Aires 1405, Argentina
2 Centro de Virología Animal CEVAN, Serrano 669, 3er piso, Buenos Aires 1414, Argentina
Correspondence
Andrea V. Gamarnik
agamarnik{at}leloir.org.ar
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ABSTRACT |
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MAIN TEXT |
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We have previously reported the complete nucleotide sequence analysis of TrV and showed that this virus belongs to the family Dicistroviridae (Czibener et al., 2000), formerly known as insect picorna-like viruses (Mayo, 2002
). The members of this family possess single-stranded, positive-sense RNA genomes with a distinctive bicistronic arrangement. The RNA genome contains two open reading frames (ORFs) each encoding a polyprotein separated by an intergenic region. The non-structural proteins are encoded in the 5'-proximal ORF and the structural proteins are encoded in the second ORF (Czibener et al., 2000
; Domier et al., 2000
; Johnson & Christian, 1998
; Sasaki et al., 1998
; Wilson et al., 2000
). For several members of this family, it has been demonstrated that the two ORFs are preceded by RNA structures that function as internal ribosomal entry sites (IRESs) for translation of the viral proteins (Domier et al., 2000
; Kanamori & Nakashima, 2001
; Sasaki et al., 1998
; Wilson et al., 2000
; Woolaway et al., 2001
). The 5'UTR and the intergenic region (IGR) IRES exhibit different sequences and presumably different mechanisms of translation initiation. An unusual feature of the IGR-IRES is that translation of the capsid proteins initiates with an amino acid other than methionine. Usually, the initiation site selection for translation involves base-pair formation between an AUG codon and the anticodon triplet of an initiator methionine tRNA. In contrast, for several members of the Dicistroviridae, different initiation codons were found: CUU for Plautia stali intestine virus (PSIV) and CCU in the case of Cricket paralysis virus (CrPV). It has been proposed that secondary and tertiary structures of the RNA within the IGR enable Met-independent initiation of translation (Domier et al., 2000
; Jan et al., 2003
; Jan & Sarnow, 2002
; Pestova et al., 2004
; Sasaki & Nakashima, 2000
; Spahn et al., 2004
; Wilson et al., 2000
).
Translation initiation mediated by the 5'UTR and the IGR of TrV has not been examined. In order to investigate the translation of the two ORFs of TrV, we generated different RNA molecules carrying the firefly luciferase gene flanked by the 5'UTR or the IGR and the 3'UTR of TrV. To this end, we obtained viral particles from infected T. infestans and purified them using sucrose gradients (1030 %) as previously described (Muscio et al., 1988). RNA extraction was performed using TRIzol and directly used for reverse transcription and PCR amplification of the 5'UTR, the IGR and the 3'UTR. According to our previous sequencing and alignment analysis, we defined the 3' boundary of the 5'UTR IRES at nt 549 and the IGR-IRES spanning nt 59346111 (GenBank accession no. AF178440). From sequence alignments, we deduced that the initiator triplet of ORF2 is CUC (Czibener et al., 2000
). Both the 5'UTR and the IGR (including the first 40 nt of the respective viral-coding sequences) were fused in-frame with the luciferase-coding region. Amplification of the viral sequences was performed using the primers indicated in Fig. 1
(a). In vitro transcriptions were performed to generate the RNA 5'UTR-TrV-Luc and IGR-TrV-Luc (Fig. 1b
). Translation was evaluated by microinjecting the RNAs into Xenopus oocytes. This system has proved to be a useful tool to analyse IRES-dependent translation, since, in contrast to in vitro translation systems, it does not initiate translation of uncapped RNAs (Fig. 1c
) (Gamarnik & Andino, 1996
; Gamarnik et al., 2000
).
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To extend these studies, we tested the ability of the 5'UTR and IGR of TrV to direct translation of the reporter in different cell types. RNA was transfected into baby hamster kidney (BHK) and insect (C6/36) cells using Lipofectamine 2000 (Invitrogen). The RNAs were in vitro transcribed and purified (RNeasy; Qiagen). In contrast to microinjection into oocytes, in which precise volumes of RNA can be delivered inside the cell, transfection of RNA into cells grown in culture required normalization. Thus, we co-transfected quantified RNAs with a second capped mRNA encoding Renilla luciferase. Translation efficiencies were expressed as the ratio of the activities measured for the firefly and Renilla luciferases in each case. Similar to the results observed in oocytes, translation of the RNA mediated by the 5'UTR or IGR of TrV was efficient, while the uncapped RNA control only showed background levels (Fig. 1d). In addition, in all the systems used, the TrV IGR was 3050 % more efficient in directing translation than the viral 5'UTR.
The RNA molecules carrying the 5'UTR or IGR of TrV at the 5' end also contained the complete 3'UTR sequence of TrV (295 nt) after the stop codon of luciferase. It has previously been shown that sequences and RNA structures present at the 3'UTR of viral and cellular mRNAs can modulate cap- and IRES-mediated translation initiation (reviewed by Mazumder et al., 2003). To test whether the 3'UTR was important for efficient IRES activity, we replaced the 3'UTR of TrV with unrelated 3'UTRs [3'UTR of dengue virus (DV) or the 3'UTR of
-globin]. Translation of the RNAs carrying the 5'UTR or IGR of TrV was efficient for both TrV and the unrelated 3'UTRs (data not shown), suggesting that translation initiation mediated by the two putative IRESs of TrV is not modulated by specific 3'UTR elements.
