Institute of Biochemistry, Faculty of Medicine, Friedrichstrasse 24, 35392 Giessen, Germany
Correspondence
Michael Niepmann
michael.niepmann{at}biochemie.med.uni-giessen.de
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ABSTRACT |
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Present address: Friedrich-Miescher Institute, 4058 Basel, Switzerland.
Present address: EISAI GmbH, Lyoner Strasse 14, D-60528 Frankfurt, Germany.
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INTRODUCTION |
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A key role in translation initiation is attributed to eIF4G, which is a large, multifunctional adaptor protein that is a component of the cap-binding complex eIF4F and connects the RNA that is to be translated to the ribosome (Hentze, 1997; Prevot et al., 2003
). In contrast to the indirect interaction of eIF4G with capped cellular mRNAs via the cap-binding protein eIF4E, eIF4G binds directly to the IRES 3' regions of FMDV (López de Quinto & Martínez-Salas, 2000
; López de Quinto et al., 2001
; Saleh et al., 2001
), EMCV (Pestova et al., 1996b
) and poliovirus (Ochs et al., 2003
), probably synergistically with the associated ATP-dependent RNA helicase eIF4A and its stimulating cofactor eIF4B (Meyer et al., 1995
; Kolupaeva et al., 1998
; Ochs et al., 1999
, 2002
; Rust et al., 1999
). In the FMDV strain O1K IRES, the binding site for eIF4G comprises stemloops 4 and 5 (Fig. 1
; Pilipenko et al., 2000
; Saleh et al., 2001
), whereas in a study with the FMDV C-S8c1 strain, only stemloop 4 was required (López de Quinto & Martínez-Salas, 2000
).
At the primary sequence level, only some short sequence stretches are conserved among the type II IRES elements of cardio- and aphthoviruses (Jackson & Kaminski, 1995). Two of these comprise a characteristic, discontinuous sequence element (Fig. 1b
, boxed) that resides in the stem of the apical subdomain 4-1 (named J in the related EMCV IRES) within the Y-shaped domain 4 of the IRES. This sequence element is discontinuous at the primary sequence level, but forms a compact, continuous element at the secondary structure level, which consists of an apical 2 bp stack and two unpaired dinucleotide stretches flanking a 4 bp stack (Fig. 1
). The absolute conservation of both its primary sequence and secondary structure among all cardio- and aphthoviruses (Jackson & Kaminski, 1995
) points to an essential role of this element in the virus life cycle.
As IRES domain 4 carries the determinants for its interaction with eIF4G, we suspected that this element may be involved in the interaction with this initiation factor that is crucial for FMDV translation. Therefore, we have mutated this element in the context of the complete FMDV IRES and analysed the effects of these mutations on eIF4G binding and IRES activity.
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METHODS |
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Preparation of RNAs and in vitro translation.
The pSP449 series plasmids were linearized with SmaI in the linker downstream of the FMDV sequence. Labelled RNAs were synthesized by using SP6 RNA polymerase in the presence of 2·5 µM [-32P]UTP (400 Ci mmol1; Amersham Biosciences) plus 10 µM unlabelled UTP. For in vitro translations, the pM12 and pD128 series plasmids were linearized with SmaI downstream of the luciferase gene and mRNA was synthesized in the presence of 500 µM unlabelled nucleotides. Then, 0·2 µg RNA was used in a 10 µl reaction that contained 4·4 µl rabbit reticulocyte lysate (RRL; Promega) and 0·2 µl [35S]methionine. In addition to the 50 mM endogenous potassium acetate that is added to RRL by the supplier, KCl was added to a final potassium concentration of 125 mM, unless otherwise indicated. Reactions were incubated at 30 °C for 60 min and 5 µl of the reaction was analysed by gel electrophoresis and autoradiography. FMDV L-protease was translated in RRL from plasmid pFMDV14 linearized with BamHI (Saleh et al., 2001
). Purified eIF4F (Scheper et al., 1992
) was kindly provided by Adri Thomas or prepared freshly as described by Grifo et al. (1983)
.
Transfections.
