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Animal: RNA Viruses |
Department of Microbiology and Immunology and the Co-operative Research Centre for Vaccine Technology, The University of Melbourne, Australia1
The Walter and Eliza Hall Institute of Medical Research, PO The Royal Melbourne Hospital, VIC 3050, Australia2
Author for correspondence: Brendan Crabb (at The Walter and Eliza Hall Institute of Medical Research). Fax +61 3 9347 0852. e-mail crabb{at}wehi.edu.au
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Introduction |
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; Pringle, 1999 ). The closest known relatives are the members of the Cardiovirus and Aphthovirus genera. Although the nucleotide sequence of the ERBV genome is known (Wutz et al., 1996 ) the virus has not been extensively studied. ERBV infection of horses can result in an acute febrile respiratory tract disease characterized by coughing and lymphadenitis (Mumford & Thomson, 1978 ) and horses can carry ERBV for long periods of time (Burrows, 1978 ; Thorsen, 1991 ). Recent studies have suggested that ERBV may be an important cause of respiratory disease in horse populations (Carman et al., 1997 ; McCollum & Timoney, 1992 ). Further investigation into the epidemiology and pathogenesis of ERBV infections is clearly required.
The positive-sense RNA genome of picornaviruses is not capped but instead contains a long 5'-nontranslated region (NTR) which allows cap-independent, internal translation initiation. This process is facilitated by an internal ribosomal entry site (IRES) which forms an extensive and highly stable secondary structure that interacts with both canonical and non-canonical translation factors (Andino et al., 1999 ; Belsham & Sonenberg, 1996 ). Some picornaviral IRESs are recognized as virulence determinants, perhaps through the involvement of these non-canonical factors. The predicted IRES structure and start codon usage vary considerably throughout the genera but generally picornavirus IRESs conform to one of three models known as IRES types I, II and III. Type I is found in enteroviruses and rhinoviruses and is characterized by a particular IRES fold and by translation initiation occurring a considerable distance downstream of this structure (Agol, 1991 ; Pilipenko et al., 1992 ). Cardioviruses and aphthoviruses contain a type II IRES. These fold into a distinct secondary structure, characterized by stem–loops H–L, and direct translation initiation to an AUG immediately 3' of the IRES structure some 12–15 nt downstream of the polypyrimidine tract (Hinton et al., 2000 ; Palmenberg & Sgro, 1997 ; Pilipenko et al., 1989 ; Stewart & Semler, 1997 ). Type II IRESs may also lead to translation initiation at additional downstream AUG codons such as in the genome of the demyelinating form of Theiler’s murine encephalomyelitis virus (TMEV), strain DA (Kong & Roos, 1991 ; Yamasaki et al., 1999 ) and in members of the Aphthovirus genus (Beck et al., 1983 ; Clarke et al., 1985 ; Hinton et al., 2000 ). The type III IRES model is found in the genome of Hepatitis A virus (HAV) and appears to share characteristics of both types I and II (Brown et al., 1991 ).
The cardiovirus Encephalomyocarditis virus (EMCV) strain R uses the 11th AUG codon in the 5'-NTR to initiate translation of the polyprotein. This codon is the central of three AUG codons in a region of 25 nt downstream of the polypyrimidine tract. The use of AUG11, as opposed to AUG10 and AUG12, appears to be due to a number of factors including distance from the IRES and sequence context surrounding the AUG codon (Davies & Kaufman, 1992 ; Kaminski et al., 1994 ). The ERBV genome has three similarly positioned AUG codons.
In this report we describe secondary structure modelling and functional characterization of the ERBV IRES using a bicistronic system. Structural modelling predicted that ERBV possesses a type II IRES. Full translation initiation activity required nt 189–920 downstream of the poly(C) tract, a region that includes the core type II stem–loops H–L and some additional sequence/structures upstream. We present evidence that the second of the three putative AUG codons is the major translational start site and show that the ERBV IRES is at least as efficient if not more so than the EMCV IRES in rabbit reticulocyte lysates (RRLs).
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Secondary structure determination. ) downstream of the poly(C) tract was created through use of the mFOLD program (Mathews et al., 1999 ; Zuker et al., 1998 ). The minimum free-energy structures were determined for the first 921 nt. To assist in the modelling (i.e. to choose between different thermodynamically stable structures), the corresponding nucleotide sequences of EMCV strain B (Bae et al., 1989 ), EMCV strain R (Palmenberg et al., 1984 ), TMEV strain DA (Pevear et al., 1987 ), Foot-and-mouth disease virus (FMDV) strain A12 (Robertson et al., 1985 ) and ERAV 393/76 (Li et al., 1996 ) were aligned with the use of Clustal W software (Thompson et al., 1994 ) (data not shown). The alignment was analysed for conserved regions especially within distal loops of known function.
