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• W2023671437 abstract "The leader proteinase (Lpro) of foot-and-mouth disease virus frees itself from the nascent polyprotein, cleaving between its own C terminus and the N terminus of VP4 at the sequence Lys-Leu-Lys-↓-Gly-Ala-Gly. Subsequently, the Lpro impairs protein synthesis from capped mRNAs in the infected cell by processing a host protein, eukaryotic initiation factor 4GI, at the sequence Asn-Leu-Gly-↓-Arg-Thr-Thr. A rabbit reticulocyte lysate system was used to examine the substrate specificity of Lpro and the relationship of the two cleavage reactions. We show that Lpro requires a basic residue at one side of the scissile bond to carry out efficient self-processing. This reaction is abrogated when leucine and lysine prior to the cleavage site are substituted by serine and glutamine, respectively. However, the cleavage of eIF4GI is unaffected by the inhibition of self-processing. Removal of the 18-amino acid C-terminal extension of Lpro slowed eIF4GI cleavage; replacement of the C-terminal extension by unrelated amino acid sequences further delayed this cleavage. Surprisingly, wild-type Lpro and the C-terminal variants all processed the polyprotein cleavage site in an intermolecular reaction at the same rate. However, when the polyprotein cleavage site was part of the same polypeptide chain as the wild-type Lbpro, the rate of processing was much more rapid. These experiments strongly suggest that self-processing is an intramolecular reaction. The leader proteinase (Lpro) of foot-and-mouth disease virus frees itself from the nascent polyprotein, cleaving between its own C terminus and the N terminus of VP4 at the sequence Lys-Leu-Lys-↓-Gly-Ala-Gly. Subsequently, the Lpro impairs protein synthesis from capped mRNAs in the infected cell by processing a host protein, eukaryotic initiation factor 4GI, at the sequence Asn-Leu-Gly-↓-Arg-Thr-Thr. A rabbit reticulocyte lysate system was used to examine the substrate specificity of Lpro and the relationship of the two cleavage reactions. We show that Lpro requires a basic residue at one side of the scissile bond to carry out efficient self-processing. This reaction is abrogated when leucine and lysine prior to the cleavage site are substituted by serine and glutamine, respectively. However, the cleavage of eIF4GI is unaffected by the inhibition of self-processing. Removal of the 18-amino acid C-terminal extension of Lpro slowed eIF4GI cleavage; replacement of the C-terminal extension by unrelated amino acid sequences further delayed this cleavage. Surprisingly, wild-type Lpro and the C-terminal variants all processed the polyprotein cleavage site in an intermolecular reaction at the same rate. However, when the polyprotein cleavage site was part of the same polypeptide chain as the wild-type Lbpro, the rate of processing was much more rapid. These experiments strongly suggest that self-processing is an intramolecular reaction. eukaryotic initiation factor C-terminal extension foot-and-mouth disease virus internal ribosome entry site leader proteinase polyacrylamide gel electrophoresis rabbit reticulocyte lysate polymerase chain reaction Specific proteolysis is an essential component of the life cycle of many viruses. Virally encoded proteinases are required not only to process viral protein precursors into the mature proteins but also to cleave host proteins to modulate the physiology of the infected cell (1Dougherty W.G. Semler B.L. Microbiol. Rev. 1993; 57: 781-822Crossref PubMed Google Scholar, 2Krausslich H.G. Wimmer E. Annu. Rev. Biochem. 1988; 57: 701-754Crossref PubMed Scopus (424) Google Scholar). A classic example is the inhibition of host protein synthesis from capped cellular mRNA that occurs during the replication of most picornaviruses (3Rueckert R. Fields B. Nkipe D. Howley P. Fields Virology. 