Báo cáo khoa học: The binding of foot-and-mouth disease virus leader proteinase to eIF4GI involves conserved ionic interactions ppt

The binding of foot-and-mouth disease virus leader proteinase to eIF4GI involves conserved ionic interactions Nicole Foeger*, Elisabeth Kuehnel†, Regina Cencic and Tim Skern Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Medical Biochemistry, Medical University of Vienna, Austria The eukaryotic translation initiation factor (eIF) 4F is a protein complex that mediates recruitment of ribo- somes to mRNA [1]. This event is one of the rate-lim- iting steps for translation and thus an important target for translational control. The eIF4F complex consists of several components: eIF4E, a protein recognizing the 5¢ cap structure of the mRNA; the RNA helicase eIF4A; and the bridging protein eIF4G, that brings together mRNA and ribosome via mRNA circulariza- tion [2]. eIF4G is a central part of this complex as it provides binding sites not only for the already men- tioned translation factors eIF4E and eIF4A [3], but also for the ribosome-associated eIF3 [4], the poly(A) binding protein [5] and the eIF4E kinases Mnk 1 [6] and Mnk 2 [7]. Picornavirus infection leads to the so-called host cell shut-off. Virally encoded picornaviral proteases cleave eIF4GI and eIF4GII, thereby leading to an inhibition of cap-dependent cellular protein synthesis [8,9]. Viral translation is unaffected as it initiates via an internal Keywords Foot-and-mouth disease virus; papain-like proteinase; self-processing; exosite; protein synthesis inhibition Correspondence T. Skern, Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Medical Biochemistry, Medical University of Vienna, Dr Bohr-Gasse 9 ⁄ 3, A-1030 Vienna, Austria Fax: +43 14277 9616 Tel: +43 14277 61620 E-mail: timothy.skern@meduniwien.ac.at Website: http://www.meduniwien.ac.at/ medbch Present addresses *Division of Cell Biology and †Division of Tumour Genetics, German Cancer Research Center, Im Neuheimer Feld 280, D-69120 Heidelberg, Germany (Received 22 February 2005, revised 20 March 2005, accepted 24 March 2005) doi:10.1111/j.1742-4658.2005.04689.x The leader proteinase (L pro ) of foot-and-mouth disease virus (FMDV) ini- tially cleaves itself from the polyprotein. Subsequently, L pro cleaves the host proteins eukaryotic initiation factor (eIF) 4GI and 4GII. This prevents protein synthesis from capped cellular mRNAs; the viral RNA is still trans- lated, initiating from an internal ribosome entry site. L pro cleaves eIF4GI between residues G674 and R675. We showed previously, however, that L pro binds to residues 640–669 of eIF4GI. Binding was substantially improved when the eIF4GI fragment contained the eIF4E binding site and eIF4E was present in the binding assay. L pro interacts with eIF4GI via resi- due C133 and residues 183–195 of the C-terminal extension. This binding domain lies about 25 A ˚ from the active site. Here, we examined the binding of L pro to eIF4GI fragments generated by in vitro translation to narrow the binding site down to residues 645–657 of human eIF4GI. Comparison of these amino acids with those in human eIF4GII as well as with sequences of eIF4GI from other organisms allowed us to identify two conserved basic residues (K646 and R650). Mutation of these residues was severely detri- mental to L pro binding. Similarly, comparison of the sequence between resi- dues 183 and 195 of L pro with those of other FMDV serotypes and equine rhinitis A virus showed that acidic residues D184 and E186 were highly conserved. Substitution of these residues in L pro significantly reduced eIF4GI binding and cleavage without affecting self-processing. Thus, FMDV L pro has evolved a domain that specifically recognizes a host cell protein. Abbreviations 2A pro , 2A proteinase; CTE, C-terminal extension; eIF, eukaryotic initiation factor; ERAV, equine rhinitis A virus; ERBV, equine rhinitis B virus; FMDV, foot-and-mouth disease virus; HRV, human rhinovirus; L pro , leader proteinase, containing amino acids 1–201; Lb pro , shorter form of L pro containing amino acids 29–201; RRL, rabbit reticulocyte lysate. 2602 FEBS Journal 272 (2005) 2602–2611 ª 2005 FEBS ribosome entry site (IRES) and therefore is independ- ent of the interaction between eIF4E and eIF4G [10,11]. The picornaviral proteinases such as the leader proteinase (L pro ) of foot-and-mouth-disease virus (FMDV) and equine rhinitis A virus (ERAV) or the 2A proteinase of enteroviruses and human rhinoviruses (HRVs) cleave eIF4GI and eIF4GII so that the eIF4E- binding part of the eIF4G proteins is separated from the bulk of the translation complex [4,12,13]. However, the remainder of the complex is sufficient for viral translation. FMDV L pro is the most N-terminal protein on the viral polyprotein, the primary translation product pro- duced from the viral RNA genome. As a papain-like cysteine proteinase, L pro shows a typical two