Plasmids.
Plasmids were constructed using standard methods with only slight modifications (Sambrook et al., 1989 ). The 5'-NTR of ERBV was subjected to RT–PCR amplification with specific oligonucleotides containing an AgeI restriction site (Table 1). Briefly, purified ERBV RNA was copied into cDNA with Superscript II reverse transcriptase according to the manufacturer’s instructions (Gibco-BRL). The cDNA was subjected to PCR amplification in the presence of the relevant oligonucleotides and using 35 cycles at 94 °C 45 s, 58 °C 45 s and 72 °C 45 s with Taq polymerase according to instructions (Promega). PCR products were digested with AgeI before ligation into similarly digested parental bicistronic plasmid pT7CG containing the CAT and GFP reporter genes as described previously (Hinton et al., 2000 ). These plasmids included pEB(1–920) [containing nt 1–920 downstream from the poly(C) tract], pEB(189–920) and pEB(351–920) (Table 1). Note that in these plasmids there is a 13 nt spacer between the AgeI site and the GFP initiation codon provided by the parent GFP vector (Hinton et al., 2000 ).
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. The PCR products were digested with AgeI and BsrGI, at sites located within the GFP coding sequence, before ligation into the same sites of pT7CG. This process removes the 13 nt spacer mentioned above. Plasmids containing AUG mutations were constructed by site-directed mutagenesis on pEB(186–920) through overlap extension PCR by standard techniques. For the mutation of AUG triplets to AUAs the oligonucleotides shown in Table 2 were used. PCR products were digested with AgeI and ligated into this site in pT7CG. All ERBV inserts were completely sequenced by primer extension and BigDye chain chemistry (Applied Biosystems) to confirm the correct sequence of each insert. Plasmids used for the comparative IRES analyses were constructed similarly. pE1(1–965) was described previously (Hinton et al., 2000 ) and pEB(1–920) is described above. The EMCV IRES was amplified from the pTM-1 (Moss et al., 1990 ) construct using the oligonucleotides shown in Table 1
. The EMCV IRES was then digested with AgeI and ligated into pT7CG to derive pEMCV. For the CAT cap-dependent constructs, pE1(1–965) was fully digested with XhoI and partially digested with NcoI. The insert containing the CAT-ERAV IRES-GFP cassette was ligated into NcoI/XhoI-digested pET28a (Novagen) to derive pET.E1(1–965). The ERAV IRES was then removed from pET.E1(1–965) with AgeI to derive pET.CG. AgeI fragments containing ERBV and EMCV IRESs were isolated from plasmids described above and inserted into this plasmid to derive pET.EB(1–920) and pET.EMCV.
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). This PCR product was ligated into pGEMTeasy (Promega). After sequencing, the insert was excised with NheI/XhoI and ligated into similarly digested pET28a to produce pET
CAT.GFP, a plasmid that is in effect identical to pET.CG but with most of the CAT gene removed. The IRES elements from ERAV, ERBV and EMCV were excised from pE1(1–961), pE2(1–920) and pEMCV using AgeI and ligated into similarly digested pETCAT.GFP to derive pETCAT.EA, pETCAT.EB and pETCAT.EM respectively.
Transient expression assays.
For T7-mediated transcription, 0·5 µg of plasmid DNA was transfected into BHK-21 cells which had been infected 1 h previously with recombinant vaccinia virus vTF7-3 expressing T7 RNA polymerase (Fuerst et al., 1986 ). Lipofectamine Plus (Gibco-BRL)-mediated transfection was carried out as described previously (Hinton et al., 2000 ). Cell extracts were prepared 20 h post-transfection. Each experiment was repeated at least twice. Transfected cells were harvested by trypsin treatment, resuspended in PBS and separated for the relevant assays. CAT activity was determined by thin-layer chromatography as directed by the manufacturer (Promega). GFP activity was determined by FACS analysis as described previously (Hinton et al., 2000 ). For each sample the GFP:CAT represented a ratio of total GFP fluorescence (mean) to CAT activity (% acetylation in a linear range) from an equivalent number of cells.
In vitro transcription and translation.