BHK cells were split into 12-well plates and grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10 % fetal calf serum (FCS) and 100 mg penicillin/streptomycin ml1 to 90 % confluence. One day before transfection, the medium was altered to DMEM plus FCS, but without antibiotics. For transfection, 0·5 µg of each dicistronic plasmid and 1 µl Lipofectamine (Invitrogen) were each diluted separately in 25 µl DMEM (without FCS and antibiotics) and incubated at room temperature. Within 5 min, both dilutions were mixed together, incubated for another 20 min at room temperature and then added to the cells. After 24 h, cells were washed in PBS; the PBS was then aspirated, 250 µl passive lysis buffer (Promega) was added and the cells were lysed by gentle agitation. Lysates were collected, centrifuged for 30 s at 13 000 g and 15 µl supernatant was used for luciferase and CAT reporter enzyme assays (Niepmann et al., 1997).
UV cross-linking assays.
UV cross-linking assays (Ochs et al., 1999) were performed with 4·4 µl RRL and 0·2 pmol [
-32P]UTP-labelled IRES RNA in a volume of 10 µl at 125 mM final potassium concentration, unless otherwise indicated. Competitor RNAs were added if indicated. Reactions were incubated at 30 °C for 30 min under irradiation with 254 nm UV light. Excess RNA was digested with 2 mg RNase A ml1 at 37 °C for 60 min. Proteins were separated on 8 % SDS-PAGE gels and analysed by autoradiography.
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RESULTS |
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Mutations in the 4 bp stack abolish binding of eIF4G and IRES activity
In the same way, we analysed the effect of mutations in the lower 4 bp stack of the discontinuous, conserved sequence element. This 4 bp stack is expected to contribute largely to the stability of the stem of the FMDV IRES subdomain 4-1. We introduced sequence changes separately in either (i) the left (upstream) part of the primary sequence (mutant up-4) (Fig. 3a), which disrupt the predicted structure of the wild-type (wt) sequence (data not shown), even if they still allow formation of weak base pairs with the opposite strand, or (ii) in the right (downstream) part (mutant down-4), which also disrupts the secondary structure. Both changes completely abolish in vivo translation, both in BHK cells (Fig. 3b
) and in RRL (Fig. 3c
, lanes 2 and 3), as well as binding of eIF4G to the IRES (Fig. 3d
, lanes 2 and 3). Surprisingly, even a compensatory mutant (up/down-4) in which the primary sequences of the two sides of the stack were altered to restore stable base-pairing in the dsRNA could rescue neither IRES activity (Fig. 3b, c
, lane 4) nor binding of eIF4G and eIF4B (Fig. 3d
, lane 4). Not only is base-pairing evidently important in this lower part of the conserved element, but both the integrity of the original dsRNA secondary structure and the primary sequence are crucial for eIF4G binding and IRES activity.
Sequence and structure requirements in the upper 2 bp stack of the conserved element
When primary sequence changes were introduced into the upper base stack by mutating either the left or the right part of the 2 bp (mutants up-2 or down-2, respectively) (Fig. 4a), thereby disrupting the RNA secondary structure, we found that IRES activity was reduced to levels of about 10 % compared to the wild-type (Fig. 4b, c
, lanes 2 and 3). Binding of eIF4G was also consistently reduced to levels below the detection limit (Fig. 4e
, lanes 2 and 3). However, when both primary sequence mutations were combined to yield a compensatory mutant (up/down-2) in which only the primary sequences were mutated, but the secondary structure was restored, we found, surprisingly, that IRES activity was partially restored both in BHK cells (Fig. 4b
) and in vitro (Fig. 4c
, lane 4). Accordingly, eIF4G binding was also partially restored (Fig. 4e
, lane 4). Thus, maintenance of the secondary structure of the dsRNA is important, whereas the primary sequence in this part of the conserved element is not absolutely essential for eIF4G binding and IRES activity, but confers a considerable advantage to IRES activity.