1. Lippincott-Raven, Philadelphia1996: 609-654Google Scholar). These viruses mediate this through the cleavage of the two homologues of eukaryotic initiation factor 4G (eIF4GI1 and eIF4GII) by either the Lpro of FMDV or by the 2A proteinase of human rhinoviruses, Coxsackie virus, and polioviruses (4Devaney M.A. Vakharia V.N. Lloyd R.E. Ehrenfeld E. Grubman M.J. J. Virol. 1988; 62: 4407-4409Crossref PubMed Google Scholar, 5Kräusslich H.G. Nicklin M.J. Toyoda H. Etchison D. Wimmer E. J. Virol. 1987; 61: 2711-2718Crossref PubMed Google Scholar, 6Lloyd R.E. Grubman M.J. Ehrenfeld E. J. Virol. 1988; 62: 4216-4223Crossref PubMed Google Scholar, 7Svitkin Y.V. Gradi A. Imataka H. Morino S. Sonenberg N. J. Virol. 1999; 73: 3467-3472Crossref PubMed Google Scholar, 8Gradi A. Svitkin Y.V. Imataka H. Sonenberg N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11089-11094Crossref PubMed Scopus (271) Google Scholar). As a consequence of the cleavage of the eIF4G homologues, the domain of eIF4G that binds the cap-binding protein eIF4E is severed from the domain of eIF4G which binds eIF3, so that the infected cell is unable to recruit its own capped mRNA to the 40 S ribosome (Fig.1 A (9Lamphear B.J. Kirchweger R. Skern T. Rhoads R.E. J. Biol. Chem. 1995; 270: 21975-21983Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 10Mader S. Lee H. Pause A. Sonenberg N. Mol. Cell. Biol. 1995; 15: 4900-4997Crossref Google Scholar)). Translation of viral mRNA is unaffected as it initiates internally via an IRES (11Belsham G.J. Sonenberg N. Microbiol. Rev. 1996; 60: 499-511Crossref PubMed Google Scholar, 12Jackson R. Howell M. Kaminski A. Trends Biochem. Sci. 1990; 15: 477-483Abstract Full Text PDF PubMed Scopus (280) Google Scholar). The exact mechanism of the cleavage of eIF4GI and eIF4GII by the picornaviral proteinases is still unresolved. As this cleavage occurs before measurable amounts of viral proteins can be detected in vivo (13Kirchweger R. Ziegler E. Lamphear B.J. Waters D. Liebig H.D. Sommergruber W. Sobrino F. Hohenadl C. Blaas D. Rhoads R.E. Skern T. J. Virol. 1994; 68: 5677-5684Crossref PubMed Google Scholar), it has been proposed that the viral proteinases induce cleavage of the eIF4G homologues by activating an as yet unidentified cellular proteinase (5Kräusslich H.G. Nicklin M.J. Toyoda H. Etchison D. Wimmer E. J. Virol. 1987; 61: 2711-2718Crossref PubMed Google Scholar, 14Wyckoff E.E. Lloyd R.E. Ehrenfeld E. J. Virol. 1992; 66: 2943-2951Crossref PubMed Google Scholar). This idea was supported by the inability to imitate in vitro the efficiency of eIF4GI cleavage using purified recombinant proteinases (15Bovee M.L. Marissen W.E. Zamora M. Lloyd R.E. Virology. 1998; 245: 229-240Crossref PubMed Scopus (44) Google Scholar, 16Bovee M.L. Lamphear B.J. Rhoads R.E. Lloyd R.E. Virology. 1998; 245: 241-249Crossref PubMed Scopus (42) Google Scholar, 17Belsham G.J. McInerney G.M. Ross Smith N. J. Virol. 2000; 74: 272-280Crossref PubMed Scopus (149) Google Scholar). However, we have recently shown that eIF4GI can be efficiently cleaved in RRLs by nascently translated proteinases at concentrations close to those calculatedin vivo (18Glaser W. Skern T. FEBS Lett. 2000; 480: 151-155Crossref PubMed Scopus (45) Google Scholar). The Lpro of FMDV, which carries out the cleavage of the eIF4G homologues, is a papain-like cysteine proteinase (19Gorbalenya A.E. Koonin E.V. Lai M.M. FEBS Lett. 1991; 288: 201-205Crossref PubMed Scopus (275) Google Scholar, 20Guarné A. Tormo J. Kirchweger K. Pfistermueller D. Fita I. Skern T. EMBO J. 1998; 17: 7469-7479Crossref PubMed Scopus (117) Google Scholar); it is the first protein encoded on the FMDV polyprotein (Fig. 1, Band C). Lpro frees itself by cleavage between its own C terminus and the N terminus of VP4. As the initiation of protein synthesis on the FMDV RNA can occur at one of two AUG codons lying 84 nucleotides apart, two forms of the Lpro are synthesized (designated Labpro and Lbpro). The reason for this is not clear, as both forms appear to have the same enzymatic properties (21Medina M. Domingo E. Brangwyn J.K. Belsham G.J. Virology. 