Coat protein enhances translational efficiency of Alfalfa mosaic virus RNAs and interacts with the eIF4G component of initiation factor eIF4F

Ivo M. Krab1, Christian Caldwell2, Daniel R. Gallie2 and John F. Bol1

1 Institute of Biology, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands
2 Department of Biochemistry, Boyce Hall, University of California, Riverside, CA 92521, USA

Correspondence
John F. Bol
j.bol{at}chem.leidenuniv.nl


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The three plus-strand genomic RNAs of Alfalfa mosaic virus (AMV) and the subgenomic messenger for viral coat protein (CP) contain a 5'-cap structure, but no 3'-poly(A) tail. Binding of CP to the 3' end of AMV RNAs is required for efficient translation of the viral RNAs and to initiate infection in plant cells. To study the role of CP in translation, plant protoplasts were transfected with luciferase (Luc) transcripts with 3'-terminal sequences consisting of the 3' untranslated region of AMV RNA 3 (Luc–AMV), a poly(A) tail of 50 residues [Luc–poly(A)] or a short vector-derived sequence (Luc–control). Pre-incubation of the transcripts with CP had no effect on Luc expression from Luc–poly(A) or Luc–control, but strongly stimulated Luc expression from Luc–AMV. From time-course experiments, it was calculated that CP binding increased the half-life of Luc–AMV by 20 % and enhanced its translational efficiency by about 40-fold. In addition to the 3' AMV sequence, the cap structure was required for CP-mediated stimulation of Luc–AMV translation. Glutathione S-transferase pull-down assays revealed an interaction between AMV CP and initiation factor complexes eIF4F and eIFiso4F from wheatgerm. Far-Western blotting revealed that this binding occurred through an interaction of CP with the eIF4G and eIFiso4G subunits of eIF4F and eIFiso4F, respectively. The results support the hypothesis that the role of CP in translation of viral RNAs mimics the role of the poly(A)-binding protein in translation of cellular mRNAs.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
After entry into a host cell and disassembly of virions, the genomic RNA of positive-strand RNA viruses needs to be translated into replicase proteins and possibly other viral proteins. To achieve this goal, the viral RNA has to compete with a vast excess of cellular mRNAs to gain access to the translation machinery of the cell. The translation of eukaryotic mRNAs is enhanced strongly by the formation of a closed-loop structure, due to synergistic interactions between the complex of initiation factors bound to the 5'-terminal cap structure and the poly(A)-binding protein (PABP) bound to the 3'-terminal poly(A) tail (Gallie, 1991; Preiss & Hentze, 1998; Wells et al., 1998). The cap-bound complex of initiation factors, eIF4F, contains the cap-binding protein eIF4E, the multifunctional scaffold protein eIF4G and the RNA helicase eIF4A. In addition to eIF4F, plants contain the isoform eIFiso4F, with its subunits eIFiso4E and eIFiso4G (Browning et al., 1992). An interaction between eIF4G and PABP was first demonstrated in plants and yeast (Le et al., 1997; Tarun & Sachs, 1996) and has been shown subsequently in mammalian cells (Imataka et al., 1998; Fraser et al., 1999; Piron et al., 1998). Association of eIF4B with eIF4F (or eIFiso4F) exerts a synergistic effect on PABP-binding activity (Le et al., 1997).

Although many viral RNAs lack a cap structure and/or a poly(A) tail, RNA viruses have evolved sophisticated strategies to allow their messengers to compete with those of the host. Picornaviruses do not have a cap structure, but the initiation factors bind to an internal ribosome entry site in the 5' untranslated region (UTR) of the RNA and mediate the interaction with PABP (Svitkin et al., 2001). Messengers of rotaviruses (family Reoviridae) do have a cap structure, but no poly(A) tail. The rotavirus non-structural NSP3 protein binds to the 3' end of the viral mRNA and interacts with eIF4G to enhance translation (Piron et al., 1998; Vende et al., 2000). The RNAs of Barley yellow dwarf virus (family Luteoviridae) have neither a cap structure nor a poly(A) tail, but long-distance base pairing between a stem–loop structure in the 5' UTR and a translation enhancer in the 3' UTR have been proposed to ensure circularization of the RNAs and transfer of initiation factors to the 5' end (Guo et al., 2001).

