The potyviral virus genome-linked protein VPg forms a ternary complex with the eukaryotic initiation factors eIF4E and eIF4G and reduces eIF4E affinity for a mRNA cap analogue Thierry Michon, Yannick Estevez, Jocelyne Walter, Sylvie German-Retana and Olivier Le Gall ´ ´ ´ Interactions Plante-Virus, UMR GDPP INRA-Bordeaux 2, Institut de Biologie Vegetale Moleculaire, Villenave d’Ornon, France Keywords eIF4E; eIF4G; fluorescence; interaction; VPg Correspondence ´ ´ T Michon, Virologie Vegetale, GDPP, IBVM-INRA, BP 81, 33883 Villenave d’Ornon Cedex, France Fax: +33 57 12 23 84 Tel: +33 57 12 23 91 E-mail: michon@bordeaux.inra.fr Website: http://www.bordeaux.inra.fr/ipv/ (Received 10 October 2005, revised 23 January 2006, accepted 26 January 2006) doi:10.1111/j.1742-4658.2006.05156.x The virus protein linked to the genome (VPg) of plant potyviruses is a 25-kDa protein covalently attached to the genomic RNA 5¢ end It was previously reported that VPg binds specifically to eIF4E, the mRNAcapbinding protein of the eukaryotic translation initiation complex We performed a spectroscopic study of the interactions between lettuce eIF4E and VPg from lettuce mosaic virus (LMV) The cap analogue m7GDP and VPg bind to eIF4E at two distinct sites with similar affinity (Kd ¼ 0.3 lm) A deeper examination of the interaction pathway showed that the binding of one ligand induces a decrease in the affinity for the other by a factor of 15 GST pull-down experiments from plant extracts revealed that VPg can specifically trap eIF4G, the central component of the complex required for the initiation of protein translation Our data suggest that eIF4G recruitment by VPg is indirectly mediated through VPg–eIF4E association The strength of interaction between eIF4E and pep4G, the eIF4E-binding domain on eIF4G, was increased significantly by VPg Taken together these quantitative data show that VPg is an efficient modulator of eIF4E biochemical functions Lettuce mosaic virus (LMV), is a member of the genus Potyvirus [1] Potyviruses are plant viruses with flexible rod-shaped particles packing a single-stranded, polyadenylated, positive-sense genomic RNA [2] This RNA, of about 10 kb, is linked at its 5¢ end to a viral protein, VPg (virus protein linked to the genome), through a tyrosine phosphoester covalent bound [3–5] The viral particle does not carry the molecular machinery required for its replication in the host cell, and the viral genome codes for a limited number of proteins Thus the infectious cycle needs to recruit various host factors, including the host translation apparatus Eukaryotic mRNAs are capped by the addition of a 7-methylated guanine (m7G) This post-transcriptional modification occurs in the nucleus [6,7] The 5¢-cap acts as a flag on the mRNA for cytoplasmic export and for the ribosomal translation complex assembly The recognition of this m7G functional group by a cap-binding protein (eIF4E, the eukaryotic translation initiation factor 4E, or its isoform eIFiso4E) is the first step of a complex cascade of molecular events leading to binding of the 40S ribosomal subunit to the mRNA [8] The general structural similarity between the potyvirus RNA and eukaryotic mRNAs suggests that VPg may act functionally as a cap-like structure This hypothesis gained strength when a specific interaction between eIF4E and VPg was identified in several pathosystems, such as tomato ⁄ tobacco etch virus [9] and Arabidopsis thaliana ⁄ turnip mosaic virus (TuMV) [10] In the latter case, a single amino acid replacement in Abbreviations BN, blue native; eIF4E, eukaryotic translation initiation factor 4E; GST, glutathione S-transferase; LMV, lettuce mosaic virus; PABP, poly(A)binding protein; TuMV, turnip mosaic virus; VPg, virus protein linked to the genome 1312 FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS T Michon et al VPg abolished its interaction with eIF4E, and this correlated with a defect in infectivity in Brassica perviridis [11] The simplest hypothesis is that VPg recruits eIF4E to initiate the translation of the potyvirus polyprotein [12] The involvement of eIF4E in pea seed-borne mosaic virus cell to cell movement has been suggested [13] Some lettuce cultivars display a recessive resistance to LMV As this resistance is opposed by the eIF4E isoform, we focused this study on the VPg– eIF4E interaction [14] Whatever the biochemical process involved, the VPg–eIF4E complex appears to play a crucial role in the outcome of the plant–potyvirus interaction Therefore, we developed a quantitative test to (a) measure precisely the binding strength between VPg and eIF4E and (b) assess the pathway of the interactions between VPg, eIF4E and the cap structure A glutathione S-transferase (GST) pull-down test from plant extracts was used to demonstrate that VPg can recruit the binary complex eIF4E–eIF4G, a component of eIF4F, the complex of translation initiation Finally, we evaluated the possible effect of VPg on eIF4G binding to eIF4E using a synthetic peptide that mimicks the eIF4G-binding domain Results and discussion Interaction between VPg and eIF4E We initially evaluated the perturbation of the intrinsic fluorescence of eIF4E upon its interaction with VPg As VPg contains no tryptophan, the contribution of its intrinsic fluorescence was assumed to be negligible with respect to the nine tryptophans present in eIF4E Upon the addition of VPg, a decrease in the overall fluorescence of eIF4E was observed (Fig 1) This seemed to correlate with a specific interaction between the two proteins, as the fluorescence decrease reached a plateau at high VPg concentrations The binding of VPg only slightly affected eIF4E tryptophan fluorescence The signal-to-noise ratio was poor, implying low accuracy in the determination of the dissociation constant (Kd ¼ 0.3 lm) The binding curve extrapolated to a : stoichiometry for the two proteins (Fig inset) From these data it is likely that VPg binding to eIF4E is not associated with much modification of the environment of the eIF4E tryptophans The two tryptophan residues present in the cap-binding site are accessible to the surface [15] Direct access of VPg to the pocket would probably be associated with greater modification of the fluorescence of these two