domain a-helix ⁄ b-sheet fold of a papain proteinase but is unique in bearing a so-called C-terminal extension (CTE) that protrudes from the globular structure [14]. Cleavage of eIF4GI by L pro , both in vivo and in vitro, is highly efficient [15–18]. Nevertheless, the observed L pro proteinase concentration at which eIF4G is cleaved during viral replication is much lower than that required in vitro when purified recombinant pro- teins are employed [16,18,19]. For this reason, it has been proposed that picornaviral proteinases activate cellular proteinases, which cleave eIF4G in an indirect reaction [20,21]. However, such cellular proteinases have as yet not been identified. Ohlmann et al. have previously obtained evidence that the substrate for L pro is the eIF4GI–eIF4E complex [22]. In addition, for the HRV2 2A proteinase (2A pro ), Haghihat et al. [23] demonstrated, using purified recombinant proteins, that the eIF4GI–eIF4E complex was cleaved much more efficiently than eIF4GI alone. Pertinently, it has been shown that yeast eIF4GI undergoes an unfolded- to-folded transition on binding eIF4E [24,25]. This could be a reason why the eIF4GI–eIF4E complex is the preferred substrate for picornaviral proteinases. Recently, we showed that FMDV L pro and HRV2 2A pro indeed bind directly to the eIF4GI–eIF4E com- plex, but much less well to eIF4GI alone [26]. Addi- tionally, we have shown that both of these proteinases interact with their substrate eIF4GI at a site distant from their cleavage site [26,27]. For FMDV L pro ,we minimized the binding site on eIF4GI to amino acids 640–669; in contrast, the enzyme cleaves eIF4GI between residues G674 and R675. Here we show that we can define this region further to the 13 amino acids between residues 645–657. This region in eIF4GI con- tains conserved basic residues, which are here demon- strated to be involved in binding by L pro . The L pro binding domain for eIF4GI is located 25 A ˚ from the active site of the enzyme and comprises C133 as well as residues 183–195 of the CTE. Mutations in these amino acids significantly decrease eIF4GI cleavage by L pro , without affecting L pro processing at the viral polyprotein sequence. Here we emphasize the role of two acidic residues (D184 and E186) in the CTE of FMDV L pro which are responsible for eIF4GI binding and cleavage. Results Recently, we have shown that FMDV Lb pro can bind to its substrate eIF4GI between amino acids 640–669 [26]; binding in the presence of eIF4E to eIF4GI frag- ments containing the eIF4E binding site was more efficient. However, Lb pro cleaves between amino acids G674 and R675. We wished to further define this region between amino acids 640–669 and therefore cloned shorter fragments of eIF4GI, as shown in Fig. 1A. We started with an eIF4GI fragment contain- ing amino acids 260–657; RNA from this construct was translated in vitro in rabbit reticulocyte lysate AB DC Fig. 1. Minimizing the eIF4GI binding domain of Lb pro . (A) eIF4GI fragments used. Sites for eIF4E binding and Lb pro cleavage are indi- cated. (B–D) 35 S-labelled proteins translated in vitro from the indica- ted cDNA fragments of eIF4GI (input lanes 1, corresponding to a quarter of that used in each pull-down) were incubated with GST (lanes 2) or GST-Lb pro C51A (lanes 3). Bound proteins were resolved by SDS ⁄ PAGE and detected by fluorography. All fragments were reproducibly synthesized as doublets, presumably due to initiation of translation at two AUG initiating codons in close proximity. N. Foeger et al. FMDV L proteinase eIF4GI interaction FEBS Journal 272 (2005) 2602–2611 ª 2005 FEBS 2603 (RRL) in the presence of radiolabelled methionine. The labelled protein containing eIF4GI residues 260– 657 was then incubated with the fusion protein GST– Lb pro C51A, which had been expressed in bacteria and purified by binding to glutathione-sepharose beads. The C51A mutation serves to inactivate the enzyme during the pull-down assays. Figure 1B shows that the eIF4GI fragment 260–657 is bound by GST– Lb pro C51A, but not by GST alone. When we used an eIF4GI fragment of amino acids 260–649 (Fig. 1C), binding to Lb pro could still be detected but was clearly weaker in comparison to that containing amino acids 260–657. In contrast, a protein containing amino acids 260–645 (Fig. 1D) was essentially not recognized by the GST–Lb pro C51A fusion protein. These results demonstrate that the 13 amino acids in the region of 645–657 on eIF4GI are important for binding by FMDV Lb pro . Nevertheless, the presence of eIF4E binding sequences on the eIF4G fragment and the presence of eIF4E in the binding assay are required for more efficient Lb pro binding. As a control, we examined the binding of radiola- belled cortactin, a cellular protein that we have shown to be cleaved in vitro by Lb pro but at much slower rates than eIF4GI. The radiolabelled cortactin is not bound by the GST–Lb pro C51A fusion