1993; 194: 355-359Crossref PubMed Scopus (126) Google Scholar). All work described here was carried out with the Lbpro form. Recognition and cleavage of the sites on the polyprotein and on the eIF4G homologues present a number of questions. Cleavage at the junction of Lbpro and VP4 occurs at Lys-Leu-Lys201-↓-Gly202-Ala-Gly, whereas that on eIF4GI takes place at the sequence Asn-Leu-Gly634-↓-Arg635-Thr-Thr (13Kirchweger R. Ziegler E. Lamphear B.J. Waters D. Liebig H.D. Sommergruber W. Sobrino F. Hohenadl C. Blaas D. Rhoads R.E. Skern T. J. Virol. 1994; 68: 5677-5684Crossref PubMed Google Scholar). The sequence of Lbpro cleavage on eIF4GII has not yet been determined, but it would appear by comparison to be between Asn-Phe-Gly685-↓-Arg686-Gln-Thr (22Gradi A. Imataka H. Svitkin Y.V. Rom E. Raught B. Morino S. Sonenberg N. Mol. Cell. Biol. 1998; 18: 334-342Crossref PubMed Scopus (247) Google Scholar). To achieve this unusual specificity, the Lbpro has evolved to be able to bind basic residues at P1 or P1′, while maintaining the requirement of papain-like enzymes for a hydrophobic residue at P2 (20Guarné A. Tormo J. Kirchweger K. Pfistermueller D. Fita I. Skern T. EMBO J. 1998; 17: 7469-7479Crossref PubMed Scopus (117) Google Scholar,23Guarné A. Hampoelz B. Glaser W. Carpena X. Tormo J. Fita I. Skern T. J. Mol. Biol. 2000; 302: 1227-1240Crossref PubMed Scopus (55) Google Scholar). Nevertheless, the exact determinants of specificity are still not clear. A further open question is the mechanism of self-processing (20Guarné A. Tormo J. Kirchweger K. Pfistermueller D. Fita I. Skern T. EMBO J. 1998; 17: 7469-7479Crossref PubMed Scopus (117) Google Scholar, 21Medina M. Domingo E. Brangwyn J.K. Belsham G.J. Virology. 1993; 194: 355-359Crossref PubMed Scopus (126) Google Scholar,24Belsham G.J. Brangwyn J.K. Ryan M.D. Abrams C.C. King A.M.Q. Virology. 1990; 176: 524-530Crossref PubMed Scopus (24) Google Scholar). X-ray structural data provided evidence for an intermolecular mechanism, as the CTE of one Lbpro molecule was bound by the active site of the neighboring molecule (20Guarné A. Tormo J. Kirchweger K. Pfistermueller D. Fita I. Skern T. EMBO J. 1998; 17: 7469-7479Crossref PubMed Scopus (117) Google Scholar). However, modeling studies using this structure suggested that a CTE would be able to reach into the active site of the same molecule, hinting that an intramolecular reaction was possible. Finally, it is not known whether self-processing is a prerequisite for cleavage of the eIF4G homologues. In this paper, we examine these questions by expressing various mutants of Lbpro in RRLs and investigating the kinetics of cleavage of the polyprotein and eIF4GI. The plasmid pet8cFMDV Lbpro (encoding the mature Lbpro that consists of amino acids 29–201 of the FMDV polyprotein) contains the FMDV nucleotides 892–1413 of the FMDV O1k cDNA (25Forss S. Strebel K. Beck E. Schaller H. Nucleic Acids Res. 1984; 12: 6587-6601Crossref PubMed Scopus (221) Google Scholar) followed by two stop codons, cloned into the NcoI and BamHI restriction sites of the T7 polymerase expression vector pET8c (Novagen). The plasmid pet8cFMDV LbproVP4VP2 (encoding the mature Lbpro, all 85 amino acids of VP4 and 78 amino acids of VP2) contains the FMDV nucleotides 892–1896 followed by two stop codons similarly cloned into pet8c (13Kirchweger R. Ziegler E. Lamphear B.J. Waters D. Liebig H.D. Sommergruber W. Sobrino F. Hohenadl C. Blaas D. Rhoads R.E. Skern T. J. Virol. 