residues, as happens when cap analogues penetrate into the Modulation of plant eIF4E properties by a potyvirus VPg Fig Fluorescence emission spectra of eIF4E upon VPg addition Aliquots of 2.5 lL from a 60-lM VPg stock solution were added to a 0.5-lM eIF4E solution in buffer M After each addition, the mixture was incubated for at 25 °C, and spectra were recorded (- - - -) No VPg added; (—) from top to bottom increasing amounts of VPg Inset: variation in eIF4E fluorescence as a function of VPg concentration in the medium pocket [16] The emission maximum of lettuce eIF4E was found to be at 342 nm, which is rather high It is likely that the major contribution to the intrinsic fluorescence of the protein comes from these two tryptophans in a polar environment, the fluorescence of the others being partially shielded This was also found in previous studies, although not to such an extent [16] Binding-site characterization To obtain a better fluorimetric response of eIF4E upon its interaction with VPg, we attempted to follow indirectly the complex formation using the cap analogue m7GDP There are considerable discrepancies between the affinities of the cap analogue published so far In the absence of VPg, the value of the dissociation constant obtained (Kc ¼ 0.31 ± 0.02 lm) was significantly lower than in previous reports for this type of analogue [17,18] A possible explanation is that, during the procedure used for eIF4E isolation, the elution step from m7GTP–Sepharose 4B is usually performed with free m7GTP We observed that, even after extensive dialysis, a significant fraction of active eIF4E retains m7GTP in its binding site (data not shown) In our study, all eIF4E fractions were previously eluted from m7GTP– Sepharose 4B with m KCl instead of m7GTP (see Experimental Procedures) However, it cannot be excluded that lettuce eIF4E displays a higher affinity for the FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS 1313 Modulation of plant eIF4E properties by a potyvirus VPg T Michon et al cap than its wheatgerm counterpart It is worth mentioning that, in a recent study, values in the nanomolar range were reported The authors emphasize that this could be because the recombinant eIF4E used was obtained from carefully renatured batches from inclusion bodies [19], thus avoiding affinity purification involving exposure to cap analogues The apparent dissociation constant of m7GDP to eIF4E was higher in the presence of increasing amounts of VPg (Fig 2) The plateau value (ligand saturation) was also affected (Fig 2, inset) It was suspected that VPg could remain bound to eIF4E even at high cap analogue concentrations The fact that m7GDP could not displace VPg argued in favour of the presence of a VPg-binding site on eIF4E, which is distinct but structurally related to the cap-binding site A possible mixed-type noncompetitive ligand binding of eIF4E and the cap to the VPg was reported previously [11] However, as the amount of free and bound ligand was not determined, a nonlinear double-reciprocal plot was obtained, from which it was difficult to establish an accurate pathway for the interactions [20] Complexes between proteins often involve large surface overlaps It cannot be ruled out that VPg interacts Fig Effect of VPg on m7GDP binding to eIF4E at 25 °C VPg aliquots were mixed with lM eIF4E in buffer M Before titration with m7GDP Inset: isotherms of m7GDP binding to eIF4E Solid lines represent theoretical data calculated using the best fit of the experimental data to eqn (4) (see Experimental Procedures) (d) No VPg; (s) 0.3 lM VPg; (n) 1.2 lM VPg; (h) 2.4 lM VPg 1314 with eIF4E through several domains, one of them being structurally linked to the cap site In such a case, the presence of VPg might negatively affect the binding of m7GDP To discriminate between competitive and noncompetitive interactions, we performed a m7GDP– eIF4E binding test in the presence of a large excess of VPg (30–60 lm) with respect to eIF4E (2 lm) In such conditions, the concentration of free VPg ([V]free) is assumed to be close to [V]total The same saturation behaviour was observed as for lower concentrations of VPg (Fig 3A) In the case of strict competition, the Fig (A) Binding isotherms of m7GDP with eIF4E in the presence of high VPg concentrations Experimental conditions were as in Fig Lines represent theoretical data calculated using the best fit of the experimental data to eqn (4) using either two distinct but dependent sites (solid line) or strict competition at the same site (dashed line) All measurements were made at least in triplicate (d) 30 lM VPg; (s) 60 lM VPg (B) Plots of residuals between theoretical and experimental data using either two distinct but dependent sites (solid line) or strict competition at the same site (dashed line) VPg concentration, 30 lM FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS T Michon et al Modulation of plant eIF4E properties by a potyvirus VPg A Table Equilibrium association constants for various forms of eIF4E and its ligands NB, no binding detected m7GDP eIF4E VPg Kv eIF4E-VPg B 10)6 · Ka (M)1) VPg pep4G m7GDP eIF4E eIF4E-VPg eIF4E-pep4G W123A eIF4E-m7GDP Kc 3.3 ± 0.3 – 2.9 ± 0.2 1.5 ± 0.8 4.1 ± 0.7 12.1 ± 1.5 – 3.9 ± 0.4 3.2 ± 0.3 0.2 ± 0.07 8.1 ± 0.9 NB m7GDP eIF4E eIF4E-m7GDP Kc VPg VPg αKv Kv αKc VPg-eIF4E-m7GDP eIF4E-VPg m7GDP Scheme Two plausible models for the interactions between eIF4E, VPg and the cap analogue 7mGDP (A) Strictly competitive model; (B) binding at two distinct but dependent sites apparent dissociation constant K app can be derived c according to Scheme 1A as:   ẵV app 1ị Kc ẳ Kc ỵ free Kv where [V]free is the concentration of free VPg in the medium The apparent dissociation constant for binding at two distinct but dependent sites defined in Scheme 1B is: ! Kv ỵ ẵVfree app 2ị Kc ẳ Kc ẵV Kv þ afree where a and KV are the ratio of symmetrical dissociation constants (Scheme 1B) and the VPg–eIF4E dissociation constant, respectively In this case, as eIF4E remains partitioned between the three species eIF4E–m7GDP, m7GDP–eIF4E–VPg, and eIF4E–VPg whatever the concentration of m7GDP, the plateau value is also affected and:   Kv app Ysat ẳ Ysat 3ị aKv ỵ ẵVfree Kc and Ysat were replaced in eqn (4) by the expressions app