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Short Communication |
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei Province 430072, China
Correspondence
Jiamin Zhang
jmzhang{at}whu.edu.cn
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ABSTRACT |
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A supplementary table showing primer sets used for PCR amplification is available with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Picornavirus IRES elements are grouped into two major classes according to their predicted secondary structure and activity in vitro (Belsham & Jackson, 2000; Jackson et al., 1994). The cardiovirus and aphthovirus elements represent one class that functions efficiently in the rabbit reticulocyte lysate (RRL) in vitro translation system. The enterovirus and rhinovirus IRES elements form a second class. These IRESs have low activity in the RRL system, but are stimulated by the addition of HeLa cell extracts (Brown & Ehrenfeld, 1979; Dorner et al., 1984). The hepatitis A virus IRES is distinct from those listed above and forms a minor class on its own; it can function in the RRL system, but its activity is stimulated in this system by liver cell and not HeLa cell extracts (Glass & Summers, 1993). These findings highlight the importance of cellular trans-acting factors in the mechanism of IRES action and could provide some explanation for the cellular tropism of picornaviruses. Indeed, it has been demonstrated that the intracellular activities of different picornavirus IRES elements vary in different cell types (Borman et al., 1997b; Roberts et al., 1998).
Ectropis obliqua picorna-like virus (EoPV) is an insect RNA virus that causes a lethal granulosis infection in larvae of the tea looper (Ectropis obliqua) (Wang et al., 2004). Several viruses with a genome organization similar to that of EoPV, including infectious flacherie virus (IFV), sacbrood virus of bees, Perina nuda picorna-like virus, deformed wing virus, Kakugo virus and Varroa destructor virus 1 (VDV-1), have been found in various species of insect. They have been grouped in the genus Iflavirus (Christian et al., 2005). Although the genus is not currently assigned to any virus family, the members of this genus have many characteristics in common with viruses in the families Picornaviridae and Dicistroviridae.
To date, several IRES elements isolated from members of the family Dicistroviridae have been shown to be functional in different translation systems. The 5' UTR and intergenic region (IGR) IRES of cricket paralysis virus (CrPV) and Rhopalosiphum padi virus (RhPV) are active in plant and mammalian translation systems, but have varying activities in different insect systems (Masoumi et al., 2003; Wilson et al., 2000; Woolaway et al., 2001). The 5' UTR of Triatoma virus contains an IRES element active in Xenopus oocytes (Czibener et al., 2005). In addition, the IGR IRES of Plautia stali intestine virus functions efficiently in vitro based on RRL (Sasaki et al., 1998). In the genus Iflavirus, the IFV genome is translated efficiently in mammalian RRL and plant-derived wheatgerm extract (WGE) systems (Hashimoto et al., 1984). The 5' UTR of VDV-1 is active in Lymantria dispar Ld652Y and Spodoptera frugiperda Sf21 cells of insect origin (Ongus et al., 2006). We have reported recently that an IRES mediates translation initiation of EoPV RNA (Lu et al., 2006). This study is aimed at determining the ability of the EoPV IRES to direct efficient translation in systems derived from different cell lines.
To examine the activity of the EoPV 5' IRES in the different translation systems, a set of cDNA fragments corresponding to regions of the EoPV 5' UTR were generated by PCR. EoPV cDNA (Wang et al., 2004) was used as a template in a number of PCRs. The primer sets used are shown in Supplementary Table S1 (available in JGV Online). The fragments obtained were purified and digested with the restriction enzymes XhoI and EcoRI, and the products were ligated into similarly digested plasmid pR
EF [described previously by Carter & Sarnow (2000)] to generate the pR/EoPV/F series of bicistronic plasmids shown in Fig. 1. The integrity of all constructs generated was confirmed by restriction-enzyme digestion and nucleotide sequencing. The plasmids pRE-EMCVF, containing a functional encephalomyocarditis virus (EMCV) IRES, and pRE-CrPVF, which contains a functional CrPV IGR IRES, were included in experiments as positive controls. pREF, which contains a non-functional EMCV IRES, was used as a negative control.