To confirm the internal entry of ribosomes during translation of the TrV genome, we constructed bicistronic mRNAs in which the 5'UTR and IGR of TrV were introduced preceding a second ORF. A schematic representation of the RNA constructs is shown in Fig. 2(a) (Bicis 5'UTR TrV-Luc and Bicis IGR TrV-Luc RNAs). The two bicistronic RNAs were microinjected into Xenopus oocytes or transfected into BHK and C6/36 cells as described above. Luciferase activity was observed with both RNAs in all cell types used, confirming that the 5'UTR and IGR of TrV can direct internal entry of ribosomes (Fig. 2b
). To determine the background levels of translation of the second cistron due to leaky scanning, we constructed a bicistronic RNA control carrying an unrelated sequence of 200 nt in the intergenic region preceding a firefly luciferase-coding sequence. This bicistronic construct was capped and encoded Renilla luciferase in the first ORF. Analysis of the translation efficiency of both luciferases in transfected BHK cells showed efficient translation only from the first ORF (Fig. 2c
).
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It has been reported that mutation of the initiator CCU in CrPV impairs IGR-IRES function, which is in agreement with the proposed formation of a pseudoknot structure during the initiation process (Wilson et al., 2000). In contrast, the IGR-IRES of PSIV tolerates mutations in the initiator triplet CUU (Shibuya et al., 2003
). These observations indicate that, even though many similarities exist in the mechanism of initiation mediated by the IGR of different dicistroviruses, there are some features that are different among them. To examine the requirements of the TrV IGR-IRES, we mutated the initiator codon CUC to AUG or CCU in the bicistronic RNA constructs. Translation of the three RNAs with different initiator triplets was very efficient (data not shown), suggesting that the initiation site is flexible during translation mediated by the TrV IGR-IRES, resembling the initiation of PSIV.
We observed that translation efficiency of the RNAs carrying the TrV IGR directing initiation of a second cistron, which resembles the natural position in the viral genome, was consistently three to fivefold less that that observed with the 5'UTR IRES (Fig. 2b). These observations are intriguing, as it has been noted that the capsid proteins (ORF2) are produced in large excess over the non-structural proteins in cells infected with insect picorna-like viruses (Moore et al., 1981
), suggesting that the IRES activity present in the IGR should be more efficient than the IRES located at the viral 5'UTR. It is possible that changes in the cellular translation machinery during viral infection could result in a differential modulation of the two IRESs. Indeed, it has previously been reported that stress or direct phosphorylation of initiation factor IF2-
, conditions likely to occur during viral infection, enhances translation mediated by the CrPV IGR-IRES (Fernandez et al., 2002
). It has been postulated that translation initiation by the IGR-IRES independently of the IF-2GTPtRNAi complex could explain the advantage of translation of RNAs with this IRES over translation of other mRNAs in conditions with low active IF2-
(Fernandez et al., 2002
; Thompson et al., 2001
).
In order to examine whether the IRES activities present in the TrV genome were differentially modulated by an antiviral state of the cell, we analysed the translation efficiency of the two IRESs in BHK cells pre-treated with IFN-, which is known to induce phosphorylation of IF2-
(reviewed by Katze et al., 2002
). For these experiments, we constructed a new bicistronic mRNA mimicking the genomic organization of TrV. The RNA contained: (i) the 5'UTR of TrV followed by the first ORF encoding firefly luciferase; (ii) the IGR-IRES of TrV followed by a second ORF encoding Renilla luciferase; and (iii) the 3'UTR of TrV (5'UTR-Fluc-IGR-Rluc RNA; Fig. 3a
). BHK cells were treated with IFN-
(1000 IU per 35 mm culture plate) for 24 h before transfection with the 5'UTR-Fluc-IGR-Rluc RNA. The luciferase activities obtained in the untreated control cells were arbitrarily set to 100 % and the translation of the respective RNA in the treated cells was expressed relative to the controls. A large decrease in firefly luciferase activity in treated cells indicated that translation mediated by the 5'UTR of TrV was strongly inhibited under these conditions (Fig. 3b
). In contrast, Renilla luciferase activity was higher in the treated cells, suggesting that translation by the IGR-IRES was not reduced by IFN-
(Fig. 3b
). These results indicated that the relative translation efficiency of the two IRESs drastically changes, resulting in a sixfold increase in IGR-IRES translation upon IFN-
treatment.
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Taken together our results confirm that TrV translation is mediated by two different IRESs. Translation activities of both IRESs were detected in different cell types, even in Xenopus oocytes, suggesting a minimum requirement of host factors. Furthermore, the translation efficiency of the two IRESs was differently modulated under conditions that resemble virus infection, providing a mechanism to control the relative amounts of structural and non-structural viral proteins during replication.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 17 December 2004;
accepted 19 May 2005.
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