app Kc and Ysat Assuming [V]free to be close to [V]tot, data sets of eIF4E fluorescence as a function of [m7GDP]total were fitted to models mimicking either strictly competitive binding or binding at two distinct but dependent sites Plots of residuals obtained from both fits showed unambiguously that VPg and m7GDP bind to distinct but interdependent sites (Fig 3B) Although the cap analogue and VPg displayed comparable affinity for eIF4E (Kc ¼ 0.31 ± 0.02 lm and Kv ¼ 0.3 ± 0.03 lm), binding of the first molecule affected the binding of the second one by a factor 15 (a ¼ 15 ± 3) We examined the effect of disruption of the cap-binding capacity on eIF4E association with VPg To so, we engineered W123A, an eIF4E mutant in which W123, one of the two conserved tryptophan residues involved in p-p stacking with the cap aromatic moiety [15], was substituted with an alanine As expected, this substitution abolished m7GDP binding to W123A while retaining its capacity to associate with VPg (Table 1) The VPg surface defining the zone of interaction with eIF4E spans at least two distinct domains, one of which can affect the topology of the cap-binding pocket The VPg–eIF4E interaction may be a mechanism for recruiting the host translation machinery cut off by several unrelated positive-stranded RNA viruses In a recent study, an interaction between VPg and a human enteric calcivirus was demonstrated [21] GST pull-down assay of plant proteins forming complexes with VPg Several studies have reported specific interactions between the VPg from potyvirus and eIF4E or one of its isoforms [9] A recombinant VPgỈGST fusion protein was used as a bait for trapping molecular species susceptible to be recruited by VPg in planta A soluble protein fraction was recovered after mild detergent treatment of the plant leaves This extract was submitted to a VPgỈGST pull-down procedure [22] To determine the nature of the protein complexes involved in specific interactions with VPg, the fraction recovered was analysed by electrophoresis in native conditions A high-molecular-mass (above 200 kDa) species and two minor species (below 100 kDa) were affinity-purified from the protein extract (Fig 4A, lane 2) A western blot analysis with antibodies to VPg showed that all FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS 1315 Modulation of plant eIF4E properties by a potyvirus VPg T Michon et al A B Fig Analysis of the plant soluble proteins complexed with VPg (A) BN-PAGE of the protein fraction retained on glutathione–Sepharose 4b Lane 2, the plant soluble extract was incubated with recombinant VPgỈGst fusion and mixed with glutathione–Sepharose 4b beads After extensive washing, the proteins specifically retained on the resin were eluted with glutathione (see Experimental procedures for details) The fractions obtained were loaded on to a 6–18% polyacrylamide slab gel After migration, the complexes were Coomassie stained Proteins from lane were transferred to a nitrocellulose membrane Complexes were revealed with polyclonal antibodies raised against VPg (lane 3), eIF4E (lane 4) and eIF4G (lane 5) Lane 6, same as lane except that protein extracts were from LMV-Infected plants (40 lg VPgỈGst added) (B) Sds ⁄ PAGE of the proteins retained on glutathione–Sepharose 4b Affinity chromatography was as in (A) Lane 4, electrophoretic pattern of the protein fraction under denaturing conditions; Coomassie blue staining Lanes 5, and 7: Western blot analysis of the proteins retained using polyclonal antibodies raised against eIF4G, VPg and eIF4E, respectively Lane 1, molecular mass markers Lane 2: recombinant GSTỈVPg fusion extracted from E coli and affinitypurified on glutathione–Sepharose 4b Lane 3, as a control, the plant soluble extract was incubated with nonfused recombinant GST and mixed with glutathione–Sepharose 4b After being washed, the fraction eluted with glutathione was analysed By SDS ⁄ PAGE contained this protein (Fig 4A, lane 3) As the interaction of VPg with plant translation initiation factors was suspected, a western blot was performed with specific antibodies to eIF4E (lane 4) and eIF4G (lane 5) Two of the three species (the one >205 kDa and the intermediate one) contained eIF4E (lane 4) The eIF4G forms were restricted to the high-molecular-mass species (lane 5) When pull-down assays were performed 1316 on extracts from infected leaves, the highest-molecularmass species was not detected (lane 6) The purified fraction was also analysed by SDS ⁄ PAGE to determine the composition of the complexes They mainly contained three groups of polypeptide chains according to their relative molecular masses: a group with bands in the range of 180 kDa, a 54-kDa band, and a single chain of 26 kDa (Fig 4B, lane 4) The proteins were identified by western blotting Antibodies to eIF4G revealed many polypeptides in the 70–200 kDa region (Fig 4B, lane 5) It has previously been reported that, in several organisms, eIF4G is highly susceptible to proteolysis [23] It is likely that, during the extraction step, cleavage occurred along the polypeptide chain Whereas blue native (BN)-PAGE native conditions revealed a single molecular species probably because of conformational locking, SDS ⁄ PAGE denaturing conditions revealed the proteolysis The VPg antibodies reacted with a single 54-kDa polypeptide corresponding to the GSTỈVPg fusion (Fig 4B, lane 6) The eIF4E antibodies revealed only a 26-kDa polypeptide in accordance with the expected plant eIF4E (Fig 4B, lane 7) Our GST pull-down experiment showed that VPg can recruit eIF4E (26 kDa [14]) and eIF4G (% 180 kDa according to A Thaliana eIF4G [24]) From the SDS ⁄ PAGE pattern we could determine precisely the nature of the three complexes revealed by BN-PAGE analysis The highest-molecular-mass band (>205 kDa) corresponds to the heterotrimer eIF4G–eIF4E–VPgỈGST (% 260 kDa; Fig 4A, compare lanes 3, and 5) The intermediate band (% 80 kDa) observed in native conditions contained VPg and eIF4E but not eIF4G (Fig 4A, compare lanes and with 5) It was attributed to the binary complex GSTỈVPg–eIF4E The band of lowest molecular mass, % 54 kDa, on BN-PAGE was unambiguously identified as the GSTỈVPg fusion on SDS ⁄ PAGE (Fig 4B, lane 6; see also lane for comparison with pure