The replication strategy of Alfalfa mosaic virus (AMV), a virus with a tripartite positive-strand RNA genome, was studied in this report. RNAs 1 and 2 encode the replicase proteins P1 and P2, respectively, whereas RNA 3 encodes the movement protein (MP) and coat protein (CP). CP is translated from the subgenomic RNA 4. AMV is the type species of the genus Alfamovirus and a member of the family Bromoviridae. In this family, a mixture of the three genomic RNAs of bromo-, cucumo- or oleaviruses is infectious as such, whereas the RNAs of alfamo- and ilarviruses require the addition of a few molecules of CP per RNA molecule to initiate infection (Bol, 1999, 2003; Jaspars, 1999). The 3' termini of the RNAs of bromo- and cucumoviruses contain a tRNA-like structure (TLS) that can be charged with tyrosine. It has been shown that the 3' termini of the RNAs of AMV can adopt two alternative structures: a linear array of hairpins with a high affinity for CP (CPB conformation) or a pseudoknotted structure that resembles the TLS of bromo-, cucumo- and oleaviruses, although it cannot be charged with an amino acid (TLS conformation) (Olsthoorn et al., 1999). The TLS conformation was required for minus-strand promoter activity of AMV RNA in an in vitro assay with purified AMV replicase (Olsthoorn et al., 1999, 2004). Binding of CP to the CPB conformation strongly enhanced translation of AMV RNAs in vivo (Neeleman et al., 2001). Transfection of tobacco protoplasts with AMV RNA 4 resulted in synthesis of CP only when the RNA encoded wild-type or mutant CP that was able to bind to the 3' end of its messenger (Neeleman et al., 2001). Translation of RNA 4 encoding a CP that was defective in RNA binding could be rescued by expression of functional CP in trans or by replacing the 3' UTR of the RNA with the 3' UTR of Brome mosaic virus (BMV, genus Bromovirus) (Neeleman et al., 2001, 2004). From these experiments, it was concluded that the AMV 3' UTR stimulates translation in a CP-dependent manner, whereas the BMV 3' UTR stimulates translation independently of CP. Because extension of the AMV RNAs with a poly(A) tail circumvented the requirement for CP to initiate infection, it was proposed that, in a wild-type infection, CP mimics the function of PABP (Neeleman et al., 2001).

It has been shown that introduction of the 3' UTR of BMV and Tobacco mosaic virus (TMV) downstream of a reporter gene strongly enhances translational efficiency of the chimeric RNAs in carrot protoplasts, whereas introduction of the AMV 3' UTR does not (Gallie & Kobayashi, 1994). Here, the effect of CP on message stability and translation of luciferase transcripts with 3'-terminal sequences consisting of the AMV 3' UTR or a poly(A) tail was analysed. In carrot protoplasts, CP strongly enhanced translation efficiency of the message with the AMV sequence, but not that of the polyadenylated RNA. Glutathione S-transferase (GST) pull-down and Far-Western assays revealed a specific interaction of AMV CP with eIF4G and eIFiso4G from wheatgerm. The data support the model in which CP stimulates translation by converting the viral RNAs into a closed-loop structure.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
mRNA and GST–CP constructs.
The pT7-Luc and pT7-Luc-A50 constructs, in which the firefly luciferase-coding region is under the control of the T7 promoter, have been described previously (Gallie, 1991). Derivative pT7-Luc-AMV that contains the 3'-terminal 156 nt from AMV RNA 3 has been described by Gallie & Kobayashi (1994). T7 RNA polymerase transcription was done essentially as described previously (Gallie et al., 1996), using the buffer conditions of Pokrovskaya & Gurevich (1994). pT7-Luc-A50 was linearized with BamHI for transcription of Luc–control; pT7-Luc-A50 was digested with DraI for transcription of Luc–poly(A); pT7-Luc-AMV was linearized with SmaI for transcription of Luc–AMV. The 5' UTR of these transcripts has the sequence 5'-GGCCTAAGCUUGUCGACCaug... and the 3' UTR has the sequence ...uaaAAUGUAACUCUAGAGGAUC-3' in the case of Luc–control; this 3' sequence is extended by the sequence UCCCC(A)25GUUAU(A)25 for Luc–poly(A) and the 3'-terminal 156 nt from AMV RNA 3 in the case of Luc–AMV.

The GST–CP fusion construct was created by PCR amplification of the CP gene from plasmid pAL3 (Neeleman et al., 1991), using oligonucleotide 5'-CGAGATCTGAGAACCTGTACTTCCAGAGTTCTTCACAAAAGAAAGCT-3', which specifies a BglII site (underlined), the tobacco etch virus protease-recognition motif and the N terminus of the CP gene (in italics), and oligonucleotide 5'-GGAATTCAATGACGATCAAGATCGTCA-3', which contains an EcoRI site (underlined) adjacent to the CP stop codon (italics complementary to the 5'-terminal sequence of the CP gene). The amplified fragment was introduced as a BglII–EcoRI fragment into pGEX-2T between the BamHI and EcoRI sites and verified by sequencing.