protein (data not shown), indicating that the interaction of the GST–Lb pro C51A complex protein with the eIF4GI is specific and that it is probably responsible for the rapid cleavage of Lb pro observed on eIF4GI. When we examined the 13 amino acids of eIF4GI responsible for binding Lb pro as well as those sur- rounding this sequence more closely, we found that the sequence of eIF4GI comprised three basic residues (K643, K646 and R650), which were conserved between human eIF4GI and eIF4GII (Fig. 2A). Ana- lysis of protein databases revealed that these residues were also present in eIF4GI sequences from several mammalian species such as sheep, cow, hamster, horse, mouse, pig and rabbit (EMBL accession numbers: AJ746218–AJ746224 inclusive). This conservation sug- gested that these basic residues might be recognized by residues in the Lb pro CTE and thus enable the inter- action to take place. To investigate this notion, we individually mutated these three residues to alanine in the plasmid con- taining the eIF4GI deletion 260–657 to give the cor- responding three plasmids shown in Fig. 3A,B. Radiolabelled proteins corresponding to the mutants were expressed in RRLs and their ability to be bound by GST–Lb pro C51A in the pull-down assays was then examined. The results are shown in Fig. 3 and Table 1. The quantitation shows that about 12.5% of the input material is bound by the wild-type fusion protein in this assay. The efficiency of the pull-down is probably limited by the presence of the GST part of the fusion protein, the small size of the radiolabelled fragment and the probability that a certain fraction of both binding partners are incorrectly folded. When we introduced the mutation K643A into the eIF4GI construct comprising residues 260–657, we found that binding by GST–Lb pro C51A was reduced to between 80 and 90% of the wild-type level (Fig. 3A, Table 1). However, the presence of the mutation K646A reduced binding by about 75% when com- pared to the wild type fragment (Fig. 3A, Table 1). Fig. 2. (A) Sequence alignment of human eIF4G proteins. The swissprot entries are I4G1_human (eIF4GI) and I4G3_human (eIF4GII). (B) Comparison of the C-terminal extensions of FMDV Lb pro , ERAV Lb pro and ERBV Lb pro . Asterisks in (A) and (B) indicate conserved basic residues and acidic resi- dues, respectively. Amino acids 183–195 in FMDV Lb pro were shown to be required for eIF4GI binding [26]. FMDV L proteinase eIF4GI interaction N. Foeger et al. 2604 FEBS Journal 272 (2005) 2602–2611 ª 2005 FEBS Binding to the eIF4GI protein bearing the mutation R650A was also reduced, but only by between 50 and 70% (Fig. 3A,B, Table 1). The relatively low involve- ment of K643 for binding was supported by the behaviour of the double mutant K643AK646A (Fig. 3A,D); the binding is similar to that of the single mutant K646A. Finally, no binding to the triple mutant K643AK646AR650A in which all three con- served basic residues were substituted with alanine was observed (Fig. 3E). The above results show that mutation of any of the three conserved basic residues of eIF4GI impairs its interaction with FMDV Lb pro but that the effects of the mutations differ. This implied that conserved acidic residues should be present in Lb pro that represent the interaction partners of the basic eIF4GI residues. We have shown previously that residues 183–195 of the 18 amino acid CTE as well as C133 of Lb pro were involved in the interaction of Lb pro and eIF4GI [28]. Examination of the amino acids 183–195 of the CTE revealed two residues, D184 and E186 (Fig. 2B), which are conserved in all seven serotypes, including those from Africa [29,30]. ERAV L pro is also responsible for cleavage of eIF4GI and eIF4GII [13]. Analysis of the CTE of this enzyme revealed that both residues were also present in its CTE. In contrast, in the CTE of equine rhinitis B virus (ERBV), L pro appears not to cleave eIF4GI [13]. Fittingly, there is a residue equival- ent to E186 in ERBV L pro but not to D184 (Fig. 2B). We thus investigated the role of these amino acids in their interaction with eIF4GI by mutational analysis. Figure 4A shows that mutations in the amino acid sequence of this region of Lb pro can be readily intro- duced by using an oligonucleotide cassette spanning the BsiWI and Bpu10I restriction sites. In this way, we generated the substitutions D184A, E186K and Q185RE186K, and investigated the ability of these Lb pro mutants to carry out self-processing and eIF4GI cleavage when expressed in RRLs. Figure 4B (lanes 1–5) shows that self-processing of wild-type Fig. 3. Conserved basic residues in eIF4GI are essential for Lb pro binding. (A–E) 35 S lab- elled proteins translated in vitro from the indicated cDNA fragments of eIF4GI (input lanes 1, corresponding to a quarter of that used in each pull-down) were incubated with GST (lanes 2) or GST-Lb pro C51A (lanes 3). Bound proteins were resolved by SDS ⁄ PAGE and detected by fluorography. Table 1. Efficiency of binding of mutated eIF4GI fragments to GST–Lb pro C51A. The % bound values are expressed relative to the amount bound by the wild-type, which represents about 12.5% of the total input. Experiment 2 shows the quantitation of Fig. 3. eIF4GI fragment % Bound Experiment 1 Experiment 2 Wild-type 100 100 K643A 90 80 K646A 25 30 R650A 50 30 N. Foeger et al. FMDV L proteinase eIF4GI interaction FEBS Journal 272 (2005) 2602–2611 ª 2005 FEBS 2605 Lb pro VP4VP2 into Lb pro and VP4VP2 takes place between 4 and 8 min after protein synthesis is initiated. Similar kinetics of self-processing were observed with all three mutants tested (Fig. 4B, lanes 6–27). To examine eIF4GI cleavage by the newly synthesized Lb pro , we took advantage of the presence of eIF4GI in the RRLs and examined its fate during the synthesis of Lb pro , as reported previously [15]. Accordingly, aliquots of the translation reactions were subjected to SDS ⁄ PAGE, the gels blotted onto poly(vinylidene difluoride) membranes and probed with an antiserum against the N-terminus of eIF4GI (Fig. 4C, lower pan- els). eIF4GI itself migrates as a series of bands with a molecular mass of 220 kDa [31]. The bands have dif- ferent N-termini, which arise from the use of different AUG codons during synthesis of eIF4GI [32,33]. Clea- vage of eIF4GI by Lb pro at its single recognition site between G674R (numbering according to [32]) gener- ates a series of N-terminal cleavage products that are detected by the N-terminal antiserum used here (cp N , Fig. 4C). Figure 4C (lanes 1–5) shows that 50% of eIF4GI is cleaved after 4 min with wild-type Lb pro . However, the mutation D184A (Fig. 4C, lanes 6–12) showed a delay in the cleavage of eIF4GI, the time-point of 50% eIF4GI occurring only after 12–20 min. Thus, we con- cluded that this residue is involved in eIF4GI recogni- tion. The E186 mutant (lanes 13–19) also showed a similar delay in eIF4GI cleavage, with 50% eIF4GI cleavage being observed only after 12–20 min. To investigate whether only the charges of the residues 184 and 186 were important for cleavage of eIF4GI or whether other residues in this region could exert an influence on the reaction, we constructed the double mutant Lb pro Q185RE186K and investigated its activity (lanes 20–26). Again, eIF4GI cleavage was impaired as 50% cleavage could only be seen after 20–30 min. To verify further that these residues were involved in binding to eIF4GI, we expressed the three mutants as GST-fusion proteins and examined their ability in pull-down assays to bind to eIF4GI. In this case, we used the endogenous eIF4GI present in RRLs as a source of eIF4GI to ensure the best possible binding to the GST–Lb pro complexes. In Fig. 5A, the purity of the expressed GST fusion proteins can be seen; Fig. 5B shows the results of the GST pull-down assays. All mutant proteins showed weaker binding than that found in the wild-type (lane 2); furthermore, the extent of binding correlated with the effect of these mutations on eIF4GI. The mutants GST–Lb pro C51AD184A and Fig. 4. Substitution of residues D184 and E186 in the CTE of FMDV Lb pro affect eIF4GI cleavage. (A) Structure of the expression block of Lb pro VP4VP2 showing the position of restriction sites used to introduce mutations into the CTE of Lb pro . (B) Autoradiograms of proteins syn- thesized from the indicated RNAs. Substituted residues are underlined. Samples were taken at the times indicated and Lb pro self-cleavage from VP4VP2 examined (marked with arrows). (C), Immunoblot with the samples from (B) probed with an anti-eIF4GI antiserum to monitor the eIF4GI cleavage by Lb pro ; intact eIF4GI and the cleavage products cp N are marked. FMDV L proteinase eIF4GI interaction N. Foeger et al. 2606 FEBS Journal 272 (2005) 2602–2611 ª 2005 FEBS GST–Lb pro C51AE186K (lanes 3 and 4, respectively) showed significantly less binding to eIF4GI than GST–Lb pro C51A (lane 2); with the mutant GST– Lb pro C51AQ185RE186K, we observed only very weak binding (lane 5). Similar results were also obtained when the 35 S-labelled eIF4GI fragment from 260 to 657 was employed (data not shown). We showed in Foeger et al. [26] that endogenous eIF4GII in RRLs could also be bound by the GST– Lb pro complexes. We therefore examined the ability of the mutated GST–Lb pro complexes to bind endogenous eIF4GII. Figure 5C (lane 2) confirms that the wild- type GST–Lb pro complex binds the endogenous eIF4- GII. In contrast, binding of the mutant complexes to eIF4GII is greatly reduced or almost undetectable (Fig. 5C, lanes 3–5). Furthermore, the binding of the mutant complexes to endogenous eIF4GII mirrors that to eIF4GI (Fig. 5B,C). Thus, it seems likely that resi- dues D184 and E186 are involved in recognizing both eIF4GI and eF4GII. Taken together, these results strongly suggest that there is a specific interaction between the residues K643, K646 