GSTỈVPg) We demonstrate here that the VPg–eIF4E interaction previously described in other pathosystems such as TuMV ⁄ B perviridis is also found in the LMV ⁄ lettuce system However, this is the first report of a physical interaction between VPg and eIF4G At this stage, we found no evidence for a direct interaction between VPg and eIF4G The recruitment of eIF4G by VPg is probably indirectly mediated by eIF4E as the central element of the complex This should display at least two distinct binding sites, one for VPg and the other for eIF4G In control experiments, the protein extract was incubated with unfused GST before the affinity chromatography GST alone failed to pull down any FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS T Michon et al Modulation of plant eIF4E properties by a potyvirus VPg The strength of interaction between elF4E and pep4G, the eIF4E-binding domain on eIF4G, is enhanced by VPg Fig (A) Competition experiments with the recombinant VPg The plant soluble extract was incubated with recombinant VPgỈGst fusion in the presence of 10 lg (lane 2) and 50 lg (lane 3) of pure recombinant VPg; lane 1, no VPg added After affinity chromatography on glutathione–Sepharose 4b, the proteins were submitted To BN-PAGE and analysed by western blotting with polyclonal antieIF4E (B) LMV-Infected plant extracts were incubated with increasing amounts of recombinant GSTỈVPg fusion before affinity chromatography Proteins were analyzed by western blotting with polyclonal antibodies against eIF4E Lane 1, 10 lg VPgỈGst fusion; lane 2, 30 lg VPgỈGst fusion; lane 3, 60 lg VPgỈGst fusion; lane 4, 100 lg VPgỈGst fusion; lane 5, 150 lg VPgỈGst fusion other species from the plant extract, confirming that the formation of specific complexes with eIF4E and eIF4G only involved the VPg domain of the GSTỈVPg fusion (Fig 4B, lane 3) In another set of experiments, the GSTỈVPg pull-down was challenged by mixing increasing amounts of pure recombinant VPg with the plant extracts The presence of VPg weakened the interactions between the bait and eIF4E (Fig 5A) To study the interaction in the context of infection, extracts from LMV-infected plants were incubated with increasing amounts of purified GSTỈVPg before affinity chromatography on glutathione–Sepharose 4B A 10-fold excess of GSTỈVPg was necessary to recover an amount of eIF4E–GSTỈVPg complex comparable to that observed from uninfected plants (Fig 5B) GSTỈVPg may be strongly challenged by the presence of viral VPg forms involved in complexes with the initiation factors in planta A very small amount of free eIF4E may be accessible to GSTỈVPg, most of it associated with the viral form It was not possible to detect the eIF4E–eIF4G complex from infected plant extracts whatever the amount of GSTỈVPg added (Fig 4A, lane 6) If VPg binds strongly to the binary complex eIF4E–eIF4G, it may hardly be displaced by GSTỈVPg Moreover, VPg can exist in several molecular forms in the infected cell Sequential processing of the polyprotein may be linked to a specific requirement during each phase of the viral cycle Of the molecular forms of VPg, 6K2ỈVPgỈPro and VPgỈPro copurify with eIF4E in complexes associated with membrane fractions [25] In infected cells, a substantial amount of eIF4G may be involved in complexes with endogenous forms of VPg If these complexes are tightly bound to membranes, they may be less efficiently extracted under our conditions The eukaryotic eIF4F initiation complex is a heterotrimer consisting of eIF4E, eIF4A (an RNA helicase) and eIF4G [26] In mammals, the central part of eIF4G contains three evolutionary conserved hydrophobic amino acids, a tyrosine and two consecutive leucines separated by four less well conserved residues (YxxxxLL) This motif is associated with eIF4E binding [27] Small oligopeptides that mimick this eIF4E recognition motif can bind to eIF4E This recognition motif is also highly conserved in the plant kingdom The lettuce eIF4G sequence is not available Knowing the high homology of this sequence between wheat (Q03387) and A thaliana (NP567095), the oligopeptide KKYSRDFLLKF from A thaliana (pep4G) was synthesized and tested for its ability to bind to lettuce eIF4E In the mammalian 3D structure, Trp73 is in close contact with the peptide [28] Lettuce eIF4E was modelled on the basis of its homology with the known structure of its mammalian counterpart [14] We hypothesized that the fluorescence of Trp94 (Trp73 in mouse) may be affected by pep4G binding This feature has been used successfully to monitor pep4G binding to murine eIF4E [19] When eIF4E was incubated either alone or after saturation with VPg, a fluorescence decrease proportional to the amount of complex formed was observed, leading to a saturation plateau (Fig 6) Interestingly the strength of binding of pep4G to the preformed eIF4E–VPg complex (Kd ¼ 0.083 ± 0.016 lm) was significantly higher than that to the free eIF4E (Kd ¼ 0.24 ± 0.03 lm) The reciprocal was not observed, as there was no effect of increasing concentrations of pep4G on the binding strength between VPg and eIF4E (Fig inset) As found above for VPg and m7GDP, one could expect the interdependence of VPg and pep4G with respect to their binding to eIF4E It cannot be ruled out that the association of VPg with eIF4E induces a change in its conformation, enhancing the fit of the eIF4E-binding domain to pep4G The interaction of VPg with eIF4E affects the properties of both the cap-binding pocket and the eIF4G-binding domain Our observation is consistent with VPg–eIF4E interactions mediated over a large area Long-range effects on the conformation of eIF4E probably occur upon VPg binding The data should be interpreted with caution, as we cannot presume that the effect of VPg is the same for binding of whole eIF4G The association of pep4G with eIF4E has been shown not to be accompanied by detectable changes in the crystallographic structure of eIF4E [28] FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS 1317 Modulation of plant eIF4E properties by a potyvirus VPg T Michon et al eIF4G-eIF4E-cap ⇔ eIF4E ⇔ ⇔ VPg eIF4E-VPg ⇔ eIF4E-cap VPg cap-eIF4E-VPg Scheme Hypothetical pathways of eIF4E and eIF4G recruitment Large arrows highlight the connections that, according to the