Protein extracts.
Expression of GST–CP fusion protein from pGEX-CP was done in Escherichia coli strain DH5{alpha} at 19 °C. Expression was induced with 7·5 µM IPTG at a starting OD600 of 0·04 and the suspension was further incubated until saturation of the culture. Cells were disrupted by sonication in 25 mM HEPES/KOH (pH 7·5), 100 mM potassium acetate, 2 mM EDTA, 5 mM dithiothreitol (DTT), 0·2 % Sarkosyl and 0·5 mM PMSF. These conditions yielded >90 % soluble GST–CP in the supernatant. Cell debris was removed by centrifugation at 30 g for 20 min and the supernatant was mixed with glutathione–Sepharose 4B (Pharmacia). After 30 min incubation with gentle shaking at 4 °C, the Sepharose was pelleted by centrifugation and washed five times with the same buffer as above without Sarkosyl. Finally, the pelleted Sepharose was mixed with 4 vols 25 mM HEPES/KOH (pH 7·5), 100 mM potassium acetate, 5 mM DTT and 75 % glycerol. The excess supernatant was removed and the beads with bound GST–CP were stored at –20 °C. The unfused GST control was prepared similarly from E. coli strain DH5{alpha} containing pGEX-2T.

Expression of wheat eIF4E, eIFiso4E, eIF4G and eIFiso4G in E. coli strain DH5{alpha} and the preparation of S30 extracts was done as described previously (Gazo et al., 2004; van Heerden & Browning, 1994). AMV CP was purified from virus particles as described previously (Neeleman et al., 1993) and kept frozen in small aliquots at –80 °C. Wheatgerm extract (WGE) was purchased from Promega.

Carrot-cell electroporation and luciferase or {beta}-glucuronidase (GUS) assays.
Carrot (Daucus carota) protoplasts were prepared from a carrot-cell suspension as described previously (Gallie et al., 1995). Electroporation was done as described previously (Gallie et al., 1995) with the following modifications: RNA samples (9 µg or approx. 2·5 pmol) in a small volume of water (2–4 µl) were mixed with 10 µl of a dilution of purified AMV CP (2·5–30·0 pmol) in 5 mM HEPES (pH 7·5) and pre-incubated for 5 min on ice before mixing with 400 µl protoplast suspension and immediate electroporation (settings: 350 µF, 400 V with a 5 mm gap electrode). Luciferase and GUS assays were performed essentially as described previously (Gallie et al., 1995).

Luciferase-expression kinetics and functional half-life determination.
The rate of Luc translation was determined from the kinetics of luciferase production in carrot cells following delivery of mRNA constructs by electroporation, during the phase of linear increase after recruitment of RNA onto polysomes. This rate was used as a measure of translational efficiency and the length of time over which Luc continued to accumulate was used to determine message stability, as described previously (Gallie et al., 1996; Ling et al., 2002).

Pull-down assays and Western analysis.
Our standard pull-down buffer P was 25 mM HEPES/KOH (pH 7·5), 100 mM potassium acetate, 2·5 mM magnesium acetate, 0·5 mM EDTA, 5 mM DTT and 0·02 % Triton X-100. Glutathione–Sepharose 4B containing GST–CP or GST in 50 % glycerol buffer was prepared by adding 2·6 vol. buffer P and 0·4 vol. 10 % solution of blocking reagent (Roche) in 100 mM maleate (pH 7·5) plus 150 mM NaCl and incubating for 30 min on ice. Excess supernatant was then removed and, for the GST–CP/native CP pull-downs, 35 µg purified CP was added per 50 µl resin [containing 0·75 µg GST–CP (µl resin)–1]; preparations were incubated for 30 min at 30 °C, then washed three times with 1·3 ml buffer P containing 1 % blocking reagent. WGE (9 µl; 350 µg total protein) and 0·5 µl 100 mM PMSF were added to each 50 µl aliquot of glutathione–Sepharose resin. After 30 min incubation on ice with occasional mixing, resin aliquots were washed two to four times with 1 ml buffer P as indicated. GST or GST–CP was eluted from each washed aliquot with 3x100 µl 10 mM reduced glutathione in 5 mM HEPES (pH 7·5), incubating for 2 min at room temperature before taking the supernatant. Combined successive eluates were precipitated with 1·5 ml acetone at –20 °C, incubating for 45 min at –20 °C before centrifuging for 20 min at 10 000 r.p.m. and 4 °C. Pellets were air-dried and redissolved in 35 µl SDS-PAGE loading buffer at 80 °C. Eight microlitres of each sample was separated by SDS-PAGE and transferred to Hybond P membrane (Amersham Biosciences). Detection of initiation factors by Western analysis was done as described by Ling et al. (2002), using antisera detecting eIF4E, eIF4G, eIFiso4G, eIF4A, eIF4B or eIF3 (Browning et al., 1990; Gallie et al., 1998). The antisera are specific and no cross-reactivity is observed (Gallie et al., 1997, 1998; Le et al., 1998).