To confirm that the loss of detection of eIF4G in the UV cross-linking assay with the above IRES mutants is indeed due to loss of binding, we performed competition reactions (Fig. 4f). The unlabelled, wt competitor IRES competed very well with binding of eIF4G to the radiolabelled IRES RNA (Fig. 4f
, lanes 24). In contrast, even a 64-fold excess of the up-2 or down-2 mutant competitor RNAs (lanes 512) did not compete with binding of eIF4G to the wt IRES, whereas the compensatory up/down-2 mutant competed with intermediate efficiency (lanes 1316), indicating that the loss of eIF4G detection is indeed caused by loss of binding to the mutant IRES, rather than by impaired label transfer from RNA to protein by UV cross-linking.
We then used another set of mutants to further analyse the influence of primary sequence and secondary structure alterations in the 2 bp stack on eIF4G binding and IRES activity. In mutant up-2AU, the upstream CC sequence was now altered to AU. Correspondingly, the downstream GG sequence was mutated to AU in mutant down-2AU, and both mutations were performed in combination in the compensatory mutant up/down-2AU (Fig. 4a). Mutant down-2AU showed greatly reduced IRES activity in vivo and in vitro (Fig. 4b, c
, lane 6) and also showed markedly reduced eIF4G binding (Fig. 4e
, lane 6). In contrast, to our surprise, mutant up-2AU, as well as the compensatory mutant up/down-2AU, was quite active in translation (Fig. 4b, c
, lanes 5 and 7) and in eIF4G binding (Fig. 4e
, lanes 5 and 7). However, taking the possible formation of standard cis-WatsonCrick/WatsonCrick AG and UG base pairs into account (Leontis et al., 2002
), as illustrated in Fig. 4d
, these results are in accordance with the idea that the intact secondary structure of the RNA double strand is important, whereas the primary sequence is not. In mutant up-2AU, an AG and a UG base pair (both of which involve two normal hydrogen bonds plus a bridging water; Leontis et al., 2002
) may allow retention of the dsRNA secondary structure (Fig. 4d
), whereas in mutant down-2AU, the weak AC interaction (involving only two normal hydrogen bonds) in combination with the weak UC interaction (involving only one normal hydrogen bond and a bridging water; Leontis et al., 2002
) may not be sufficient to allow stable helix formation in the apical part of the IRES subdomain 4-1.
The relationship between the integrity of the RNA secondary structure in the upper part of the conserved element and IRES activity was also evident when eIF4G interaction and IRES activity were investigated with the up-2 and down-2 mutants at different potassium concentrations (Fig. 5). With the wt IRES, eIF4G binding decreased only slightly when the potassium concentration was increased from 50 to 150 mM (Fig. 5a
, lanes 15), but wt IRES activity was optimal at 100 and 125 mM (Fig. 5b
, lanes 15). This enhanced activity is due to the stimulation of ribosome association by potassium ions, reflecting adaptation of the FMDV IRES activity to the high intracellular potassium concentration, whereas the decrease of translation activity at higher KCl concentrations is due to chloride anions (Weber et al., 1977
; Niepmann, 2003
).
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To check the activities of the IRES mutants in relation to the expression of a control gene, we cloned selected IRES mutants into a dicistronic mRNA system with CAT as the first gene and firefly luciferase as the second gene, which is under IRES control (Niepmann et al., 1997). Expression results after transfection in BHK cells (Fig. 6a
) and in vitro (Fig. 6b
) with the mutations in the 4 bp stack and the 2 bp stack confirmed the results obtained above.
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Functional IRES defects cannot be relieved by increased eIF4F concentrations
In order to analyse whether increased initiation factor concentrations could compensate for IRES defects, we performed translation reactions with the wt FMDV IRES and a selected set of IRES mutants that affected both the 4 bp and 2 bp stacks (up-4, up-2 and up/down-2). To these reactions, we added increasing amounts of purified eIF4F, the cap-binding protein complex that contains eIF4G as its major component, as present in the living cell (Fig. 7a). eIF4F was freshly purified before the experiments. On addition of eIF4F, activity of the wt IRES increased by 40 %, according to a shift in the chemical equilibrium caused by the increased eIF4F concentration. However, concentrations of up to 40 nM eIF4F (0·4 pmol eIF4F in a 10 µl reaction) could not relieve the disadvantage of the IRES mutations.