and R650 in eIF4GI, and the amino acids D184 and E186 in the CTE of FMDV Lb pro .Itis worth noting, however, that none of the mutations affected the ability of Lb pro to carry out the self-pro- cessing reaction. Discussion The cleavage by viral proteinases of the host cell pro- teins eIF4GI and eIF4GII is a central event during picornaviral replication. We have shown recently that this cleavage is mediated by regions distant from the active site of two different picornaviral proteinases, namely the papain-like Lb pro of FMDV and the chym- otrypsin-like 2A pro of HRV2 [26,27]. Furthermore, these regions interact on eIF4GI with amino acids that are not identical with the cleavage site of the particular enzyme. In this paper, we define the binding site recognized by the Lb pro of FMDV to the 13 amino acids from residues 645–657 of eIF4GI. This sequence is separated by 17 amino acids from the cleavage site of Lb pro between residues 674 and 675. In contrast, the site on eIF4GI that is bound by HRV2 2A pro lies between resi- dues 600–674 [27], with the C–terminal boundary of this binding region only seven amino acids away from its cleavage site. Furthermore, HRV2 2A pro only binds to eIF4GI when the binding site for eIF4GI is present and eIF4E is included in the binding assay. Although these two parameters increase Lb pro binding, Lb pro is still capable of binding to eIF4GI fragments in their absence. Thus, the two picornaviral proteinases recog- nize different sequences on eIF4GI; given their quite different structures, this is not unexpected. Investigation of the amino acids both within and adjacent to the region of eIF4GI to which FMDV Lb pro binds revealed three basic amino acids which were conserved between human eIF4GI and eIF4GII, as well as between the eIF4GI proteins of other mam- mals. In addition, two acidic residues (D642 and D653) also appeared to be conserved in human eIF4GI and eIF4GII (Fig. 2A). Furthermore, both of these aspartic acid residues are found in the seven animal eIF4GI sequences available in the database. As it seemed possible that conserved charged residues might be involved in the interaction, we examined the Lb pro CTE sequence from amino acids 183–195. These resi- dues had been shown previously to be important in the recognition of eIF4GI by Lb pro [26,28]. In this sequence, we indeed noted a number of acidic and basic residues. Close investigation of the sequences of CTEs from other FMDV serotypes revealed that only residues D184 and E186 were present in all other serotypes, including the more distant South African Fig. 5. Specific mutations in the CTE of Lb pro reduce binding to both eIF4GI and eIF4GII. (A) Coomassie Brilliant Blue staining of purified GST (lane 1) and modified GST-Lb pro C51A fusion proteins. (B and C), 8 lL RRL (input lanes 0, corresponding to 2 lL of RRL) were incubated with GST (lane 1) or the different GST-Lb pro C51A fusion proteins (lanes 2–5). Bound eIF4GI (B) and eIF4GII (C) were detected by immunoblotting using anti-eIF4GI and anti-eIF4GII sera. N. Foeger et al. FMDV L proteinase eIF4GI interaction FEBS Journal 272 (2005) 2602–2611 ª 2005 FEBS 2607 Territories (SAT) serotypes. In addition, comparison with the CTE sequence of ERAV L pro , which has been shown to be responsible for eIF4GI cleavage [13], showed that D184 and E186 were also present in its CTE. In contrast, only E186 was present in the ERBV L pro sequence [34]; however, this enzyme appears not to be responsible for eIF4GI cleavage [13]. Thus, the presence of D184 and E186 in the CTE of all L pro shown to be responsible for cleavage of eIF4G pro- teins suggested to us that these residues might be involved in interacting with the conserved basic resi- dues of eIF4GI. To investigate this, we individually substituted the three conserved basic residues in eIF4GI with alanine residues. Replacement of K646 reduced binding by about 75% whereas replacement of R650 only reduced binding by between 50 and 75%. The replacement of K643 had the least effect, with binding only being reduced by 10–20%. These results strongly suggested an involvement of residues K646 and R650 in the interaction with Lb pro . This encouraged us to investi- gate the role of amino acids D184 and E186 in the cleavage of eIF4GI. The substitution of D184 with alanine or that of E186 with lysine both severely delayed eIF4GI cleavage and led to a concomitant decrease in binding of both eIF4GI and eIF4GII. Interestingly, the introduction of a second basic resi- due (arginine in place of glutamine at 185) delayed cleavage even further, suggesting that the overall charge of this region is important for eIF4GI recogni- tion. Figure 4 shows clearly, however, that eIF4GI clea- vage still occurs when both D184 and E186 are replaced by alanine. One reason for this is the presence of Lb pro residue C133. This residue is not part of the CTE but lies close to it in the three-dimensional