Coupled transcription and RRL translation reactions were performed essentially as recommended by the supplier (Promega). Briefly, 0·5 µg of plasmid DNA, except where indicated, and [35S]methionine (10 µCi) were added to a methionine-free RRL reaction mixture and the reaction incubated at 30 °C for 90 min. In reactions where two plasmids were combined, 0·25 µg of each was added. If a single plasmid was to be compared with samples containing two plasmids, 0·25 µg of parental pET28a was added to the mix such that the final amount of DNA in all reactions was 0·5 µg. The samples were separated on 15% SDS–PAGE gels. Gels were fixed for 30 min at room temperature (7% acetic acid, 10% methanol), scintillated for 30 min at room temperature (1 M sodium salicylate, 50% methanol), dried and radioactivity detected by phosphoimager analysis for isotope quantification and exposed to X-ray film for preparation of figures.
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). The model incorporates 15 stem–loop structures to a position just downstream of the polypyrimidine tract that demonstrates the conserved characteristics of a type II IRES. This includes the G, H, I, J, K and L stem–loops. As with ERAV (Hinton et al., 2000 ) and according to an EMCV model proposed by Palmenberg & Sgro (1997) , stem–loop I was found to be more thermodynamically stable when separated into three smaller stem–loops, Ia, Ib and Ic. Alignment of the 5'-NTR sequence of ERBV with members of the Cardiovirus and Aphthovirus genera revealed a low overall sequence identity but showed conservation of many of the unpaired regions predicted to be present in the stem–loop structures (Fig. 2).
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). In this plasmid the reporter genes are both placed under the control of the T7 RNA polymerase promoter (Hinton et al., 2000 ). Initially, the entire 5'-NTR was inserted into pT7CG with the 3' boundary placed 22 nt downstream of the first putative initiation codon, corresponding to nt 920. This region incorporated the three potential AUG codons in-frame with the GFP gene (Fig. 3A). Plasmids containing progressive 5' and 3' deletions of the ERBV 5'-NTR insert were made and the sequence of each fragment was determined to confirm that the overlapping regions were identical. When transfected into BHK-21 cells infected with a recombinant vaccinia virus that expresses T7 RNA polymerase (vTF7-3), the full-length construct pEB(1–920) showed strong expression of GFP compared to the parental vector pT7CG (Fig. 3B). This indicated that an active IRES was present in this viral sequence. Deletion of the 5'-end to nt 189 [pEB(189–920)] had no apparent effect on GFP expression. A further 5' truncation to nt 351 [pEB(351–920)] caused a substantial decrease in GFP expression indicating that sequence between nt 186–351 contained an element important to translation initiation. Truncation of the 3' end of the IRES to AUG1 [pEB(189–895)] also led to a major reduction in GFP expression; however, truncation to AUG2 [pEB(189–904)] or AUG3 [pEB(189–916)] had little effect. This indicates that sequence between AUG1 and AUG2 is important for translation initiation. All plasmids were transfected on at least two separate occasions with very similar results obtained. Each plasmid was also assayed in vitro using a coupled transcription/translation system. In this system, a similar IRES activity pattern to that seen in transfected the BHK-21 cells was observed (Fig. 3C). However, in the RRLs both pEB(189–904) and pEB(189–916), which appeared to be fully active in BHK-21 cells, had substantially reduced activity (
40%) relative to the parental pEB(189–920). This result may reflect a difference in the detection systems between the two assays. It is possible that the GFP species with no or few amino acids fused to the N terminus may fluoresce more brightly, perhaps leading to artificially high GFP to CAT ratios for pEB(189–904) and pEB(189–916) in the BHK-21 cells where fluorescence was used as a measure of GFP expression [note that pEB(189–920) contains a 13 nt spacer upstream of the GFP AUG that is not present in pEB(189–904) and pEB(189–916) and hence has additional amino acids fused to the N terminus]. These results imply a role for residues between 916 and 920 in translation efficiency.
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a difference in migration of the GFP species produced by plasmids with deletion to AUG1 and AUG2 was apparent. It was expected that the GFP species produced here would most likely represent wild-type GFP with no residues fused to the N terminus. Somewhat surprisingly, this species migrated more slowly than the GFP species with N-terminal fusions, presumably due to charge and/or structural differences between these species. The plasmid that contained the deletion to AUG3 pEB(189–916) demonstrated no alteration in GFP migration from that of the parental pEB(189–920), indicating that the GFP species produced by this construct possessed residues fused the N terminus and hence initiation was occurring from a codon upstream of AUG3. This is addressed further below.
