1 School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
2 RiNA GmbH, Takustraße 3, 14195 Berlin, Germany
3 BBSRC Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK
Correspondence
Lisa O. Roberts
l.roberts{at}surrey.ac.uk
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ABSTRACT |
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E. R. and K. E. W. contributed equally to this work.
Present address: BBSRC Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK.
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MAIN TEXT |
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The best-characterized IRES elements are those from the mammalian picornaviruses (reviewed by Belsham & Jackson, 2000). The picornavirus IRES elements are grouped into two major classes based on their predicted secondary structure and their activity in vitro. One class contains IRES elements from the entero- and rhinoviruses (e.g. poliovirus) while the second contains the cardio- and aphthovirus IRES elements (e.g. encephalomyocarditis virus, EMCV). The cardio-/aphthovirus IRES elements function efficiently in the rabbit reticulocyte lysate (RRL) translation system. However, the poliovirus and rhinovirus IRES elements are inefficient in this system unless the reaction is supplemented with HeLa cell extracts (Brown & Ehrenfeld, 1979
; Dorner et al., 1984
). Similarly, the activity of the hepatitis A virus IRES (which forms a third class of IRES) is stimulated by the addition of liver cell, but not HeLa cell, extracts (Glass & Summers, 1993
). It is clear that cellular trans-acting factors play an important role in the mechanism of IRES action and may contribute to the cellular tropism of picornaviruses. Indeed, it has been demonstrated that different IRES elements function with different efficiencies in different cell types (Borman et al., 1997
; Roberts et al., 1998
).
We have previously demonstrated that the 5' UTR of Rhopalosiphum padi virus (RhPV) mRNA contains an IRES element (Woolaway et al., 2001). This virus belongs to the family Dicistroviridae, which also includes cricket paralysis virus (CrPV), Drosophila C virus (DCV) and Plautia stali intestine virus (PSIV). The single-stranded, positive-sense RNA genome of these viruses contains two open reading frames (ORFs) that encode two polyproteins (Moon et al., 1998
). ORF1 encodes the non-structural proteins and ORF2 the structural proteins. All of these proteins possess sequence similarity with mammalian picornavirus proteins. It has been shown that the 5' UTRs and the intergenic regions (IGRs) of these virus genomes contain IRES elements (Sasaki & Nakashima, 1999
; Domier et al., 2000
; Wilson et al., 2000a
; Woolaway et al., 2001
). The IGR IRES elements are unusual in that they direct translation initiation from non-AUG codons and they do not require any of the canonical initiation factors for assembly of initiation complexes on the mRNA (Wilson et al., 2000b
; Nishiyama et al., 2003
). In contrast, the 5' IRES of RhPV directs initiation from AUG codons. It has been shown to function in a Drosophila cell-based in vitro translation system and also functions efficiently in RRL and wheat germ lysates (Woolaway et al., 2001
). The ability of the RhPV 5' IRES to function in insect translation systems suggests potential utility of this IRES in insect cell expression systems. To explore this further, we have tested the function of this IRES in Sf21 cells and in a novel in vitro Sf21 cell-based lysate system (Kubick et al., 2003
). These cells are commonly used with baculovirus expression systems. We report here that the RhPV 5' IRES displays efficient activity in these cells and in the in vitro system.
To examine the activity of the RhPV 5' IRES within Sf21 cells, reporter plasmids of the form T7:CAT/IRES/LUC containing the RhPV IRES (previously referred to as RhPV1; nt 1 to 579) or the mammalian picornavirus EMCV IRES were used, as described by Woolaway et al. (2001)
. Cap-dependent translation was monitored by measuring chloramphenicol acetyltransferase (CAT) expression and activity of the IRES element was assessed from the expression of luciferase (LUC). Mutated versions of the RhPV IRES plasmid containing 5' and 3' end deletions [named RhPV
2 (with nt 1 to 463),
3 (nt 1 to 374) and
4 (nt 100 to 588)] have also been described previously (Woolaway et al., 2001
). Sf21 cells were grown at 28 °C in TC100 medium (Gibco-BRL) supplemented with fetal bovine serum (FBS; 10 %), penicillin (5000 units penicillin G sodium ml1), streptomycin (5000 µg streptomycin sulphate µl1 in 0·85 % saline) and 2 mM L-glutamine. The dicistronic reporter plasmids were assayed by transfection into Sf21 cells (60 mm dishes) using lipofectin (20 µl; 1 mg ml1 stock; Gibco-BRL). T7 RNA polymerase was expressed in the cells by prior infection with a recombinant baculovirus (AcT7N; gift from Dr J. Vlak, University of Wageningen, The Netherlands; van Poelwijk et al., 1995
). After 48 h incubation at 28 °C, the cells were harvested in 400 µl cell lysis buffer (Promega) and the lysates clarified by centrifugation at 14 000 r.p.m. for 5 min at 4 °C. CAT assays were performed using the CAT ELISA kit (Boehringer Mannheim) as described in the manufacturer's instructions. Colour development was measured on an ELISA plate reader (Labsystems Multiscan Bichromatic) at 405 nm. As expected, each of the constructs induced a similar level of CAT expression (Fig. 1
). LUC expression was measured using a LUC assay kit (Promega) and a Bio-orbit luminometer. The RhPV 5' IRES was able to direct LUC expression at a level 23-fold higher than the background LUC expression seen with pGEM-CAT/LUC (no IRES) and 400-fold higher than seen from the construct containing the EMCV IRES (Fig. 1
). Presumably, the presence of the structured EMCV RNA sequences inhibited any translational read-through and hence LUC expression. As we have seen previously in in vitro translation systems (Woolaway et al., 2001
), the 5' and 3' end deletion mutants directed less-efficient expression of LUC in cells. However, the mutants still retained about 40 to 50 % activity of the wild-type RhPV IRES within cells (Fig. 1
). The antisense version of the RhPV IRES was unable to direct LUC expression as expected.
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Insertion of the RhPV 5' IRES into an uncapped monocistronic mRNA increased the translational efficiency in the Sf21 lysate by fourfold (Kubick et al., 2003). In this study we have also demonstrated that the RhPV IRES directed LUC expression between three- and 10-fold over that seen from the CAT/no IRES/LUC control in vitro. However, within Sf21 cells, the increase in expression from the RhPV IRES was 23-fold. This is likely due to a lower background of internal initiation from the no IRES dicistronic mRNA within cells compared with that seen in vitro. This is analogous to the mammalian systems in which a mutant EMCV IRES displayed around 0·25 % of the activity of a wt EMCV IRES within cells, whereas in the in vitro system the activity was around 5 % (van der Velden et al., 1995
).
These data suggest that use of this IRES element within an insect cell-based expression vector can enhance translation, without the need for making capped transcripts in vitro. Similarly, insertion of the IRES between two ORFs may allow for expression within the baculovirus expression system of a protein of interest along with a reporter or selectable marker, or expression of two subunits of a protein within the same cells at the same time.
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ACKNOWLEDGEMENTS |
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Received 27 January 2004;
accepted 19 February 2004.