struc- ture [14]; we showed previously that replacement of this residue affects both the binding to and cleavage of eIF4GI [26]. However, the results here also do not rule out further interactions between the CTE of Lb pro and the region 645–657 of eIF4GI involving other sequence motifs or hydrophobic interactions not considered here. L188, found in the CTEs of FMDV and ERAV, may be important in this respect. How can mutations in the amino acids 184–186 affect eIF4GI cleavage without affecting self-process- ing? Examination of the structure of the Lb pro (Fig. 6) shows that residues D184 and E186 are at the opposite side of the molecule from the active site and lie about 12 A ˚ from C133, a residue which has also been shown to be important for binding eIF4GI [26]. Furthermore, both residues protrude away from the globular domain of the enzyme and do not appear to interact with any residues in the globular domain or in the CTE. Indeed, they are well positioned to interact with residues from another protein. Thus, it seems that Lb pro , despite being one of the smallest papain-like enzymes, has been able to evolve a site which can significantly accel- erate cleavage of a host cell molecule without reducing the self-processing reaction. Of the two reactions, the eIF4GI cleavage reaction appears to be more sensitive to mutation than the self-processing reaction. This emphasizes the importance of the interaction of Lb pro with the eIF4G proteins for the successful replication of FMDV. In summary, we have defined closely the regions on eIF4GI and Lb pro that enable them to interact with each other. A minimal binding site on eIF4GI between residues 645 and 657 has been identified, although binding is more efficient when eIF4G fragments con- tain the eIF4E binding site and eIF4E is present in the binding assay. Once again, the versatility of viral pro- teins is amply illustrated. Although viral proteins must remain small in order to limit genome size, they are still able to evolve domains away from the canonical active site which can interact with a second substrate and contribute to the efficiency of viral replication. Experimental procedures Reagents The FMDV L pro is the most N-terminal protein on the FMDV polyprotein. L pro frees itself by cleavage between its own C-terminus and the N-terminus of VP4. As the initi- ation of protein synthesis on the FMDV RNA can occur at one of two AUG codons lying 84 nucleotides apart, two forms of L pro (designated Lab pro and Lb pro ) are synthesized in the infected cell. The reason for this is not clear, as both Fig. 6. Arrangement of Lb pro residues involved in recognizing eIF4GI. Stereo diagram of Lb pro (green, a -helices; purple, b-sheets; yellow, coils) showing C133, D184, Q185 and E186 as balls-and- sticks. The catalytic residues C51 (alanine in the crystal structure [14]) and H148 are also shown. The drawing was produced using the program MOLSCRIPT [37,38] and rendered with RASTER3D [39]. The PDB coordinates used for Lb pro were 1QOL, molecule G. FMDV L proteinase eIF4GI interaction N. Foeger et al. 2608 FEBS Journal 272 (2005) 2602–2611 ª 2005 FEBS forms appear to have the same enzymatic properties [35]. All work described here was carried out with the Lb pro form. Plasmid pCITELb pro VP4VP2, which encodes FMDV amino acids 29–364 corresponding to the Lb pro form (29– 201), VP4 (202–286) and part of VP2 (287–364) has been des- cribed previously [28]. Fragments of Lb pro to be expressed as GST fusions were introduced as EcoRI ⁄ XhoI fragments into the plasmid pGEX5X (Amersham Biosciences, Little Chal- font, Buckinghamshire, UK) as required [26]. Fragments of eIF4GI for in vitro translation were amplified from plasmid pSKHC1, which contains the human eIF4GI cDNA from amino acid 197–1600 [36], and cloned as EcoRI ⁄ HincII frag- ments into pBluescriptKS (Stratagene, La Jolla, CA, USA). Mutations were introduced into the cDNAs for Lb pro and eIF4GI using standard PCR mutagenesis except for the amino acid substitutions described in Fig. 4 which were introduced by replacing the 36 bp BsiWI and BpuI0I frag- ment [28] of Lb pro VP4VP2 with the appropriate synthetic oligonucleotides. The following antibodies were used. Rabbit polyclonal antiserum raised against the N-terminus of eIF4GI (kindly provided by R. Rhoads, Shreveport, LA, USA) was diluted 1 : 8000. Rabbit polyclonal antiserum raised against the C-terminus of eIF4GII (kindly provided by N. Sonenberg, Montreal, Quebec, Canada) was diluted 1 : 2000. Secon- dary horse radish peroxidase (HRP)-conjugated antibodies were diluted 1 : 10000 (BioRad, Hercules, CA, USA), and second alkaline peroxidase (AP)-conjugated antibodies were diluted 1 : 5000 (Sigma, St Louis, MO, USA). Purification of GST fusion proteins E. coli JM101 cells were transformed with plasmids enco- ding the GST-Lb pro fusion proteins or GST alone. To express GST-Lb pro , an overnight culture