Identification of the ERBV initiation codon
A site-directed mutational approach was performed on the parental plasmid pEB(189–920) to determine which of the three AUG codons at the 3' end of the IRES initiated translation of the ERBV polyprotein. The AUG codons were mutated to AUA either individually or together (Fig. 4A) and the entire sequence of each ERBV IRES insert was determined to ensure that only the desired change had been effected. The resulting plasmids were both transfected into vTF7-3-infected BHK-21 cells (Fig. 4B) and also analysed by in vitro transcription/translation in RRLs (Fig. 4C). Plasmids containing mutations of AUG1 and/or AUG3 led to a relatively small reduction in the efficiency of GFP translation in both systems (65–95%) of parental pEB(189–920). The migration of the GFP species produced by these plasmids in RRLs was also identical to that observed in parental pEB(189–920) (Fig. 4C). In contrast, plasmids containing mutations of AUG2 either alone or in combination with mutation to AUG1 and/or AUG3 led to a more substantial reduction in GFP expression [37–68% of parental pEB(189–920)] and to shifts in the migration of the GFP species produced in the RRL. This result suggested that AUG2 played a major role in translation initiation.
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, C), although it is apparently only accessed minimally if at all in the wild-type context given the shift to the predominant use of the GFP AUG codon in the pEB(189–920)2/3 mutant (Fig. 4C). A faint band corresponding to initiation from AUG1 could be observed for this vector in the RRL and this band was not observed with the pEB(189–920)1/2/3 mutant.
Comparison of cardiovirus, aphthovirus and erbovirus IRES activity
To investigate the relative strength of three type II IRESs, a comparative assay was performed. For this we constructed a bicistronic plasmid, pET.CG, in which translation of the first reporter gene was 5'-cap-dependent (Fig. 5A). The second reporter gene was placed under the control of the IRES from ERAV, ERBV or EMCV and activity was analysed by coupled in vitro transcription and translation in RRLs. In this system, all three IRESs appeared to be similarly active although we observed 30% more GFP translated by the ERBV IRES than by the EMCV IRES (Fig. 5B). In contrast, GFP was slightly less efficiently expressed by the ERAV IRES than by the EMCV IRES in this assay. Repeat assays consistently showed that the three IRESs were of similar strength although the ERBV IRES was not always more active than that from EMCV. One such assay is shown in Fig. 5(C).
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). These plasmids were also analysed in RRLs. Again, the ERBV IRES in pEB(1–920) produced slightly more expression of GFP than did the EMCV IRES in pEMCV. In contrast, the IRES from ERAV was less efficient in this system, expressing GFP to only 31% of the EMCV IRES (Fig. 5E). This was a consistent observation over several independent assays with this particular RRL reagent (data not shown). We repeated this assay with new DNA preparations and in vitro transcription/translation reagents and while the same pattern was observed, the ERBV IRES was somewhat more active in this assay (Fig. 5F). Using the same reagents as employed in Fig. 5(C) and 5(F) we tested if similar competition was observed when the amount of DNA used in the reaction was varied (Fig. 6). At the three DNA amounts tested (0·25, 0·5 and 1·0 µg), the same pattern was observed: that is, the ERBV IRES was able to compete efficiently with the EMCV IRES for translation factors whereas the ERAV IRES was not. Interestingly, as the amount of DNA was increased, the ability of the ERBV IRES to compete with that from EMCV improved in a roughly linear fashion (Fig. 6).
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CAT.GFP was constructed (Fig. 7A). The fully active IRES elements from ERAV, ERBV and EMCV were inserted into the AgeI site of this plasmid to derive pETCAT.EA, pETCAT.EB and pETCAT.EM, respectively. All three plasmids produced similar levels of GFP in an in vitro assay, again consistent with our earlier observations that each IRES is of similar strength (Fig. 7B, right lanes). However, the addition of a second plasmid containing the CAT gene under the control of the EMCV IRES (pT7CG) in parallel reactions had a dramatic effect on the ability of the IRESs encoded in pETCAT.EA, pETCAT.EB and pETCAT.EM to translate GFP. As observed in cis (Figs 5 and 6), the ERBV IRES (in pETCAT.EB) was able to compete more efficiently with EMCV IRES for translation factors than was the ERAV IRES (in pETCAT.EA), although this effect was more pronounced using the in trans approach (Fig. 7B).
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). In contrast, transfection with plasmids that were otherwise identical except for the presence of the EMCV IRES upstream of CAT demonstrated a pattern of competition consistent with that observed in the in vitro experiments (Fig. 8B). This was the case in two independent experiments (both of which are shown) with the exception of experiment 1B (Fig. 8B), where the ERBV IRES showed similar activity to that from EMCV. Hence, in several systems, the ERBV IRES appeared to compete effectively with the EMCV IRES for translation factors and/or ribosomes whereas that from ERAV, another type II IRES, was less effective in this regard.