experimental data, would be thermodynamically favoured Fig Effect of VPg on the association between eIF4E and pep4G (s) Titration of free eIF4E by pep4G (d) The formation of the ternary complex VPg–eIF4E–pep4G was monitored after a preliminary titration of eIF4E with VPg up to saturation (eIF4E concentration lM, VPg final concentration lM) Inset: (d) The apparent dissociation constant K d app of eIF4E–pep4G was determined in the presence of increasing amounts of VPg (s) The apparent dissociation constant K d app of eIF4E–VPg was determined in the presence of increasing amounts of pep4G In fact, pep4G has a small effect on cap binding, whereas whole eIF4G is a strong enhancer of eIF4E binding to the mRNA cap structure [29] A thermodynamic analysis predicts that the cocrystal structure of the pep4G–eIF4E complex encompasses most of the energetically significant interactions between eIF4G and eIF4E [28] However, more recently, an NMR structure of the binary complex between yeast eIF4E and the eIF4G (393–490) domain was analyzed It shows unambiguously that the N-terminus of eIF4E, which interacts with the eIF4G domain, promotes discreet conformational changes in the cap-binding site, allowing tighter binding of the cap [30] The thermodynamic parameters expressed as association constants are tabulated for comparison (Table 1) VPg is an efficient modulator of eIF4E properties Scheme summarizes a hypothetical distribution of the initiation factors eIF4E and eIF4G according to the thermodynamic binding strengths measured in this study In early stages of infection, the only form of VPg present in the host cell is linked to the viral genome Its concentration is low compared with the concentration of host cap mRNAs If VPg affinity in vivo is of the same order of magnitude as in vitro, it is unlikely 1318 that it will recruit eIF4E by displacing eIF4E–mRNA complexes Instead, the newly uncoated VPg–RNA molecules will require free eIF4E to start translation and ⁄ or other steps of the virus cycle It is likely that the pull-down experiment on healthy plant extracts reveals only eIF4E and eIF4G forms that are not involved with cellular mRNA As translation proceeds, the amount of VPg increases in the infected cell The stoichiometry of the viral particle assembly is of the order of 2000 capsid proteins for one VPg [31] Therefore, the synthesis and proteolytic maturation of the polyprotein lead to a considerable excess of VPg over that strictly required for virion assembly According to our data, the association of VPg with free eIF4E should enhance its affinity for eIF4G This would account for the inability of the VPgỈGST fusion to displace viral VPg Most of the newly synthesized VPg may recruit free eIF4E and possibly eIF4G, thereby exerting a negative co-operative effect on the binding of cellular mRNAs [32] As eIF4E is the limiting factor for translation efficiency [33], such sequestration may affect host cell metabolism and perhaps contribute to disease symptoms A large amount of the VPg–Pro form accumulates in the nucleus as nuclear inclusions, in relation to a functional nuclear localization signal present in the Pro domain [34] In animal cells, a significant proportion of eIF4E itself is located in the nucleus [35] If this is also true in plants, retention of initiation factors in the nucleus through interaction with nuclear VPg may contribute to depletion of the cytoplasmic pool of free eIF4E, and disrupt mRNA translation In turn, the host cell may respond to this disruption by increasing the expression of another eIF4E isoform, as shown in B perviridis after infection with TuMV [25] In eukaryotes, translation initiation of cellular mRNAs is mediated through the closed loop model connecting the capped 5¢ end of the messenger to its polyadenylated 3¢ end Circularization is thought to FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS T Michon et al increase translation efficiency by facilitating de novo initiation and recycling of terminating ribosomes on the same mRNA [36] The 3¢ polyadenylated end of mRNAs is bound to the poly(A)-binding protein (PABP) Circularization of the potyvirus RNA could be achieved through the 5¢ VPg instead of the cap structure The VPg–eIF4E–eIF4G–PABP complex would bring together the 3¢ poly(A) and the 5¢ VPg ends of the viral RNA As it has been reported that VPgỈPro from TuMV can bind to PABP [25], another possibility is that circularization is achieved through VPgỈPro–PABP, this more direct binding skipping the eIF4G–PABP interaction [37] Both mechanisms may be present at different stages of the virus cycle It is likely that viral recruitment of the 40S ribosome subunit is mediated by the eIF3–eIF4G–eIF4E interaction as for cellular mRNAs (see [38] for a review of translation initiation factors) Although there is no evident internal ribosome entry site in the 5¢ part of the LMV genome, the possibility of internal positioning of the ribosome was demonstrated using an uncapped tobacco etch virus 143 nucleotide leader It has been shown that, in such conditions, translation still requires eIF4G [39] Taken together, the data presented here strengthen the hypothesis of a physical involvement of eIF4G in the translation initiation complex of LMV Experimental Procedures Materials Desalted water was further purified using a Milli-Q Millipore system Buffers, salts (reagent grade), and m7GDP were from Sigma Aldrich (Lyon, France) All solutions were filtered through a 0.22-lm membrane before use The peptide pep4G was synthesized by a solid-phase method using F-moc chemistry [40] and purified by reversed-phase C18 HPLC Polyclonal antibodies were raised in rabbits after injection with purified recombinant proteins Protein expression and isolation The gene coding for the eIF4E was cloned from the lettuce (Lactuca sativa) cultivar Salinas AAP86602, which is susceptible to LMV [14] The VPg coding sequence was PCRamplified from a partial LMV cDNA (isolate LMV-0, P31999) cloned in Escherichia coli Both cDNAs were inserted into the pTrcHis-C expression vector (Invitrogen) in-frame with an N-terminal hexahistidine tag, according to the manufacturer’s instructions The W123A mutant of eIF4E was built using the QuickChangeTM Site-Directed Mutagenesis