was diluted 1 : 10 in 50 mL medium, isopropyl thio-b-d-galactoside added to a final concentration of 2 mm and the cells incubated at 30 °C for 3 h. The proteins were purified on glutathione- agarose resin (Amersham Biosciences) using standard tech- niques. GST pull-down assays Glutathione-sepharose beads coated with GST fusion pro- teins were incubated in binding buffer (50 mm Tris ⁄ HCl pH 7.4, 10 mm EDTA, 150 mm NaCl) with either an ali- quot (8 lL) of RRL or with radiolabelled in vitro translated proteins for 2 h at 4 °C. The amount of radiolabelled pro- tein was adjusted so that the same amount was added in each set of binding experiments. After three washes with binding buffer, bound proteins were eluted by boiling in SDS ⁄ PAGE loading buffer, resolved by SDS ⁄ PAGE and visualized by western blotting and using the enhanced chemiluminescence system (Pierce, Rockford, IL, USA) for detection, or fluorography. Quantitation of binding of radiolabelled fragments was done with a BioRad Fluor- S TM MultiImager using quantity one 4.4.0 (Basic) soft- ware. In vitro translation In vitro expression of radiolabelled proteins for GST pull- down assays was performed in RRLs (Quick Coupled Transcription ⁄ Translation system; Promega, Madison, WI, USA) in the presence of [ 35 S]methionine (20 lCi per reaction; Hartmann Analytic, Braunschweig, Germany). Labelled proteins were resolved by SDS ⁄ PAGE and gels were dried and exposed to X-ray films. In vitro translations in RRLs (Promega) to examine Lb pro self-processing and eIF4GI cleavage were performed using in vitro transcribed RNAs as described previously [15,28]. Acknowledgements This work was supported by the Austrian Science Foundation (grants P-16189 and P-17988) to T.S. We thank Bob Rhoads and Nahum Sonenberg for reagents. References 1 Gingras AC, Raught B & Sonenberg N (1999) eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Bio- chem 68, 913–963. 2 Morley SJ, Curtis PS & Pain VM (1997) eIF4G: transla- tion’s mystery factor begins to yield its secrets. RNA 3, 1085–1104. 3 Imataka H, Sonenberg N & Olsen HS (1997) Human eukaryotic translation initiation factor 4G (eIF4G) pos- sesses two separate and independent binding sites for eIF4A. Mol Cell Biol 17, 6940–6947. 4 Lamphear BJ, Kirchweger R, Skern T & Rhoads RE (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, 21975–21983. 5 Imataka H, Gradi A & Sonenberg N (1998) A newly identified N-terminal amino acid sequence of human eIF4G binds poly(A)-binding protein and functions in poly(A)-dependent translation. EMBO J 17, 7480–7489. 6 Pyronnet S, Imataka H, Gingras AC, Fukunaga R, Hunter T & Sonenberg N (1999) Human eukaryotic translation initiation factor 4G (eIF4G) recruits mnk1 to phosphorylate eIF4E. EMBO J 18, 270–279. 7 Scheper GC, Morrice NA, Kleijn M & Proud CG (2001) The mitogen-activated protein kinase signal- N. Foeger et al. FMDV L proteinase eIF4GI interaction FEBS Journal 272 (2005) 2602–2611 ª 2005 FEBS 2609 integrating kinase Mnk2 is a eukaryotic initiation factor 4E kinase with high levels of basal activity in mamma- lian cells. Mol Cell Biol 21, 743–754. 8 Kra ¨ usslich HG, Nicklin MJ, Toyoda H, Etchison D & Wimmer E (1987) Poliovirus proteinase 2A induces clea- vage of eucaryotic initiation factor 4F polypeptide p220. J Virol 61, 2711–2718. 9 Gradi A, Svitkin YV, Imataka H & Sonenberg N (1998) Proteolysis of human eukaryotic translation initiation factor eIF4GII, but not eIF4GI, coincides with the shutoff of host protein synthesis after polio- virus infection. Proc Natl Acad Sci USA 95, 11089– 11094. 10 Jackson R, Howell M & Kaminski A (1990) The novel mechanism of initiation of picornavirus RNA transla- tion. Trends Biochem Sci 15, 477–483. 11 Belsham GJ & Jackson RR (2000) Translation initiation on picornavirus RNA. In Translational Control of Gene Expression (Sonenberg, N, Hershey, J W B & Mathews, M B, eds), pp. 869–900. Cold Spring Harbor Press, Cold Spring Harbor, NY. 12 Borman AM, Kirchweger R, Ziegler E, Rhoads RE, Skern T & Kean KM (1997) elF4G and its proteolytic cleavage products: effect on initiation of protein synth- esis from capped, uncapped, and IRES-containing mRNAs. RNA 3, 186–196. 13 Hinton TM, Ross-Smith N, Warner S, Belsham GJ & Crabb BS (2002) Conservation of L and 3C proteinase activities across distantly related aphthoviruses. J Gen- eral Virol 83, 3111–3121. 14 Guarne ´ A, Tormo J, Kirchweger K, Pfistermueller D, Fita I & Skern T (1998) Structure of the foot-and- mouth disease virus leader protease: a papain-like fold adapted for self-processing and eIF4G recognition. EMBO J 17, 7469–7479. 15 Glaser W & Skern T (2000) Extremely efficient cleavage of eIF4G by picornaviral proteinases L and 2A in vitro. FEBS Lett 480, 151–155. 16 Kirchweger R, Ziegler E, Lamphear BJ, Waters D, Lie- big HD, Sommergruber W, Sobrino F, Hohenadl C, Blaas D, Rhoads RE & Skern T (1994) Foot-and-mouth disease virus leader proteinase: Purification of the Lb form and determination of its cleavage site on eIF-4 gamma. J Virol 68, 5677–5684. 