1994; 68: 5677-5684Crossref PubMed Google Scholar). In pet8cFMDV LbproC51A VP4VP2, the active site cysteine 51 is replaced by an alanine. The plasmid pet8c ΔLbproVP was constructed by using PCR amplification to delete the amino acids 30–75 of Lbpro in pet8cFMDV LbproVP4VP2 to give pet8cFMDV ΔLbproVP4VP2. To introduce a fragment extending the number of VP2 amino acids encoded to 140, the required fragment was amplified from the original FMDV cDNA subclone p735 (25Forss S. Strebel K. Beck E. Schaller H. Nucleic Acids Res. 1984; 12: 6587-6601Crossref PubMed Scopus (221) Google Scholar). Two stop codons followed by a BamHI site were also encoded by the 3′ PCR primer. This sequence was then introduced into pet8cFMDV ΔLbproVP4VP2 as an RsrII BamHI fragment to give pet8c ΔLbproVP. Following linearization with BamHI, transcription of this DNA gives an RNA encoding an inactive Lpro lacking 46 amino acids at the N terminus followed by all 85 amino acids of VP4 and 140 amino acids of VP2. To construct pCITE LbproVP4VP2, the LbproVP4VP2 expression block was excised from pet8cFMDV LbproVP4VP2 using the XbaI site of pET8c and the BamHI site at the 3′ end of the expression block and subcloned into pBluescript. The LbproVP4VP2 was then introduced as an NcoIPstI fragment into pCITE1 which had been cleaved byNcoI and PstI. In this construction, translation initiates at an AUG codon three codons upstream from the first Lbpro codon. On linearization with SalI and transcription with T7 polymerase, an RNA is produced containing the encephalomyocarditis virus IRES which subsequently encodes the Lbpro with three extra amino acids at the N terminus, all 85 amino acids of VP4 and 23 amino acids of VP2. Restriction sites forBpu10I and SacI were introduced into pCITE LbproVP4VP2 at the positions indicated in Fig.2 A using standard methods of PCR mutagenesis; the mutations do not change the amino acid sequence. Replacement of the 38-base pair Bpu10I SacI fragment with synthetic oligonucleotides enables the amino acid substitutions indicated in Table I to be introduced at the Lpro VP4 junction. Lbpro C-terminal variants were generated in pet8cLbpro by replacing the 47-base pair C-terminal BsiWI BamHI fragment with the required synthetic oligonucleotides as described previously (23Guarné A. Hampoelz B. Glaser W. Carpena X. Tormo J. Fita I. Skern T. J. Mol. Biol. 2000; 302: 1227-1240Crossref PubMed Scopus (55) Google Scholar). All mutations were confirmed by sequencing.Table IEffect of amino acid substitutions at the Lbpro VP4 junction on self-processingSelf-processing at 12 min%LbproVP4VP2VQRKLK201 202GAGQS85.5LbproK201QVQRKLQ201 202GAGQS37.5LbproL200S,K201QVQRKSQ201 202GAGQS0LbproK201GVQRKLG201 202GAGQS38LbproK201G,G202RVQRKLG201 202 RAGQS82.5Substituted amino acids are shown in bold. % self-processing was determined as the amount of Lbpro as a percentage of the total amount of Lbpro and LbproVP4VP2 synthesized. Open table in a new tab Substituted amino acids are shown in bold. % self-processing was determined as the amount of Lbpro as a percentage of the total amount of Lbpro and LbproVP4VP2 synthesized. Plasmids were linearized with the indicated restriction enzyme and transcribed as described (18Glaser W. Skern T. FEBS Lett. 2000; 480: 151-155Crossref PubMed Scopus (45) Google Scholar). In vitro translation reactions (typically 50 μl) contained 70% RRL (Promega), 20 μCi of [35S]methionine (1000 Ci/mmol, American Research Company), and amino acids (except methionine) at 20 μm. After preincubation for 2 min at 30 °C, translation was started by addition of RNA. RNA concentrations (typically about 10 ng/μl) were adjusted so that the Lbpro concentration reached between 15 and 20 pg/μl after 8 min of incubation, unless otherwise stated. Aliquots (10 μl) were removed at the designated time points, and the reaction was stopped by immediate transfer to ice and the addition of unlabeled methionine and cysteine to a final concentration of 2 mm and Laemmli sample buffer. The PAGE system of Dasso and Jackson (26Dasso M.C. Jackson R.J. Nucleic Acids Res. 1989; 17: 6485-6497Crossref PubMed Scopus (49) Google Scholar) was used for separation of translation products (gels contained 15% acrylamide) and for monitoring the state of eIF4GI (gels contained 6% acrylamide). Translation products were detected by fluorography; the state of eIF4GI was determined