Kit developed by Stratagene E coli (strain Modulation of plant eIF4E properties by a potyvirus VPg BL21) was transformed with pTrcHisC-eIF4E or pTrcHisC-VPg Overnight Luria–Bertani broth starters were inoculated with single colonies and cultured at 37 °C in the presence of 50 lgỈmL)1 ampicillin Larger volumes (2 L) of Luria–Bertani broth containing ampicillin were inoculated with 50 mL of the overnight culture starter and grown at 37 °C to reach A600 of 0.5 Gene expression was induced by addition of 0.5 mm isopropyl b-d-thiogalactoside, and cells were incubated at 30 °C with shaking for another h DNA sequences encoding GST and GSTỈVPg fusion were cloned in pDEST 15 according to the GatewayTM strategy (Invitrogen) The expression vectors were introduced into E coli (BL21AI strain) After induction with lgỈmL)1 arabinose, the expression vector was run in the same conditions as above All proteins were submitted to the same extraction procedure and kept at °C Phenylmethanesulfonyl fluoride (100 lL of a 200-mm stock solution in methanol) was added at each step of the extraction Cells were centrifuged and suspended in 30 mL HEX buffer (20 mm Hepes ⁄ KOH, pH 8, mm dithiothreitol, 250 lm EDTA, 0.25% Tween 20, 0.4 m KCl) Lysozyme (0.5 mgỈmL)1) was added, and the suspension was incubated for 45 at °C with gentle stirring DNase and RNase (100 lg each) were added and the suspension was incubated at °C for another 45 The lysate was sonicated in ice for (1 s cycles) The crude extract was centrifuged at 20 000 g, °C for 30 The supernatant was recovered and centrifuged at 100 000 g, °C for 45 The clarified supernatant was incubated for 45 with mL Ni ⁄ nitrilotriacetate ⁄ Sepharose CL-6B (Qiagen) equilibrated in HEX buffer The beads were centrifuged at low speed and packed into a mL column The beads were washed with HEX containing 10 mm imidazole until the A280 reached a constant value Proteins were eluted by a 10–250 mm imidazole step gradient (1 mLỈmin)1 flow rate) The eluted fractions were submitted to SDS ⁄ PAGE, and the material from the fractions containing proteins was pooled Specific isolation procedures After being desalted, VPg fractions were refined by anionexchange chromatography in 20 mm Tris ⁄ HCl, pH 8, on a mono Q column (Amersham Biotech) Pure VPg was concentrated and stored in 50% glycerol at )20 °C until use (yield % 10 mg per litre of culture) The eIF4E fractions recovered from ion metal affinity chromatography were pooled, and 20 mm Hepes ⁄ KOH (pH 7.5) ⁄ mm dithiothreitol was added to achieve a final concentration of 100 mm KCl This protein extract was incubated for 45 at °C with mL m7GTP–Sepharose 4B (Amersham Biotech) equilibrated in buffer A (20 mm Hepes ⁄ KOH, pH 7.5, 100 mm KCl, mm dithiothreitol) The beads were centrifuged at low speed, packed into a 2-mL column, and washed with buffer A Protein elution was performed with FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS 1319 Modulation of plant eIF4E properties by a potyvirus VPg T Michon et al m KCl in buffer A After SDS ⁄ PAGE analysis, the fractions containing eIF4E were pooled and concentrated under nitrogen on an Amicon ultrafiltration cell equipped with a YM10 membrane (Millipore) After being desalted, 1-mL aliquots (0.5 mgỈmL)1) were recovered in 20 mm Hepes ⁄ KOH (pH 7.5) ⁄ mm dithiothreitol ⁄ 20 mm KCl and stored at )20 °C (yield 1–2 mg per litre of culture) Protein concentration was determined spectrophotometrically using the following absorption coefcients: e280 ẳ 60 400 m)1ặcm)1 and e280 ẳ 11 520 m)1Ỉcm)1 for eIF4E and VPg, respectively GST and GSTỈVPg fusions were affinitypurified on glutathione–Sepharose 4B beads (Amersham Bioscience) as described in the following section Pull-down assays Fresh leaves (1 g) were frozen in liquid nitrogen and ground The powder was suspended in mL cold HEX buffer Then 10 lL 200 mm phenylmethanesulfonyl fluoride stock solution in methanol and 25 lL Sigma P8340 protease cocktail inhibitor were added The suspension was filtered through glass wool The filtrate was centrifuged at 10 000 g for 10 at °C Phenylmethanesulfonyl fluoride and protease cocktail inhibitor were added Typically 2.5 mg soluble proteins were recovered by this procedure The extract was incubated with 100 lL glutathione–Sepharose 4B beads and 25 lg GST for h at °C The supernatant was mixed with 100 lL glutathione–Sepharose 4B beads, and mL fractions were incubated with either 10 lg GST or 20 lg GSTỈVPg fusion for h at °C After centrifugation at 10 000 g for min, °C, the beads were recovered and washed extensively with ice-cold HEX buffer The proteins were eluted with 100 lL 10 mm reduced glutathione in 20 mm Hepes ⁄ NaOH, pH Aliquots of volume 10 lL were used for PAGE analysis BN-PAGE was carried out using 6–18% polyacrylamide slab gels as described previously [41] Proteins were revealed by western immunoblotting with specific polyclonal antibodies ting the sample at 258 nm, the Raman peak contribution to the emission spectrum was prevented In water, Raman scattering occurs % 30 nm above the excitation wavelength In the buffer used, the excitation at 258 nm was accompanied by a Raman peak at 282 nm, which was far from the maximum of emission observed (343 nm) The nine tryptophan residues in eIF4E make the intrinsic fluorescence important, allowing the recovery of a very good signal even when exciting at 258 nm These conditions proved in preliminary experiments to give the best ratio of signal to noise for the observation of tryptophan fluorescence quenching at 342 nm This set-up was in accordance with the original studies of eIf4E titration with cap analogues [16,42] Measurements were made in mL buffer M (20 mm Hepes ⁄ KOH, pH 7.6, 25 mm Kcl, mm dithiothreitol and 10% glycerol) The concentration of eIF4E was between 0.5 and 2.5 lm Ligand stock solutions were adjusted in such a way that the volume added upon titration never exceeded 5% of the total volume After addition of the ligand, emission at 342 nm was recorded over a period suitable for reaching a constant fluorescence value (steady-state usually after min) The decrease in fluorescence with respect to the initial fluorescence was recorded, and corrections were made for dilution BSA was used as a control under the same