17 Thomas AAM, Scheper GC, Kleijn M, Deboer M & Voorma HO (1992) Dependence of the adenovirus tri- partite leader on the p220 subunit of eukaryotic initia- tion factor-4F during in vitro translation – effect of p220 cleavage by foot-and-mouth-disease-virus L-pro- tease on in vitro translation. Eur J Biochem 207, 471–477. 18 Belsham GJ, McInerney GM & Ross-Smith N (2000) Foot-and-mouth disease virus 3C protease induces clea- vage of translation initiation factors eIF4A and eIF4G within infected cells. J Virol 74, 272–280. 19 Bovee ML, Lamphear BJ, Rhoads RE & Lloyd RE (1998) Direct cleavage of elF4G by poliovirus 2A pro- tease is inefficient in vitro. Virology 245, 241–249. 20 Lloyd RE, Toyoda H, Etchison D, Wimmer E & Ehren- feld E (1986) Cleavage of the cap binding protein com- plex polypeptide p220 is not effected by the second poliovirus protease 2A. Virology 150, 299–303. 21 Wyckoff EE, Lloyd RE & Ehrenfeld E (1992) Relation- ship of eukaryotic initiation factor 3 to poliovirus- induced p220 cleavage activity. J Virol 66, 2943–2951. 22 Ohlmann T, Pain VM, Wood W, Rau M & Morley SJ (1997) The proteolytic cleavage of eukaryotic initiation factor (eIF) 4G is prevented by eIF4E binding protein (PHAS-I; 4E-BP1) in the reticulocyte lysate. EMBO J 16, 844–855. 23 Haghighat A, Svitkin Y, Novoa I, Kuechler E, Skern T & Sonenberg N (1996) The eIF4G-eIF4E complex is the target for direct cleavage by the rhinovirus 2A protei- nase. J Virol 70, 8444–8450. 24 Hershey PE, McWhirter SM, Gross JD, Wagner G, Alber T & Sachs AB (1999) The Cap-binding protein eIF4E promotes folding of a functional domain of yeast translation initiation factor eIF4G1. J Biol Chem 274, 21297–21304. 25 Gross JD, Moerke NJ, von der Haar T, Lugovskoy AA, Sachs AB, McCarthy JE & Wagner G (2003) Ribo- some loading onto the mRNA cap is driven by confor- mational coupling between eIF4G and eIF4E. Cell 115, 739–750. 26 Foeger N, Glaser W & Skern T (2002) Recognition of eukaryotic initiation factor 4G isoforms by picornaviral proteinases. J Biol Chem 277, 44300–44309. 27 Foeger N, Schmid EM & Skern T (2003) Human rhino- virus 2, 2A pro recognition of eukaryotic initiation factor 4GI: Involvement of an exosite. J Biol Chem 278, 33200–33207. 28 Glaser W, Cencic R & Skern T (2001) Foot-and-mouth disease Leader proteinase: involvement of C-terminal residues in self-processing and cleavage of eIF4GI. J Biol Chem 276, 35473–35481. 29 George M, Venkataramanan R, Gurumurthy CB & Hemadri D (2001) The non-structural leader protein gene of foot-and-mouth disease virus is highly variable between serotypes. Virus Genes 22, 271–278. 30 van Rensburg H, Haydon D, Joubert F, Bastos A, Heath L & Nel L (2002) Genetic heterogeneity in the foot-and-mouth disease virus Leader and 3C protei- nases. Gene 289, 19–29. 31 Etchison D, Milburn SC, Edery I, Sonenberg N & Hershey JWB (1982) Inhibition of HeLa cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide asso- ciated with eucaryotic initiation factor 3 and a cap binding protein complex. J Biol Chem 257, 14806– 14810. FMDV L proteinase eIF4GI interaction N. Foeger et al. 2610 FEBS Journal 272 (2005) 2602–2611 ª 2005 FEBS 32 Byrd MP, Zamora M & Lloyd RE (2002) Generation of multiple isoforms of eukaryotic translation initiation factor 4GI by use of alternate translation initiation codons. Mol Cell Biol 22, 4499–4511. 33 Bradley CA, Padovan JC, Thompson TL, Benoit CA, Chait BT & Rhoads RE (2002) Mass spectrometric ana- lysis of the N terminus of translational initiation factor eIF4G-1 reveals novel isoforms. J Biol Chem 277, 12559–12571. 34 Skern T, Fita I & Guarne A (1998) A structural model of picornavirus leader proteinases based on papain and bleomycin hydrolase. J General Virol 79, 301–307. 35 Cao X, Bergmann IE, Fullkrug R & Beck E (1995) Functional analysis of the two alternative translation initiation sites of foot-and-mouth disease virus. J Virol 69, 560–563. 36 Yan RQ, Rychlik W, Etchison D & Rhoads RE (1992) Amino acid sequence of the human protein synthesis initiation factor-eIF-4gamma. J Biol Chem 267, 23226– 23231. 37 Kraulis PJ (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr 24 , 946–950. 38 Esnouf R (1997) An extensively modified version of Molscript that includes greatly enhanced colouring capacities. J Mol Graphics 15, 133–138. 39 Merrit E & Murphy M (1994) Raster3d, Version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr D50, 869–873. N. Foeger et al. FMDV L proteinase eIF4GI interaction FEBS Journal 272 (2005) 2602–2611 ª 2005 FEBS 2611 . The binding of foot-and-mouth disease virus leader proteinase to eIF4GI involves conserved ionic interactions Nicole Foeger*,. by the presence of the GST part of the fusion protein, the small size of the radiolabelled fragment and the probability that a certain fraction of both binding