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; Pestova et al., 1996 ; Pilipenko et al., 1989 ; Robertson et al., 1999 ; Stewart & Semler, 1997 ). Sequence comprising these elements (encompassed by nt 189–920) plus additional sequence/structures upstream of the ERBV H stem–loop were required for maximal IRES activity in our system. This is in broad agreement with what is required for activity in other type II IRESs (Belsham & Brangwyn, 1990 ; Duke et al., 1992 ; Hinton et al., 2000 ; Jang & Wimmer, 1990 ).
Site-directed mutagenesis experiments and 3'-deletion analysis clearly identified ERBV AUG2 as the major initiation codon. However, AUG1 was used to initiate translation, albeit inefficiently, when all other AUG codons were removed either through mutagenesis or deletion to AUG1. Two attributes appear to be important in initiation codon site selection in type II IRESs: the distance between the end of the polypyrimidine tract and first utilized AUG codon, which is generally 12–18 nt, and sequence context surrounding the AUG (Table 3). With respect to the first of these, shortening the spacer region between the polypyrimidine tract and the AUG in EMCV result in an inefficient shift to the use of downstream AUG codons (Kaminski et al., 1994 ). Conversely, these authors also showed that the addition of sequence in the spacer region results in a 15-fold increase in initiation at an upstream AUG codon. ERBV AUG1 begins 16 nt downstream from the polypyrimidine tract (Table 3); however, it does not appear to be easily accessed by ribosomes. Modification of sequence surrounding the initiating AUG codon to a less favoured Kozak context has been shown to lead to increased initiation at downstream AUG codons (Davies & Kaufman, 1992 ). In ERBV, AUG2 is surrounded by a favourable Kozak consensus sequence with an A at -3 and a G at +4 (Table 3). This is not the case with AUG1 which has the less favoured T nucleotide at the -3 position. A relatively poor sequence context surrounding AUG1 is probably the reason that it is poorly accessed. However, it should be recognized that Human parechovirus 1 (HPeV1), which has also recently been shown to contain a type II IRES (Nateri et al., 2000 ), has a sequence context surrounding AUG1 that is similar to that found in ERBV but in this instance AUG1 is the major codon (Table 3). Some initiation can occur at a downstream AUG codon in HPeV1. Hence, the possibility remains that a broader sequence surrounding the start codon also plays a role in initiation from type II IRESs (Le et al., 1993 ). Our observation that AUG1 can be accessed by ribosomes suggests that two forms of the ERBV L proteinase may be synthesized in infected cells, although one would predict that the smaller of these, initiating from AUG2, would be in vast excess of the larger form.
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; Hinton et al., 2000 ; Piccone et al., 1995 ). However, it has been shown for EMCV that the inclusion of 14 nt downstream of the initiating AUG to a naturally occurring NcoI site improves translation initiation efficiency approximately 2-fold (Table 3; Davies & Kaufman, 1992 ). Similarly, in RRLs, we observed that the inclusion of some residues downstream of the major start site also led to an increase in activity of approximately the same magnitude.
IRES elements have been widely used for heterologous gene expression both in in vitro cultured cells and in transgenic animals. The use of an IRES element in these vectors allows for expression of two (or more) genes from the one transcriptional cassette. This has significant benefits in terms of allowing co-regulation of these genes in particular cell/tissue types and also in simplifying construct design. At present, the EMCV IRES is almost exclusively used for this purpose due to the fact that it is one of the most powerful elements known for internal initiation of translation and that it has activity in a broad range of host cells. Interestingly, when compared directly to the EMCV IRES, the ERBV and ERAV IRESs were similarly active both in vitro (Fig. 5B, C and Fig. 6) and in vivo (Fig. 8A). In IRES competition experiments, however, the ERBV IRES was generally effective at sequestering translation factors in the presence of the EMCV IRES. Again, this was true both in vitro (Fig. 5E, F and Fig. 6) and in vivo (Fig. 8) and was also the case when the IRES competition was performed in trans (Fig. 7). In contrast, the ERAV IRES was generally less effective at competing with the EMCV IRES for translation factors in all systems.
This work suggests that the ERBV IRES may prove to be a useful tool in gene expression vectors either as an alternative to EMCV or when several IRES elements are required in the one plasmid. Further work is needed to address the reason(s) for the functional differences observed between the type II IRESs. It will be of particular interest to identify the limiting initiation factors that are involved in the observed competition between these IRESs as well as to identify any factors that are possibly unique to the function of particular type II IRES elements.
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References |
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Received 29 January 2001;
accepted 5 June 2001.
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