conditions to test for nonspecific binding The fluorescence signal did not show significant changes upon BSA Addition (data not shown) In some experiments, it was necessary to perform emission measurements in the presence of high VPg concentrations Quantum yield loss was estimated according to the equation Fcor ẳ Fobs 100:5Aexị where Fcor and Fobs are the intensity of fluorescence corrected and observed, and Aex is the absorbance of the solution at 258 nm [43] As the quantum yield decrease was less than 3%, no inner filter correction was necessary VPg emission was checked at high VPg concentration No significant light scattering was observed For each ligand concentration, three data acquisitions were made Fluorescence measurements All spectra were acquired at 25 °C on a SAFAS Xenius spectrophotometer (Monaco) Excitation and emission slits were set to a 10-nm path The photomultiplier’s power was set at 600–800 V (of a maximum possible value of 1200 V) The photons emitted were collected at right angles from the vertical excitation beam The geometry of the device allowed us to set the optical path length of the emitted light mm above the excitation source With this set-up, fluorescence emission was linear up to 0.7 absorbance units at the wavelength of excitation (data not shown) The excitation wavelength was set at 258 nm Although there is a contribution of m7GDP absorption at this wavelength, with our optical system the inner filter effect was about 2% at the highest concentration and no correction was made By exci- 1320 Data analysis Let us consider the simple bimolecular association between eIF4E and one ligand: EỵC ! EC Kc According to this scheme, the saturation function of eIF4E with its ligand follows an hyperbola: ẵEC ẳ ẵEtot ẵC Kc ỵ ẵC 4ị The experimental procedures used in this work not allow direct determination of bound and free ligand concentrations without access to [Ec] and [C] eIF4E fluorescence decreases together with the eIF4E–ligand FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS T Michon et al Modulation of plant eIF4E properties by a potyvirus VPg complex formation The association is followed by monitoring Fmax ) F as a function of the amount of ligand added For the sake of comparison between various sets of data, the fluorescence decrease was normalized to Y ¼ ) (F ⁄ Fmax) At ligand saturation, a maximum for Dfmax is obtained, and we can define the plateau value as Ysat ¼ ) (Fmin ⁄ Fmax) Accordingly we can write: Y Ysat 5ị ẵC Kc ỵ ẵC 6ị ẵEC ẳ ẵEtot Eqn (1) becomes: Y ¼ Ysat An interesting feature of eqn (6) is that it expresses a saturation function that does not require the true concentration of the active protein ([E]tot) in the sample to be known to determine Kc Eqn (7) relates the observed signal [Y ¼ ) (F ⁄ Fmax)] to [C]tot, the total ligand concentration [44] q Kc ỵ ẵCtot ỵ Ysat 4ẵCtot Ysat Y ẳ Kc ỵ ẵCtot ỵ Ysat À ð7Þ Hypothetical models of interaction were examined by fitting the associated equations to experimental data using nonlinear regression Affinity constants were determined from the model giving the best fit to the data Acknowledgements We would like to thank Genevieve Roudet for her skilful assistance, and Dr T Candresse and Dr T Delaunay for stimulating discussions Many thanks to Professor K S Browning for providing us with eIF4G polyclonal antibodies We thank Dr J F Bussotti from SAFAS S.A for his skilful assistance in optical device optimization We are grateful to C Manigand (UMR 5471 CNRS-Bordeaux 1) for pep4G synthesis We are indebted to region Aquitaine for its financial support for this work References Le Gall O (2003) Lettuce mosaic virus In CMI ⁄ AAB Description of Plant Viruses Association of Applied Biologists, Wellesbourne Riechmann JL, Lain S & Garcia JA (1992) Highlights and prospects of potyvirus molecular biology J Gen Virol 73, 1–16 Puustinen P, Rajamaki ML, Ivanov KI, Valkonen JP & Makinen K (2002) Detection of the potyviral genomelinked protein VPg in virions and its phosphorylation by host kinases J Virol 76, 12703–12711 Murphy J, Rychlik W, Rhoads R, Hunt A & Shaw J (1991) A tyrosine residue in the small nuclear inclusion protein of Tobacco Vein Mottling Virus links the VPg to the viral-Rna J Virol 65, 511–513 Murphy J, Klein P, Hunt A & Shaw J (1996) Replacement of the tyrosine residue that links a potyviral VPg to the viral RNA Is lethal Virology 220, 535–538 Wen Y, Yue Z & Shatkin AJ (1998) Mammalian capping enzyme binds RNA and uses protein tyrosine phosphatase mechanism Proc Natl Acad Sci USA 95, 12226–12231 Itoh N, Yamada H, Kaziro Y & Mizumoto K (1987) Messenger RNA Guanylyltransferase from Saccharomyces cerevisiae Large scale purification, subunit functions, and subcellular localization J Biol Chem 262, 1989–1995 Pestova TV, Kolupaeva VG, Lomakin Ib, Pilipenko EV, Shatsky IN, Agol VI & Hellen CU (2001) Molecular mechanisms of translation initiation in eukaryotes Proc Natl Acad Sci USA 98, 7029–7036 Schaad M, Anderberg R & Carrington J (2000) Strainspecific interaction of the tobacco etch virus Nla protein with the translation initiation factor elF4E in the yeast two-hybrid system Virology 273, 300–306 10 Wittmann S, Chatel H, Fortin M & Laliberte J (1997) Interaction of the viral protein genome linked of turnip mosaic potyvirus with the translational eukaryotic initiation factor (iso) 4e of Arabidopsis thaliana using the yeast two-hybrid system Virology 234, 84–92 11 Leonard S, Plante D, Wittmann S, Daigneault N, Fortin M & Laliberte J (2000) Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4e correlates with virus infectivity J Virol 74, 7730–7737 12 Lellis AD, Kasschau KD, Whitham SA & Carrington JC (2002) Loss-of-susceptibility mutants of Arabidopsis thaliana reveal an essential role for Eif (iso) 4e during potyvirus infection Curr Biol 12, 1046–1051 13 Gao Z, Eyers S, Thomas C, Ellis N & Maule A (2004) Identification of markers tightly linked to sbm recessive genes for resistance to Pea seed-borne mosaic virus Theor Appl Genet 109, 488–494 Epub April 2004 14 Nicaise V, German-Retana S, Sanjuan R, Dubrana MP, Mazier M, Maisonneuve B, Candresse T, Caranta C & Le Gall O (2003) The eukaryotic translation initiation factor 4e controls lettuce susceptibility to the Potyvirus