Far-Western assays.
Proteins were run in 10 % acrylamide gels made in 375 mM Tris/HCl (pH 8·8) containing 0·02 % SDS. A relatively low concentration of SDS was used to facilitate renaturing of the proteins. Gels were blotted onto Hybond P membrane (Amersham Biosciences) and the membranes were probed with GST–CP fusion protein or native CP and analysed with GST or CP antisera, using a previously described procedure (Le et al., 1997).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
CP stimulates expression of luciferase mRNA with the AMV 3' UTR in protoplasts
The standard reporter construct used in our studies was a luciferase (Luc) mRNA flanked by short plasmid-derived 5' and 3' UTRs of 17 and 19 nt, respectively (Luc–control). This transcript and its derivatives have been used in several previous studies on the translation of plant virus RNAs (Gallie & Kobayashi, 1994; Gallie et al., 1989). The 3' end of this transcript was extended by a poly(A) tail of 50 residues [Luc–poly(A)] or the 3'-terminal 156 nt of AMV RNA 3 (Luc–AMV) and carrot protoplasts were transfected with capped Luc transcripts pre-incubated with increasing amounts of AMV CP. After a 20 h incubation period, the relative Luc expression of Luc–control, Luc–poly(A) and Luc–AMV was 1 : 18 : 5 (Fig. 1). Addition of CP did not increase Luc expression by Luc–control or Luc–poly(A), but did increase Luc expression from Luc–AMV by about 20-fold (Fig. 1). Luc expression by Luc–AMV reached a maximum upon addition of approximately six CP molecules per mRNA molecule.



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Fig. 1. Relative luciferase activity in carrot protoplasts 20 h after electroporation with capped luciferase mRNAs in the presence of various amounts of AMV CP. The mRNAs corresponded to Luc–control ({lozenge}), Luc–poly(A) ({square}) and Luc–AMV ({triangleup}). Error bars show SD calculated from three independent experiments.

 
Transfection of protoplasts with AMV RNA 4 results in the synthesis of detectable amounts of CP (Neeleman et al., 2001, 2004) and RNA 4 can replace CP in the initiation of infection by the three genomic RNAs (Bol et al., 1971). Fig. 2 shows a comparison of Luc expression in carrot protoplasts induced by a mixture of Luc transcripts with CP [6 mol CP (mol transcript)–1] or RNA 4 [1 mol RNA 4 (mol transcript)–1]. RNA 4 did not stimulate expression of Luc–poly(A), but did stimulate Luc–AMV expression by five- to sixfold. Whether higher amounts of RNA 4 would have further stimulated Luc expression was not investigated. However, data indicate that CP does not stimulate Luc expression by affecting uptake of Luc–AMV by protoplasts.



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Fig. 2. Stimulation of Luc expression from Luc–poly(A) and Luc–AMV by AMV CP [6 mol CP (mol mRNA)–1; hatched bars] or AMV RNA 4 [1 mol RNA 4 (mol mRNA)–1; shaded bars]. Luc expression was measured in carrot protoplasts 20 h after electroporation.

 
CP enhances the efficiency of translation of Luc–AMV mRNA in protoplasts
The stimulation of Luc expression by CP, as observed in Figs 1 and 2, could be due to an effect on the stability of the Luc–AMV transcript, to an increase in the translational efficiency of this mRNA or both. To distinguish between these possibilities, the kinetics of Luc accumulation in carrot protoplasts transfected with Luc–AMV plus or minus CP were analysed. Fig. 3(a) shows Luc accumulation plus and minus CP at the same scale; Fig. 3(b) shows Luc accumulation minus CP on an enlarged scale. Once the mRNA has been loaded onto polyribosomes, translation proceeds at a rate that reflects its translational efficiency and for a period of time that is determined by the stability of the mRNA (Gallie et al., 1996; Ling et al., 2002). The plateau of the curve represents cessation of Luc accumulation due to eventual degradation of the mRNA.



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Fig. 3. Time course of Luc expression in carrot protoplasts electroporated with Luc–AMV transcripts with (filled symbols) or without (open symbols) pre-incubation of the transcripts with AMV CP [6 mol CP (mol RNA)–1]. Diamonds and circles represent data from two independent experiments. Curved lines give the mean of the two experiments. Straight lines represent the rate of increase of Luc accumulation during the steady-state phase of translation. Dotted lines show the time at which Luc accumulation is at half of its maximum value. Luc expression in the absence of CP as shown in (a) is plotted on an extended scale in (b).