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In conclusion, these functional assays demonstrate that disturbances in the structure of this conserved element in the IRES affect eIF4G binding in a way that cannot be overcome by increased eIF4G concentrations.
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DISCUSSION |
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Among the type II IRES elements of members of the cardio-/aphthovirus group in the picornavirus family, the Y-shaped domain 4 (Fig. 1) in the IRES 3' region carries the determinants for recruitment of eIF4G to the virus RNA. In previous studies, either after optimization of the spurious detection of eIF4G in the UV cross-linking assay or by using purified initiation factors, eIF4G was found to contact the type II IRES elements (FMDV or EMCV, respectively) at many different sites. There are many contacts in the base of domain 4 (Kolupaeva et al., 1998
, 2003
; López de Quinto & Martínez-Salas, 2000
; Pilipenko et al., 2000
). Detailed mutagenesis of the lower stem of domain 4 revealed that the secondary structure, rather than the primary sequence, appears to be important for FMDV IRES activity, whereas in the first small, unpaired bulge at the base of domain 4, the primary sequence is important (López de Quinto & Martínez-Salas, 2000
). Virtually all nucleotides of the unpaired, A-rich bulge (subdomain 4-3 in Fig. 1
) are contacted by eIF4G (Pilipenko et al., 2000
; Kolupaeva et al., 2003
), an observation that is consistent with the extreme sensitivity of this bulge to changes in the number of A residues (Kaminski & Jackson, 1998
). Other contact sites are in the base of subdomain 4-2 (named K in EMCV) (Kolupaeva et al., 2003
). Also, the lower stem and apical loop of domain 5 appear to be involved in the contacts of eIF4G with the type II IRES (Pilipenko et al., 2000
; Saleh et al., 2001
; Kolupaeva et al., 2003
), although another study found that eIF4G also bound well to the FMDV CS8 strain IRES in the absence of domain 5 (López de Quinto & Martínez-Salas, 2000
).
Several contacts for eIF4G were also detected in the stem of subdomain 4-1 (named J in EMCV; Kolupaeva et al., 1998, 2003
; Pilipenko et al., 2000
). In this subdomain, a characteristic, conserved, discontinuous sequence element is present (Jackson & Kaminski, 1995
) (Fig. 1
). The absolute conservation of both secondary structure and primary sequence within this sequence element among all members of the cardio-/aphthovirus group argues strongly for an essential function of this element. We and others have identified subdomain 4-1 to be essential for binding of eIF4G (Saleh et al., 2001
; Stassinopoulos & Belsham, 2001
). Interestingly, the unpaired dinucleotides are contacted directly by the eIF4G protein. In structure-probing experiments (Pilipenko et al., 2000
; Kolupaeva et al., 2003
), eIF4G protects the unpaired A residues from dimethyl sulphate or RNase T1 treatment and eIF4G protects the 4 bp stack between the unpaired residues from hydroxyl radical treatment (Kolupaeva et al., 2003
), indicating that this conserved element within subdomain 4-1 is an essential determinant for direct proteinRNA contacts between eIF4G and the IRES RNA.
Here, we show that this conserved sequence is essential for recruitment of eIF4G to the FMDV IRES, as well as for IRES activity. Several mutations in this element abrogate eIF4G binding and IRES activity. Consistently, additional eIF4F in the translation system cannot restore full IRES activity of the mutants, although additional eIF4F stimulates wt IRES activity. In particular, mutations in two unpaired dinucleotide stretches almost completely abolish eIF4G binding and IRES activity. The evolutionary importance of the conservation among cardio- and aphthoviruses of these unpaired dinucleotide stretches, which appear to be exposed by the remainder of the stemloop 4-1 structure, is also supported by a recent study on the closely related EMCV IRES (Clark et al., 2003). This study showed that almost any mutation in the two unpaired dinucleotide stretches affects IRES activity, particularly mutations of those residues that are contacted by eIF4G: the A in the upstream unpaired dinucleotide stretch (Kolupaeva et al., 2003
) and both nucleotides of the downstream dinucleotide stretch (Kolupaeva et al., 1998
, 2003
; Pilipenko et al., 2000
). Together with these studies on the EMCV IRES, our results obtained with the FMDV IRES highlight the evolutionary importance of the conserved element for eIF4G binding and IRES function among the type II IRES elements of the cardio- and aphthovirus group of picornaviruses.