Lettuce mosaic virus Plant Physiol 132, 1272–1282 15 Marcotrigiano J, Gingras AC, Sonenberg N & Burley SK (1997) Cocrystal structure of the messenger RNA 5¢-cap-binding protein (eIF4E) bound to 7-methyl-Gdp Cell 89, 951–961 16 Carberry SE, Rhoads RE & Goss DJ (1989) A spectroscopic study of the binding of m7GTP and m7GpppG to human protein synthesis initiation factor 4e Biochemistry 28, 8078–8083 FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS 1321 Modulation of plant eIF4E properties by a potyvirus VPg T Michon et al 17 Sha M, Wang Y, Xiang T, van Heerden A, Browning KS & Goss DJ (1995) Interaction of wheat germ protein synthesis initiation factor Eif-(iso) 4f and its subunits p28 and p86 with m7GTP and Mrna analogues J Biol Chem 270, 29904–29909 18 Wei CC, Balasta ML, Ren J & Goss DJ (1998) Wheat germ poly (A) binding protein enhances the binding affinity of eukaryotic initiation factor 4f and (iso) 4f for cap analogues Biochemistry 37, 1910–1916 19 Niedzwiecka A, Marcotrigiano J, Stepinski J, Jankowska-Anyszka M, Wyslouch-Cieszynska A, Dadlez M, Gingras AC, Mak P, Darzynkiewicz E, Sonenberg N, et al (2002) Biophysical studies of eIF4E cap-binding protein: recognition of Mrna 5¢-cap structure and synthetic fragments of eIF4G and 4e-Bp1 proteins J Mol Biol 319, 615–635 20 van Holde KE, Johnson WC & Ho Ps (1998) Chemical equilibria involving macromolecules Principles of Physical Biochemistry, pp 587–628 Prentice Hall, London 21 Goodfellow I, Chaudhry Y, Gioldasi I, Gerondopoulos A, Natoni A, Labrie L, Laliberte JF & Roberts L (2005) Calicivirus translation initiation requires an interaction between VPg and Eif E EMBO Report 6, 968–972 22 Kaelin WG Jr, Pallas DC, DeCaprio JA, Kaye FJ & Livingston DM (1991) Identification of cellular proteins that can interact specifically with the T ⁄ E1A-binding region of the retinoblastoma gene product Cell 64, 521– 532 23 Prevot D, Darlix JL & Ohlmann T (2003) Conducting the initiation of protein synthesis: the role of eIF4G Biol Cell 95, 141–156 24 Yoshii M, Nishikiori M, Tomita K, Yoshioka N, Kozuka R, Naito S & Ishikawa M (2004) The Arabidopsis cucumovirus multiplication and loci encode translation initiation factors 4e and 4g J Virol 78, 6102–6111 25 Leonard S, Viel C, Beauchemin C, Daigneault N, Fortin MG & Laliberte JF (2004) Interaction of VPg-Pro of Turnip mosaic virus with the translation initiation factor 4e and the poly (A)-binding protein in planta J Gen Virol 85, 1055–1063 26 Browning KS (1996) The plant translational apparatus Plant Mol Biol 32, 107–144 27 Mader S, Lee H, Pause A & Sonenberg N (1995) The translation initiation factor Eif-4e binds to a common motif shared by the translation factor Eif-4 gamma and the translational repressors 4e-binding proteins Mol Cell Biol 15, 4990–4997 28 Marcotrigiano J, Gingras AC, Sonenberg N & Burley SK (1999) Cap-dependent translation initiation in eukaryotes is regulated by a molecular mimic of eIF4G Mol Cell 3, 707–716 29 Haghighat A & Sonenberg N (1997) eIF4G dramatically enhances the binding of eIF4E to the Mrna 5¢-cap structure J Biol Chem 272, 21677–21680 1322 30 Gross JD, Moerke NJ, von der Haar T, Lugovskoy AA, Sachs AB, McCarthy JE & Wagner G (2003) Ribosome loading onto the Mrna cap is driven by conformational coupling between eIF4G and eIF4E Cell 115, 739–750 31 Shukla DD, Ward CW & Brunt AA (1994) The Potyviridae, pp 114 CAB International, Wallingford 32 Aranda M & Maule A (1998) Virus-induced host gene shutoff in animals and plants Virology 243, 261–267 33 Mader S & Sonenberg N (1995) Cap binding complexes and cellular growth control Biochimie 77, 40–44 34 Schaad M, HaldemanCahill R, Cronin S & Carrington J (1996) Analysis of the VPg-proteinase (Nia) encoded by tobacco etch potyvirus: effects of mutations on subcellular transport, proteolytic processing, and genome amplification J Virol 70, 7039–7048 35 Lejbkowicz F, Goyer C, Darveau A, Neron S, Lemieux R & Sonenberg N (1992) A fraction of the Mrna 5¢-cap-binding protein, eukaryotic initiation factor 4e, localizes to the nucleus Proc Natl Acad Sci USA 89, 9612–9616 36 Welch EM, Wang W & Ps (2000) Translation termination: it’s not the end of the story In Translation Control of Gene Expression (Sonenberg N, Hershey JWB & Mathews MB, eds), pp 467–486 Cold Spring Harbor Laboratory Press, New York 37 Thivierge K, Nicaise V, Dufresne PJ, Cotton S, Laliberte J-F, Le Gall O & Fortin MG (2005) Plant virus Rnas Coordinated recruitment of conserved host functions by (+) ssrna viruses during early infection events Plant Physiol 138, 1822–1827 38 Gingras AC, Raught B & Sonenberg N (1999) Eif4 initiation factors: effectors of Mrna recruitment to ribosomes and regulators of translation Annu Rev Biochem 68, 913–963 39 Gallie DR (2001) Cap-independent translation conferred by the 5¢ leader of tobacco etch virus is eukaryotic initiation factor 4g dependent J Virol 75, 12141–12152 40 Barany G & Merrifield RB (1979) In The Peptides (Udenfriend S & Meienhofer J, eds) Academic Press Inc., New York 41 Schagger H, Cramer WA & von Jagow G (1994) Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis Anal Biochem 217, 220–230 42 Carberry SE, Darzynkiewicz E, Stepinski J, Tahara SM, Rhoads RE & Goss DJ (1990) A spectroscopic study of the binding of N-7-substituted cap analogues to human protein synthesis initiation factor 4e Biochemistry 29, 3337–3341 43 Lakowicz JR (1999) Protein fluorescence In Principles of Fluorescence Spectroscopy, 2nd edn, pp 445–473 Plenum, New York 44 Weber G (1992) Protein Interactions, pp 14–18 Chapman & Hall, Inc, London FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS ... Michon et al eIF4G -eIF4E- cap ⇔ eIF4E ⇔ ⇔ VPg eIF4E -VPg ⇔ eIF4E- cap VPg cap -eIF4E -VPg Scheme Hypothetical pathways of eIF4E and eIF4G recruitment Large arrows highlight the connections that, according... mgỈmL)1) was added, and the suspension was incubated for 45 at °C with gentle stirring DNase and RNase (100 lg each) were added and the suspension was incubated at °C for another 45 The lysate was sonicated... sets of data, the fluorescence decrease was normalized to Y ¼ ) (F ⁄ Fmax) At ligand saturation, a maximum for Dfmax is obtained, and we can define the plateau value as Ysat ¼ ) (Fmin ⁄ Fmax) Accordingly