 
From the difference in the slope of the curves observed during the transient steady-state phase of translation in the presence and absence of CP, it was calculated that CP stimulated translational efficiency of Luc–AMV in protoplasts approximately 40-fold. The time required to reach half of the maximum accumulation of Luc can be taken as the functional half-life of the mRNA (Gallie et al., 1996; Ling et al., 2002). It was calculated that, in the absence of CP, the half-life of Luc–AMV was 2·5 h whereas, in the presence of CP, the half-life increased by about 20 % to 3·1 h (Fig. 3).

Stimulation of translational efficiency by CP is cap-dependent
The experiments described so far were done with transcripts containing a 5'-cap structure. To see whether the stimulatory effect of CP is cap-dependent, the experiments shown in Figs 1 and 2 were repeated with uncapped Luc–AMV and Luc–poly(A) transcripts. In the absence of CP or RNA 4, Luc expression in protoplasts by uncapped Luc–AMV and Luc–poly(A) was 28-fold and 93-fold lower, respectively, than expression observed with the corresponding capped transcripts (Fig. 4). Addition of CP or RNA 4 only marginally affected (approx. twofold) Luc expression by uncapped Luc–AMV (Fig. 4). This demonstrates that efficient (approx. 40-fold) CP-mediated stimulation of translation is cap-dependent.



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Fig. 4. Luc activity in carrot protoplasts 20 h after electroporation with non-capped Luc–poly(A) ({square}) or Luc–AMV ({triangleup}) mRNAs in the presence of increasing amounts of AMV CP (left) or AMV RNA 4 [right; 1 mol RNA 4 per mol Luc–poly(A) or Luc–AMV].

 
AMV CP interacts with the cap-binding complex of initiation factors
Previously, it has been proposed that AMV CP enhances translational efficiency of viral RNAs by acting as a PABP mimic (Neeleman et al., 2001). This model would predict an interaction of CP with one or more subunits from the eIF4F/eIFiso4F complex. Wheat eIF4F consists of the subunits eIF4E (26 kDa) and eIF4G (165 kDa), whereas wheat eIFiso4F consists of eIFiso4E (28 kDa) and eIFiso4G (86 kDa) (Browning, 1996; Gallie & Browning, 2001). Association of eIF4A in eIF4F and eIFiso4F complexes is relatively weak and eIF4A is easily lost during purification of these complexes (Browning, 1996; Lax et al., 1985). Sequence similarity between eIF4E and eIFiso4E is approximately 50 % (Browning, 1996). The main difference between eIFiso4G and eIF4G is the presence of an ~700 aa N-terminal region in eIF4G that is absent in eIFiso4G (Gallie & Browning, 2001). No serological cross-reaction between 4E and iso4E or 4G and iso4G isoforms is observed with available antisera (Browning et al., 1990; Gallie et al., 1998). eIF4G is highly susceptible to degradation in WGE, even with the addition of numerous protease inhibitors. This degradation follows a specific pattern in which degradation intermediates of 100–165 kDa accumulate initially and are followed by the appearance of ~75 and ~45 kDa stable end products. The ~75 kDa end product retains the ability to bind eIF4E, indicating that it represents the N-terminal region of the protein (reviewed by Browning, 1996).

To analyse a possible interaction between CP and initiation factors, an N-terminal GST fusion with CP was bound to glutathione–Sepharose 4B and used to pull down possible initiation factors from WGE. This extract contains five to ten times more eIFiso4F than eIF4F (Browning, 1996; Browning et al., 1990). In solution, CP occurs as a dimer (Jaspars, 1985) and dimer formation is required for stimulation of translation of RNA 4 by CP (Neeleman et al., 2004). Therefore, two different baits were used in the pull-down assays: the glutathione–Sepharose 4B/GST–CP complex and the same complex pre-incubated with unfused CP. Elution of this pre-incubated complex with glutathione after it had been washed several times resulted in elution of both the GST–CP fusion protein and unfused CP (data not shown). This indicates that pre-incubation resulted in the interaction of at least some of the added CP dimers with the GST–CP fusion protein. Unfused GST bound to glutathione–Sepharose 4B was used as a negative control. The baits were incubated with WGE, Sepharose beads were washed two, three or four times with washing buffer and, finally, proteins bound to the beads were eluted with buffer containing glutathione. Analysis of the eluate on a stained gel showed bands corresponding to GST–CP, unfused CP when added during pre-incubation and a few, hardly visible bands of proteins selected from the WGE (data not shown). Fig. 5 shows Western blotting analysis of the eluted proteins using antisera that specifically recognized eIF4G, eIFiso4G and eIF4A (Browning et al., 1990; Gallie et al., 1998).