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EF-based plasmids were linearized with HindIII and transcripts were made by using the T7 RiboMAX Express Large Scale RNA Production system (Promega). These were analysed by Northern blotting using a probe specific for the firefly luciferase (Fluc) sequence. Digoxigenin (DIG) labelling and detection were performed, following the protocol of a DIG High Prime DNA Labelling and Detection starter kit II (Roche). A single species of RNA of the expected size was detected in each instance (Fig. 2a), indicating that the EoPV sequence did not contain a cryptic T7 promoter or induce RNA cleavage, which could have generated monocistronic Fluc transcripts.
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) were used to programme in vitro coupled transcription/translation (TNT) reactions based on RRL (Promega). Reactions contained [35S]methionine and the products were analysed by using SDS-PAGE and autoradiography. As expected, cap-dependent translation of the upstream Renilla luciferase (RLuc) open reading frame (ORF) was efficient from all plasmids (Fig. 2b). Translation was also mediated efficiently by the EMCV IRES (pRE-EMCV/F) (Fig. 2b, lane 3), but little Fluc was detected from the plasmid pREF, which contains a non-functional EMCV IRES element (Fig. 2b, lane 1). It could be seen (Fig. 2b, lanes 2) that Fluc was translated efficiently when an intact EoPV 5' UTR was present. Thus, we concluded that the EoPV 5' UTR contained an IRES element that was active in the in vitro mammalian RRL system. To determine the boundaries of the EoPV IRES, truncated versions of the EoPV 5' UTR were produced and inserted into the bicistronic vectors. These were also analysed in the RRL system. The constructs pR/EoPV2/F and pR/EoPV3/F were notably less efficient at mediating translation of FLuc than constructs containing the EMCV IRES or full-length EoPV 5' UTR (Fig. 2b, lanes 5, 6). However, Fluc was produced efficiently from the constructs pR/EoPV4/F and pR/EoPV6/F (Fig. 2b, lanes 7, 9). These results were coincident with those reported previously (Lu et al., 2006). We also assessed the function of the EoPV IRES in a plant translation system. The bicistronic plasmids (2 µg) were assayed in the TNT T7 Coupled WGE system (Promega) essentially as described by the manufacturer. Rluc and Fluc were assayed separately by using the Dual Luciferase Reporter (DLR) assay system (Promega) and a Turner Designs TD-20/20 luminometer according to the manufacturers' protocols.
CrPV is a picorna-like insect virus that contains two IRES elements. The one used for our studies is found in the IGR and has been reported to function efficiently in the WGE and RRL systems. The other is found in the 5' UTR of the viral RNA and is inactive in WGE (Wilson et al., 2000). The activity of the EoPV IRES was examined in the WGE system and compared with that of the CrPV IGR IRES. Our data revealed that, in contrast to the CrPV IGR IRES, the EoPV 5' IRES functions inefficiently in the WGE system (Fig. 3a).
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). This allowed efficient cytoplasmic transcription of transfected DNA. After 2 h at 27 °C, the medium was removed and the cell monolayer was washed with Grace's insect cell culture medium (Gibco). DNA transfection was carried out using 0.8 µg of each plasmid DNA (Fig. 1), mixed with Grace's insect cell culture medium and Cellfectin transfection reagent (Invitrogen) as described in the supplier's protocol. After 72 h incubation at 27 °C, cell lysates were prepared and Rluc and Fluc were assayed separately by using the DLR assay system. Results showed that the EoPV IRES functioned efficiently in Sf9 and Tn368 cells. The activities of the truncated versions were similar between the two insect cell lines (Fig. 3b, c). The CrPV IGR IRES was active in Tn368 cells, but inactive in Sf9 cells.