by immunoblotting using the anti-eIF4GI peptide 7 antiserum (27Yan R.Q. Rychlik W. Etchison D. Rhoads R.E. J. Biol. Chem. 1992; 267: 23226-23231Abstract Full Text PDF PubMed Google Scholar) as described (18Glaser W. Skern T. FEBS Lett. 2000; 480: 151-155Crossref PubMed Scopus (45) Google Scholar). Quantification of protein synthesis using an Instant Imager (Canberra Packard) was as described previously, except that calculations were adjusted for the use of [35S]methionine alone (18Glaser W. Skern T. FEBS Lett. 2000; 480: 151-155Crossref PubMed Scopus (45) Google Scholar). Briefly, radioactivity in a particular band in the dried polyacrylamide gel was counted. By taking into account the counting efficiency (0.8%), the specific activity of the methionine in the assay (163 dpm/fmol), the number of methionines in Lbpro, and the molecular weight of Lbpro (19.8 kDa), the amount of protein present in a particular band can be estimated. To investigate whether self-processing of the Lbpro from the growing polypeptide chain was a prerequisite for the cleavage of eIF4GI, it was necessary to produce mutant enzymes that could not process themselves from the growing polypeptide chain but that still possessed an intact active site and a correctly folded structure. Therefore, we introduced specific mutations into the cleavage site between Lbproand VP4VP2. To simplify this, we noted that restriction sites forBpu10I and SacI could be introduced before and after the site of Lbpro cleavage without affecting the amino acid sequence (Fig. 2 A). However, the previously employed transcription vector pet8c also contained a Bpu10I site, making it necessary to move the LbproVP4VP2 expression cassette into pCITE1 which has itself no restriction sites for Bpu10I and SacI. The ensuing plasmid was designated pCITE LbproVP4VP2 (Fig. 2 A). To examine Lbpro self-processing and eIF4GI cleavage using pCITE LbproVP4VP2, a standard translation reaction was performed (Fig. 2, B and C, left panels). The cleavage products Lbpro and VP4VP2 are visible on this fluorogram after 8 min. Lbpro encoded by pCITE LbproVP4VP2 has four methionines, whereas VP4VP2 has only two; thus, the intensity of the Lbpro band is about twice that of VP4VP2. The high efficiency of self-processing is indicated by the low amount of the uncleaved precursor (LbproVP4VP2). Table I shows the extent of self-processing at 12 min. The cleavage of the endogenous eIF4GI in the RRL under these conditions occurs rapidly (Fig. 2 C, left panel); 50% cleavage was observed between 4 and 8 min. The concentration of Lbpro at 8 min was determined to be 15 pg/μl by counting the band in the Instant Imager. These results are almost identical to those previously reported, using pet8c LbproVP4VP2 driven from an uncapped RNA that did not contain an IRES (18Glaser W. Skern T. FEBS Lett. 2000; 480: 151-155Crossref PubMed Scopus (45) Google Scholar). The mutations indicated in Table I were then introduced at the Lbpro VP4 cleavage site using synthetic oligonucleotides. First, the P1 lysine was replaced by glutamine (LbproVP4VP2(K201Q)), as the Lbprothree-dimensional structure (20Guarné A. Tormo J. Kirchweger K. Pfistermueller D. Fita I. Skern T. EMBO J. 1998; 17: 7469-7479Crossref PubMed Scopus (117) Google Scholar) had revealed that the P1 lysine residue is bound within a deep groove consisting of three glutamate residues. Their aliphatic side chains bind the lysine side chain, whereas the amino group of the lysine interacts with the carboxyl groups of the glutamates. We reasoned that the uncharged glutamine residue would lack the electrostatic interaction and that the shorter side chain would further cause the amide group to clash with the glutamate side chains. Indeed, the pattern of proteins synthesized from this RNA is different from that of the wild-type RNA (Fig. 2 B, middle panel). After 12 min, self-processing drops from over 85% in the wild-type to 37.5% in the mutant protein (Table