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Fig. 5. Interaction of AMV CP with eIF4F and eIFiso4F as revealed by GST pull-down assays. Glutathione–Sepharose 4B beads were complexed with unfused GST (lanes 2 and 3) or GST fused to AMV CP (lanes 4–9). In lanes 4, 5 and 6, complexes were pre-incubated with additional CP purified from AMV virions. Complexes were incubated with WGE and washed two, three or four times with washing buffer as indicated on top of the lanes. Subsequently, bound proteins were eluted from the beads with a buffer containing glutathione and the eluted proteins were analysed by Western blotting with antisera detecting the eIF4G subunit (a), the eIFiso4G subunit (b) or the eIF4A subunit (c) of the eIF(iso)4F complexes. Lane 1, total WGE.

 
After three washings, no eIF4G or eIFiso4G was eluted from control beads to which unfused GST was bound (Fig. 5a, b; lane 3). However, eIFiso4G and fragments of eIF4G were both pulled down by beads complexed with GST–CP fusion protein (Fig. 5a, b; lanes 7, 8 and 9). Interestingly, pre-incubation of the GST–CP beads with unfused CP significantly increased the stability of binding of eIF4G and, to a lesser extent, eIFiso4G, as they could be still eluted after the beads had been washed four times (Fig. 5a, b; lane 6). When pre-incubation with unfused CP was omitted, little or no initiation factor could be eluted after four washings (Fig. 5a, b; lane 9).

The lane loaded with total WGE shows that eIF4G in the extract is mostly degraded to the ~75 kDa product, whereas little degradation of eIFiso4G is observed (Fig. 5a, b; lane 1). Longer exposure times revealed minor amounts of the ~45 kDa fragment of eIF4G in the total WGE. It is not known whether these fragments of eIF4G are still present in an eIF4F-like complex. Thus, it is possible that all eIF4A detected in Fig. 5(c) is pulled down as part of the EIFiso4F complex and that the eIF4G fragments in Fig. 5(a) are pulled down by direct interaction with CP. Enrichment of the 45 kDa fragment in the pull-down assay may reflect a relatively high affinity of CP for this fragment or an enhanced degradation of eIF4G during the pull-down assay.

As with eIF4G and eIFiso4G, eIF4A was pulled down with the GST–CP bait, but not with the GST control (Fig. 5c). eIF4A is associated weakly with the eIF4F and eIFiso4F complexes (Browning, 1996; Lax et al., 1985) and was washed off more readily than eIF4G and eIFiso4G. Initiation factors eIF4B and eIF3 interact with eIF4F and eIFiso4F (Gallie, 2002), but these two factors were not pulled down by the GST–CP bait above background levels (results not shown).

AMV CP interacts with eIF4G and eIFiso4G
Initiation factors involved in the interaction of the eIF4F and eIFiso4F complexes with CP were identified by Far-Western assays. eIF4E, eIFiso4E, eIF4G and eIFiso4G were expressed separately in E. coli from a pET3d vector and eIF4E and eIFiso4E were purified from a bacterial S30 extract by binding the recombinant proteins to m7GTP–Sepharose beads as described previously (van Heerden & Browning, 1994). Fig. 6(a) shows analysis on a stained gel of eIF4E (lane 1) and eIFiso4E (lane 2) eluted from the beads with m7GTP. In the gel system used, eIFiso4E co-migrates with the 34 kDa marker, whereas eIF4E migrates ahead of this marker. Beads complexed with eIF4E were mixed with an S30 extract of E. coli expressing eIF4G and the eIF4E–eIF4G complex was eluted from the beads with m7GTP (van Heerden & Browning, 1994). The eIF4E protein selected a few proteins migrating in the 200 kDa region of the gel that are expressed specifically from the eIF4G vector (Fig. 6a, lane 3). By using a similar procedure, beads complexed with eIFiso4E were used to purify eIFiso4G from an S30 extract of E. coli expressing this recombinant protein (Fig. 6a, lane 4). Identities of the purified eIF4E, eIF4G and eIFiso4G were confirmed by Western blotting (Fig. 7).