The activity of the EoPV IRES was then examined in a variety of different mammalian cell lines. Human uterine cervical adenocarcinoma cells (HeLa), African green monkey kidney cells (COS-7) and baby hamster kidney cells (BHK-21) were seeded into 24-well plates and grown to 90 % confluence. The bicistronic constructs (Fig. 1) were transfected into cells that had been infected 1 h previously with the recombinant vaccinia virus vTF7-3, which expresses T7 RNA polymerase (Fuerst et al., 1986). Transfections were performed by using Lipofectamine 2000 reagent essentially as described by the manufacturer (Invitrogen); 0.8 µg of the appropriate plasmid DNA was transfected into semiconfluent monolayers. At 48 h post-transfection, cell lysates were prepared and Rluc and Fluc were assayed separately by using the DLR assay system. Results showed that the EoPV IRES had a slight activity in BHK-21 and HeLa cells (Fig. 3d, f), but higher activity in COS-7 cells (Fig. 3e). However, the CrPV IGR IRES was active in BHK-21 and COS-7 cells, but inactive in HeLa cells. All of the truncated versions of the EoPV IRES demonstrated less activity than the full EoPV 5' UTR and the EMCV IRES in these systems (Fig. 3d, e, f), suggesting that the intact 5' UTR was important for activity of the EoPV IRES in mammalian cells.
The results presented here demonstrate that the 5' UTR of EoPV contains an IRES element. This IRES functions efficiently in the RRL in vitro translation system, albeit displaying less activity than the well-studied EMCV IRES. However, in addition, the EoPV 5' UTR IRES functions well in other systems in which the EMCV IRES is essentially inactive. The activity of the EoPV IRES in Sf9 and Tn368 cells was about 10-fold greater than that of the pREF vector alone (Fig. 3b, c). The lack of EMCV IRES activity observed with this system is in line with data of Finkelstein et al. (1999), who found that the EMCV IRES was inefficient at directing internal initiation in a range of different insect cells. The data obtained with the EoPV IRES in Sf9 cells are consistent with those reported for the RhPV 5' IRES elements (Domier & McCoppin, 2003; Royall et al., 2004). IRES activity was reported for both the 5' UTR and the IGR of CrPV in the RRL system, although the IGR demonstrated higher activity than the 5' UTR. However, the 5' UTR of CrPV was inactive in WGE, although the CrPV IGR was active in this system (Wilson et al., 2000). Moreover, these IRES elements were active in Tn368 cells, but inactive in Sf9 cells (Masoumi et al., 2003). The RhPV 5' IRES functioned efficiently in the plant (WGE), insect (Sf9 and Sf21 cells) and mammalian (RRL) translation systems (Domier & McCoppin, 2003; Royall et al., 2004; Sasaki et al., 1998). The EoPV 5' IRES was shown here to function inefficiently in the plant translation system (WGE), but efficiently in insect (Sf9 and Tn368 cells) and mammalian (RRL; BHK-21, COS-7 and HeLa cells) translation systems. These observations were in general agreement with the findings of Borman et al. (1997a), who showed that IRES elements of mammalian picornaviruses perform differently depending on the host.
The EoPV IRES continued to function, albeit at a reduced efficiency, when almost 112 bases were removed from the 5' end of the 5' UTR (Fig. 2b). Deletion of 178 nt from the 5' end of the EoPV 5' UTR had a significant negative effect on IRES activity. It appears that nt 63–178 are essential for EoPV IRES activity. However, constructs pR/EoPV4/F and pR/EoPV6/F showed an increased IRES activity compared with the full 5' UTR in RRL, indicating that nt 299–390 may decrease the efficiency of IRES activity in RRL, presumably due to disruption of ribosomal scanning and subsequent correct initiation of protein synthesis. These results were also observed in Sf9 insect cells, but not in mammalian cells, indicating that EoPV IRES activity was dependent on the full 5' UTR to form the correct IRES structure in mammalian hosts.
Taken together, the ability of the EoPV 5' UTR to function not only in mammalian systems, but also in insect translation systems, suggests the potential utility of this element in insect and mammalian expression. In addition, this study indicates that there are important differences in IRES function between the EoPV IRES and other previously characterized picorna-like insect viral IRESs.
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ACKNOWLEDGEMENTS |
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EF, pRE-EMCVF and pRE-CrPVF and Professor J. Vlak and Dr Marcel Westenberg (Laboratory of Virology, Wageningen University, Wageningen, The Netherlands) for the provision of the recombinant baculovirus AcT7N. We also thank Professor Congyi Zheng for providing excellent laboratory facilities and Dr Louisa S. Chard for critical reading of the manuscript. |
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Received 31 May 2007;
accepted 15 June 2007.
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