Mutations in a 4 bp stack almost completely abolish eIF4G binding and IRES activity, even if only the primary sequence is affected, but the dsRNA secondary structure is maintained in the compensatory mutant up/down-4 and, with weakened base-pairing, in mutant up-4. This result is consistent with the finding that eIF4G contacts the upstream AGGU sequence of the 4 bp stack in the conserved sequence element (Kolupaeva et al., 2003), pointing to an absolute primary sequence requirement that is probably conferred by the need for direct recognition of particular bases in this sequence by the eIF4G protein. In contrast, mutations in the 2 bp stack that affect the primary sequence, but not the secondary structure, impair, but do not completely abolish, IRES activity. Consistently, no direct contacts of eIF4G with specific bases of this 2 bp stack have been observed. In contrast to the lower 4 bp stack, in this 2 bp stack, IRES activity and eIF4G binding correlate preferentially with the stability of base-pairing (Leontis et al., 2002
), supporting the hypothesis that exposure of the unpaired upstream AC dinucleotide stretch from the RNA double strand may be the main task of this dsRNA stem region. Nevertheless, the observation that changes in the primary sequence that keep the secondary structure in the 2 bp stack intact do not completely abolish eIF4G binding and IRES activity was a surprise, as the absolute primary sequence conservation of this sequence among type II IRES suggested that not only the RNA structure, but also the exact sequence of this region, was important. We note that, in this case, a certain preference for a distinct sequence has resulted in conservation of the primary sequence among this subgroup of the picornaviruses during virus evolution.
Taken together, the binding site for eIF4G in the picornavirus type II IRES is composed of several essential determinants that cover almost the entire domain 4 (named JK in EMCV). Although the exact tertiary structure of the IRES domain 4 is not yet known, these determinants appear to act synergistically in the binding of eIF4G, as mutations in any of these determinants seriously affect both eIF4G binding and IRES activity. Moreover, the cellular proteins that assemble on the IRES also bind by synergistic interactions. First, several interaction sites within the large eIF4G protein are involved in contacting the virus IRES (Kolupaeva et al., 2003). Second, the initiation factors eIF4G, eIF4A and eIF4B form a multiprotein complex that requires synergistic involvement of all three components. Binding of eIF4B to the IRES is ATP-dependent (Meyer et al., 1995
), although only eIF4A is an RNA helicase that works ATP-dependently (Rozen et al., 1990
). This RNA helicase component of the multiprotein complex that forms on the virus RNA may introduce changes in secondary structure (Kolupaeva et al., 2003
), which perhaps contribute to the translation initiation process. When the initiation factors are added separately to the IRES RNA, they bind only weakly to the IRES and only their synergistic interaction results in stronger binding of the entire eIF4 complex to the virus RNA (Kolupaeva et al., 1998
). Consistently with this, we found here that binding of eIF4B is affected in parallel with binding of eIF4G.