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Fig. 6. Far-Western analysis of the interaction of AMV CP with wheat initiation factors. Recombinant eIF4E and eIFiso4E were purified from bacterial extracts by binding the proteins to m7GTP–Sepharose beads. Bound eIF4E (lane 1) and eIFiso4E (lane 2) were eluted with m7GTP and analysed on a stained gel (a). m7GTP–Sepharose beads complexed with eIF4E or eIFiso4E were mixed with bacterial extracts containing recombinant eIF4G or eIFiso4G, respectively, and proteins eluted from the beads with m7GTP were analysed in lanes 3 (eIF4E and eIF4G) and 4 (eIFiso4E and eIFiso4G). Gels in (a) were blotted onto membranes and used in Far-Western assays (b, c). In (b), the membrane was probed with GST–CP fusion protein and analysed with GST antiserum. In (c), the membrane was probed with native CP and analysed with CP antiserum. Lanes marked M indicate molecular-mass markers with sizes (kDa) indicated on the left; the positions of the markers in (b) and (c) are indicated by horizontal bars. Putative bacterial proteins selected through their affinity for eIF4G and eIFiso4G are indicated by asterisks.

 


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Fig. 7. Western blot analysis of purified recombinant initiation factors. A gel as shown in Fig. 6(a) was blotted onto Hybond P membrane and the membrane was probed with antisera containing a mixture of antibodies against eIF4E and eIF4G (a) or eIFiso4G (b). Lanes marked M indicate molecular-mass markers with sizes (kDa) indicated on the left.

 
Gels similar to the gel shown in Fig. 6(a) were blotted onto nitrocellulose for Far-Western assays. One blot was probed with GST–CP fusion protein and analysed with GST antiserum (Fig. 6b). Another blot was probed with native CP and analysed with CP antiserum (Fig. 6c). Results of the two assays were similar. Neither GST–CP nor native CP bound to eIF4E or eIFiso4E (Fig. 6; lanes 5, 6, 9 and 10). However, GST–CP and native CP showed a prominent interaction with a band corresponding to the 165 kDa eIF4G expressed from the pET vector (Fig. 6; lanes 7 and 11). Moreover, GST–CP and native CP both showed a prominent interaction with a band corresponding to the 86 kDa eIFiso4G expressed from the pET vector (Fig. 6, lanes 8 and 12). In addition, GST–CP and native CP interacted with three low-molecular-mass proteins (indicated by asterisks) in lanes 7, 8, 11 and 12 of Fig. 6, one of which co-migrated with the dye front. These are either E. coli proteins selected through an affinity for eIF4G and eIFiso4G or degradation products of eIF4G and eIFiso4G, which are poorly recognized by antisera against these initiation factors (Fig. 7). These low-molecular-mass proteins have not been further analysed.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The 3' UTR of AMV RNA 3 is 182 nt long and contains at least two independent CP-binding sites (Reusken et al., 1994). Previously, it has been shown that a 3' sequence of 112 nt of this UTR, containing one of the two binding sites, is sufficient for wild-type levels of translation of RNA 4 in tobacco protoplasts (Neeleman et al., 2004). This sequence is fully present in the AMV 3' UTR sequence that was fused to the 3' end of the Luc reporter transcript in the present study. In addition to the 3' UTR, the 5' UTR also contributes to efficient translation of RNA 4 in protoplasts (Neeleman et al., 2004). In vitro, this 5' UTR stimulates translation independently of the 3' UTR of RNA 4 (Jobling & Gehrke, 1987). The use of the Luc–AMV reporter permitted analysis of the role of the 3' UTR independently from other elements that might affect translation of AMV RNAs. Incubation of the chimeric Luc–AMV RNA with CP prior to transfection of protoplasts increased the half-life of the mRNA from 2·5 to 3·1 h, i.e. by 20 %, but this increase is not at all sufficient to explain the strong increase in Luc expression mediated by CP. Because the increase in Luc expression is also observed upon co-transfection of protoplasts with Luc–AMV and the CP messenger, RNA 4, a major effect of CP on the uptake of mRNA by protoplasts can be ruled out.

From the time-course studies presented in Fig. 3, it can be concluded that CP enhances the translational efficiency of Luc–AMV in plant cells by about 40-fold. CP did not stimulate translation of Luc–poly(A) or Luc–control transcripts, demonstrating that the effect of CP is specific for messengers with the AMV 3' UTR. CP mutants defective in RNA binding did not stimulate translation (Neeleman et al., 2001). Apparently, CP has to bind to the 3' end of the viral RNA to enhance translation efficiency. Addition of RNA 4 to Luc–AMV in a molar ratio of 1 : 1 stimulated Luc expression by approximately sixfold (Fig. 2). In a previous study, it was reported that addition of a 50-fold molar excess of RNA 4 to Luc–AMV only marginally affected Luc expression in carrot protoplasts (Gallie & Kobayashi, 1994). However, in this earlier study, the RNA 4 was not capped. More recently, it has been shown that uncapped RNA 4 is translated poorly in protoplasts (Neeleman et al., 2001). The observation that uncapped RNA 4 does not stimulate Luc–AMV expression is in line with the conclusion that RNA 4 stimulates translation through its encoded CP (Neeleman et al., 2001). As with the RNA 4 transcript, the Luc–AMV transcript must be capped to allow stimulation of its translation by CP (Fig. 4). In the absence of CP, Luc expression from capped Luc–AMV is not zero, but amounts to 25–30 % of the Luc expression obtained with Luc–poly(A) (Fig. 2). After co-transfection of protoplasts with Luc–AMV and RNA 4, both RNAs are probably translated initially at relatively low levels until CP expressed from RNA 4 stimulates translation of the two RNAs. The observation that Luc expression from Luc–AMV is stimulated more efficiently by CP than by RNA 4 (Fig. 2) is consistent with the notion that translation of RNA 4 must occur prior to stimulation of translation of Luc–AMV.