I). Thus, although the self-processing reaction of Lbpro is impaired by this mutation, it is not completely inhibited. The activity of this mutant protein on the cleavage of endogenous eIF4GI in the RRL was then examined by immunoblotting (Fig. 2 C, middle panel). In contrast to the effect on self-processing, no effect on the processing of eIF4GI was observed. Reduction of the amount of RNA in the RRL also failed to reveal any differences in the rate of eIF4GI processing between the wild-type and the mutant protein (data not shown). As the K201Q mutant protein still possessed some self-processing activity, we wished to examine whether a complete inhibition of self-processing would eliminate the mature Lbpro from the reaction and thus affect the kinetics of eIF4GI processing. Therefore, we substituted the P2 leucine with serine in addition to the P1 lysine to glutamine substitution; the encoded mutant protein was designated LbproVP4VP2(L200S,K201Q). As both the Lbpro and eIF4GI cleavage sites have leucine at the P2 position (13Kirchweger R. Ziegler E. Lamphear B.J. Waters D. Liebig H.D. Sommergruber W. Sobrino F. Hohenadl C. Blaas D. Rhoads R.E. Skern T. J. Virol. 1994; 68: 5677-5684Crossref PubMed Google Scholar) and the Lbpro structure shows a deep hydrophobic pocket for the binding of the P2 residue, we reasoned that a P2 serine residue would not be accepted and that its presence in addition to the P1 mutation would inhibit any self-processing activity. Fig. 2 B(right panel) shows that this is indeed the case. After 12 min, only the uncleaved product was visible. Remarkably, however, no effect on the cleavage of eIF4GI was observed (Fig. 2 C, right panel); the kinetics of cleavage are almost identical to those of the wild-type enzyme. The above changes in the amino acid sequence at the cleavage junction clearly affected the self-processing reaction but not the cleavage of eIF4GI by Lbpro. To produce a second mutant protein that was handicapped in self-processing, we chose to replace the P1 lysine with glycine (LbproVP4VP2(K201G); Table I) and then examined the effect on eIF4GI cleavage. Fig.3 A (compare leftand center panels) shows that the absence of a basic group before the scissile bond reduced self-processing to about the level seen with the K201Q mutant protein (Table I). Once again, however, no reduction in the activity of the Lbpro on eIF4GI was observed (Fig. 3 B, left and center panels). Finally, to confirm the hypothesis that the presence of a basic residue at either P1 or P1′ was sufficient to allow cleavage by the Lbpro, we used the oligonucleotide cassette to introduce an arginine residue into the K201G mutant protein at the P1′ position (i.e. to replace residue glycine 1 of VP4 with arginine) to give the mutant protein LbproVP4VP2(K201G, G202R) (Table I). As can be seen in Fig. 3 A (right panel), the presence of P1′ arginine in the K201G,G202R mutant protein restored the cleavage to almost wild-type levels (compare Fig.3 A, left and right panels and Table I). Once again, no effect on the cleavage of eIF4GI could be observed (Fig.3 B, right panel). It might be argued that the enzyme is recognizing the newly introduced Arg202 as the P1 residue, with cleavage occurring between Arg202 and Ala203. However, we believe this to be unlikely as this would imply that Leu200 would be the P3 residue and Gly201 the P2 residue. As the three-dimensional structure of Lbpro shows that a basic residue is preferred at P3 and a hydrophobic residue at P2, it is improbable that leucine and glycine could provide sufficient binding energy at these positions. In summary, these results support the predictions of the three-dimensional structure about the substrate specificity of Lbpro and demonstrate clearly that Lbpro which is still covalently connected to the capsid proteins can efficiently cleave eIF4GI. Thus, self-processing of the Lbpro is not a prerequisite for the efficient cleavage of eIF4GI. The