In addition, so-called RNA chaperones, such as PTB, may augment the interaction of initiation factors with the IRES, although it is not known how they act at the molecular level. PTB stimulates the activity of the FMDV IRES (Niepmann, 1996; Niepmann et al., 1997
) and the EMCV IRES (Kaminski et al., 1995
; Kaminski & Jackson, 1998
) and the RNA determinants that are contacted by PTB appear to be interspersed between the sites that are contacted by the standard factors. Accordingly, it is not surprising that the mutations in the eIF4G-binding site that most seriously affect the assembly of the complex of initiation factors on the virus RNA may, in turn, also affect binding of PTB, such as deletion of the unpaired dinucleotide stretches (Fig. 2d
, lanes 24) and mutations in the 4 bp stack (Fig. 3d
, lanes 24). Nevertheless, among all RNA determinants that provide contact sites for proteins, the highly conserved sequence element in subdomain 4-1 is involved directly in recruitment of eIF4G to the picornavirus RNA during the initial steps of translation of the virus genome.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Belsham, G. J. (1992). Dual initiation sites of protein synthesis on foot-and-mouth disease virus RNA are selected following internal entry and scanning of ribosomes in vivo. EMBO J 11, 11051110.[Abstract]
Blyn, L. B., Towner, J. S., Semler, B. L. & Ehrenfeld, E. (1997). Requirement of poly(rC) binding protein 2 for translation of poliovirus RNA. J Virol 71, 62436246.[Abstract]
Boussadia, O., Niepmann, M., Créancier, L., Prats, A.-C., Dautry, F. & Jacquemin-Sablon, H. (2003). Unr is required in vivo for efficient initiation of translation from the internal ribosome entry sites of both rhinovirus and poliovirus. J Virol 77, 33533359.
Brown, F. (1999). Control of foot-and-mouth disease by vaccination. Dev Biol Stand 100, 131135.[Medline]
Carrasco, L. & Smith, A. E. (1976). Sodium ions and the shut-off of host cell protein synthesis by picornaviruses. Nature 264, 807809.[Medline]
Clark, A. T., Robertson, M. E. M., Conn, G. L. & Belsham, G. J. (2003). Conserved nucleotides within the J domain of the encephalomyocarditis virus internal ribosome entry site are required for activity and for interaction with eIF4G. J Virol 77, 1244112449.
Devaney, M. A., Vakharia, V. N., Lloyd, R. E., Ehrenfeld, E. & Grubman, M. J. (1988). Leader protein of foot-and-mouth disease virus is required for cleavage of the p220 component of the cap-binding protein complex. J Virol 62, 44074409.[Medline]
Ehrenfeld, E. & Teterina, N. L. (2002). Initiation of translation of picornavirus RNAs: structure and function of the internal ribosome entry site. In Molecular Biology of Picornaviruses, pp. 159170. Edited by B. L. Semler & E. Wimmer. Washington, DC: American Society for Microbiology.
Evdokimova, V. M. & Ovchinnikov, L. P. (1999). Translational regulation by Y-box transcription factor: involvement of the major mRNA-associated protein, p50. Int J Biochem Cell Biol 31, 139149.[CrossRef][Medline]
Ferguson, N. M., Donnelly, C. A. & Anderson, R. M. (2001). The foot-and-mouth epidemic in Great Britain: pattern of spread and impact of interventions. Science 292, 11551160.
Grifo, J. A., Tahara, S. M., Morgan, M. A., Shatkin, A. J. & Merrick, W. C. (1983). New initiation factor activity required for globin mRNA translation. J Biol Chem 258, 58045810.
Hentze, M. W. (1997). eIF4G: a multipurpose ribosome adapter? Science 275, 500501.
Hunt, S. L., Hsuan, J. J., Totty, N. & Jackson, R. J. (1999). unr, a cellular cytoplasmic RNA-binding protein with five cold-shock domains, is required for internal initiation of translation of human rhinovirus RNA. Genes Dev 13, 437448.
Jackson, R. J. (2002). Proteins involved in the function of picornavirus internal ribosomal entry sites. In Molecular Biology of Picornaviruses, pp. 171186. Edited by B. L. Semler & E. Wimmer. Washington, DC: American Society for Microbiology.
Jackson, R. J. & Kaminski, A. (1995). Internal initiation of translation in eukaryotes: the picornavirus paradigm and beyond. RNA 1, 9851000.[Medline]
Kaminski, A. & Jackson, R. J. (1998). The polypyrimidine tract binding protein (PTB) requirement for internal initiation of translation of cardiovirus RNAs is conditional rather than absolute. RNA 4, 626638.