How does binding of CP to the viral RNA stimulates translation? It has been reported previously that extension of the 3' termini of AMV genomic RNAs with a poly(A) tail of 40 or 80 residues permitted initiation of infection of tobacco plants and protoplasts at a level that was 5 % of the CP-mediated initiation of infection (Neeleman et al., 2001). Here, it has been shown that, in the presence of CP, the AMV 3' UTR stimulates Luc expression in plant cells much more efficiently than a poly(A) tail of 50 residues does. After entry into a plant cell, CP may allow the AMV RNAs to compete efficiently with the approximately 300 000 cellular messengers for the translation machinery. The poly(A) tail of cellular messengers binds PABP, which enhances translation efficiency via interaction with the eIF4G subunit of eIF4F. Our pull-down and Far-Western assays revealed that AMV CP interacts specifically with the eIF4G and eIFiso4G subunits from wheat eIF4F and eIFiso4F, respectively. This supports the proposal that CP acts as a PABP mimic (Neeleman et al., 2001).

The role of AMV CP in the translation of viral RNA may resemble the function of the rotavirus NSP3 protein. Both proteins have to bind as dimers to the 3' end of their non-polyadenylated mRNAs to enhance translation and both proteins interact with components of the eIF4F complex (Neeleman et al., 2004; Piron et al., 1998; Vende et al., 2000). The N-terminal domains of NSP3 and AMV CP are involved in binding of the cognate viral RNAs (Bol, 1999; Deo et al., 2002). NSP3 evicts PABP from eIF4F during rotavirus infection, leading to enhanced translation of viral mRNAs and the concomitant inhibition of the translation of cellular mRNAs (Piron et al., 1998). AMV infection does not inhibit cellular protein synthesis (Hooft van Huijsduijnen et al., 1986). Preliminary experiments showed that, when GST–CP (or GST–CP plus native CP) was used to pull down eIF4G and eIFiso4G, PABP was also bound to the Sepharose beads (results not shown). This indication that AMV CP does not evict PABP from eIF4F or eIFiso4F will be further investigated. In addition to the similarity with NSP3, the early function of AMV CP may resemble the role of the stem–loop-binding protein (SLBP), which binds to the 3' end of non-polyadenylated animal histone mRNAs. Interaction of SLBP with the mRNA and with eIF4G is required for efficient translation of the histone mRNA (Ling et al., 2002).

Like bromo- and cucumoviruses in the family Bromoviridae, the 3' termini of the RNAs of TMV (genus Tobamovirus) and Turnip yellow mosaic virus (TYMV, genus Tymovirus) can be folded into a TLS that can be charged with an amino acid. The TLS of TMV and TYMV have been shown to bind the elongation factor eEF1A (Matsuda & Dreher, 2004; Zeenko et al., 2002) and, for TYMV, this interaction enhanced translation of the viral RNA in plant cells (Matsuda & Dreher, 2004). By analogy, eEF1A may stimulate translation of the RNAs of bromo-, cucumo- and possibly oleaviruses in the family Bromoviridae. CP inhibits AMV minus-strand RNA synthesis in vitro (Houwing & Jaspars, 1986), whereas eEF1A inhibits TYMV minus-strand RNA synthesis in vitro (Matsuda et al., 2004). CP and eEF1A may have similar regulatory roles in the early steps of the replication cycles of CP-dependent and CP-independent plant viruses, respectively (Olsthoorn et al., 1999; Matsuda et al., 2004).


   ACKNOWLEDGEMENTS
 
Thanks are due to Drs V. V. Zeenko (Riverside) for help with the experimental work and Drs H. J. M. Linthorst, C. W. A. Pleij and R. C. L. Olsthoorn (Leiden) for stimulating discussions. This work was sponsored in part by the division Chemical Sciences (CW) of the Netherlands Organization for Scientific Research (NWO) and grants from the NSF (MCB-0130664) and USDA (03-35100-13375) to D. R. G.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 30 November 2004; accepted 8 February 2005.