Kaminski, A., Hunt, S. L., Patton, J. G. & Jackson, R. J. (1995). Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA. RNA 1, 924938.[Abstract]
Kolupaeva, V. G., Hellen, C. U. T. & Shatsky, I. N. (1996). Structural analysis of the interaction of the pyrimidine tract-binding protein with the internal ribosomal entry site of encephalomyocarditis virus and foot-and-mouth disease virus RNAs. RNA 2, 11991212.[Abstract]
Kolupaeva, V. G., Pestova, T. V., Hellen, C. U. T. & Shatsky, I. N. (1998). Translation eukaryotic initiation factor 4G recognizes a specific structural element within the internal ribosome entry site of encephalomyocarditis virus RNA. J Biol Chem 273, 1859918604.
Kolupaeva, V. G., Lomakin, I. B., Pestova, T. V. & Hellen, C. U. T. (2003). Eukaryotic initiation factors 4G and 4A mediate conformational changes downstream of the initiation codon of the encephalomyocarditis virus internal ribosomal entry site. Mol Cell Biol 23, 687698.
Korneeva, N. L., Lamphear, B. J., Hennigan, F. L. C. & Rhoads, R. E. (2000). Mutually cooperative binding of eukaryotic translation initiation factor (eIF) 3 and eIF4A to human eIF4G-1. J Biol Chem 275, 4136941376.
Lamphear, B. J., Kirchweger, R., Skern, T. & Rhoads, R. E. (1995). Mapping of functional domains in eukaryotic protein synthesis initiation factor 4G (eIF4G) with picornaviral proteases. Implications for cap-dependent and cap-independent translational initiation. J Biol Chem 270, 2197521983.
Leontis, N. B., Stombaugh, J. & Westhof, E. (2002). The non-WatsonCrick base pairs and their associated isostericity matrices. Nucleic Acids Res 30, 34973531.
López de Quinto, S. & Martínez-Salas, E. (2000). Interaction of the eIF4G initiation factor with the aphthovirus IRES is essential for internal translation initiation in vivo. RNA 6, 13801392.
López de Quinto, S., Lafuente, E. & Martínez-Salas, E. (2001). IRES interaction with translation initiation factors: functional characterization of novel RNA contacts with eIF3, eIF4B, and eIF4GII. RNA 7, 12131226.
Luz, N. & Beck, E. (1991). Interaction of a cellular 57-kilodalton protein with the internal translation initiation site of foot-and-mouth disease virus. J Virol 65, 64866494.[Medline]
Meerovitch, K., Svitkin, Y. V., Lee, H. S., Lejbkowicz, F., Kenan, D. J., Chan, E. K. L., Agol, V. I., Keene, J. D. & Sonenberg, N. (1993). La autoantigen enhances and corrects aberrant translation of poliovirus RNA in reticulocyte lysate. J Virol 67, 37983807.[Abstract]
Meyer, K., Petersen, A., Niepmann, M. & Beck, E. (1995). Interaction of eukaryotic initiation factor eIF-4B with a picornavirus internal translation initiation site. J Virol 69, 28192824.[Abstract]
Niepmann, M. (1996). Porcine polypyrimidine tract-binding protein stimulates translation initiation at the internal ribosome entry site of foot-and-mouth-disease virus. FEBS Lett 388, 3942.[CrossRef][Medline]
Niepmann, M. (1999). Internal initiation of translation of picornaviruses, hepatitis C virus and pestiviruses. Recent Res Devel Virol 1, 229250.
Niepmann, M. (2003). Effects of potassium and chloride on ribosome association with the RNA of foot-and-mouth disease virus. Virus Res 93, 7178.[CrossRef][Medline]
Niepmann, M., Petersen, A., Meyer, K. & Beck, E. (1997). Functional involvement of polypyrimidine tract-binding protein in translation initiation complexes with the internal ribosome entry site of foot-and-mouth disease virus. J Virol 71, 83308339.[Abstract]
Ochs, K., Rust, R. C. & Niepmann, M. (1999). Translation initiation factor eIF4B interacts with a picornavirus internal ribosome entry site in both 48S and 80S initiation complexes independently of initiator AUG location. J Virol 73, 75057514.
Ochs, K., Saleh, L., Bassili, G., Sonntag, V. H., Zeller, A. & Niepmann, M. (2002). Interaction of translation initiation factor eIF4B with the poliovirus internal ribosome entry site. J Virol 76, 21132122.
Ochs, K., Zeller, A., Saleh, L., Bassili, G., Song, Y., Sonntag, A. & Niepmann, M. (2003). Impaired binding of standard initiation factors mediates poliovirus translation attenuation. J Virol 77, 115122.[CrossRef][Medline]
Paul, A. V. (2002). Possible unifying mechanism of picornavirus genome replication. In Molecular Biology of Picornaviruses, pp. 227246. Edited by B. L. Semler & E. Wimmer. Washington, DC: American Society for Microbiology.
Pestova, T. V., Hellen, C. U. T. & Shatsky, I. N. (1996a). Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry. Mol Cell Biol 16, 68596869.[Abstract]
Pestova, T. V., Shatsky, I. N. & Hellen, C. U. T. (1996b). Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes. Mol Cell Biol 16, 68706878.[Abstract]
Pilipenko, E. V., Blinov, V. M., Chernov, B. K., Dmitrieva, T. M. & Agol, V. I. (1989). Conservation of the secondary structure elements of the 5'-untranslated region of cardio- and aphthovirus RNAs. Nucleic Acids Res 17, 57015711.[Abstract]
Pilipenko, E. V., Pestova, T. V., Kolupaeva, V. G., Khitrina, E. V., Poperechnaya, A. N., Agol, V. I. & Hellen, C. U. (2000). A cell cycle-dependent protein serves as a template-specific translation initiation factor. Genes Dev 14, 20282045.
Prevot, D., Darlix, J. L. & Ohlmann, T. (2003). Conducting the initiation of protein synthesis: the role of eIF4G. Biol Cell 95, 141156.[CrossRef][Medline]
Rozen, F., Edery, I., Meerovitch, K., Dever, T. E., Merrick, W. C. & Sonenberg, N. (1990). Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F. Mol Cell Biol 10, 11341144.[Medline]
Rust, R. C., Ochs, K., Meyer, K., Beck, E. & Niepmann, M. (1999). Interaction of eukaryotic initiation factor eIF4B with the internal ribosome entry site of foot-and-mouth disease virus is independent of the polypyrimidine tract-binding protein. J Virol 73, 61116113.
Saleh, L., Rust, R. C., Füllkrug, R., Beck, E., Bassili, G., Ochs, K. & Niepmann, M. (2001). Functional interaction of translation initiation factor eIF4G with the foot-and-mouth disease virus internal ribosome entry site. J Gen Virol 82, 757763.
Sangar, D. V., Newton, S. E., Rowlands, D. J. & Clarke, B. E. (1987). All foot and mouth disease virus serotypes initiate protein synthesis at two separate AUGs. Nucleic Acids Res 15, 33053315.[Abstract]
Scheper, G. C., Voorma, H. O. & Thomas, A. A. M. (1992). Eukaryotic initiation factors-4E and -4F stimulate 5' cap-dependent as well as internal initiation of protein synthesis. J Biol Chem 267, 72697274.
Stanway, G., Hovi, T., Knowles, N. J. & Hyypiä, T. (2002). Molecular and biological basis of picornavirus taxonomy. In Molecular Biology of Picornaviruses, pp. 1726. Edited by B. L. Semler & E. Wimmer. Washington, DC: American Society for Microbiology.
Stassinopoulos, I. A. & Belsham, G. J. (2001). A novel proteinRNA binding assay: functional interactions of the foot-and-mouth disease virus internal ribosome entry site with cellular proteins. RNA 7, 114122.
Stone, R. (2002). Foot-and-mouth disease: report urges U.K. to vaccinate herds. Science 297, 319321.
Weber, L. A., Hickey, E. D., Maroney, P. A. & Baglioni, C. (1977). Inhibition of protein synthesis by Cl. J Biol Chem 252, 40074010.[Abstract]
Received 11 March 2004;
accepted 12 May 2004.