above experiments confirmed the high efficiency of eIF4GI cleavage in RRLs by Lbpro, regardless of whether self-processing had occurred or not. What are the features of the Lbpro that enable it to cleave eIF4GI so efficiently? One unique feature of Lbpro, which is not found in papain, is the presence of the 18-amino acid CTE in Lbpro. We wished to examine whether this feature was involved in some way in eIF4GI cleavage. To investigate this, we used a second construction pet8cLbpro that contains a synthetic stop codon after the Lys201 residue, so that a mature Lbpro is expressed without the need for self-processing (Fig.4 A). The kinetics of eIF4GI cleavage are the same as those with pet8c LbproVP4VP2 (18Glaser W. Skern T. FEBS Lett. 2000; 480: 151-155Crossref PubMed Scopus (45) Google Scholar). C-terminal deletions were introduced into pet8cLbpro by replacing the wild-type BsiWI BamHI fragment with synthetic oligonucleotides. Initially, two deletions in Lbpro were made; the first (designated Lbpro-6; Table II) lacked the six most C-terminal amino acids, i.e. just those which had been found in the active site of the neighboring molecule in the crystal structure. In the second (Lbpro-18), the entire 18 amino acids of the CTE were removed. RNAs were prepared from both deletion mutants and were used to drive protein synthesis in RRLs; synthesis of Lbpro (Fig. 4 B) and cleavage of eIF4GI (Fig. 4 C) were analyzed as before. The mutant protein Lbpro-6 cleaved eIF4GI with kinetics similar to that of the wild-type enzyme (Fig. 4, B and C, left andmiddle panels); however, the mutant protein Lbpro-18 had a reduced ability to cleave eIF4GI, cleavage was still incomplete after 12 min (Fig. 4, B andC, left and right panels). This was despite the fact that about twice as much Lbpro-18 had been synthesized than in the wild-type reaction.Table IIAmino acid sequence of the wild-type Lbpro, CTE, and deletion mutantsCTELbpro179VFVPYDQEPL NGEWKAKVQR KLK201Lbpro− 6179VFVPYDQEPL NGEWKAK195Lbpro− 18179VFVPY183Lbpro− 11*179VFVPFNASSN DK191Lbpro+ 9*179VFVPFNASSN DKDPAANKAR KEAELAAATA EQ210Underlined sequences are identical. The start of the CTE in the wild-type sequence is at Tyr183. Open table in a new tab Underlined sequences are identical. The start of the CTE in the wild-type sequence is at Tyr183. Fig. 4 B shows that the deletion Lbpro-6 migrates more slowly than the wild-type Lbpro. This is reminiscent of an observation of Sangar et al. (28Sangar D.V. Clark R.P. Carroll A.R. Rowlands D.J. Clarke B.E. J. Gen. Virol. 1988; 69: 2327-2333Crossref PubMed Scopus (10) Google Scholar) who demonstrated that prolonged incubation (i.e. longer than 1 h) of Lbpro in RRLs led to a modification of the protein which caused it to migrate more slowly on SDS-PAGE. This modified form appeared to be deleted at its C terminus because it could also be generated by carboxypeptidase A digestion (28Sangar D.V. Clark R.P. Carroll A.R. Rowlands D.J. Clarke B.E. J. Gen. Virol. 1988; 69: 2327-2333Crossref PubMed Scopus (10) Google Scholar). Thus, removal of a small number of amino acids decreases the mobility of Lbproon SDS-PAGE. We believe that the missing 6 amino acids of the Lbpro-6 deletion are responsible for the reduced mobility of this protein. The above experiment suggested that the presence of the CTE was required for full enzymatic activity. This notion was further strengthened by a serendipitous observation made during the synthesis of the above deletion mutants. A variant plasmid was obtained in which an incorrect, truncated oligonucleotide was inserted between theBsiWI site and the BamHI site at the 3′ end of the Lbpro (Fig. 4 A and Table II). As a consequence, the reading frame is shifted from amino acid 183 onwards and there are only 8 amino acids before the BamHI site. Thus, translation of an RNA transcribed from a template linearized withBamHI produces a variant encoding a C-terminal extension of 8 amino acids, all differing from the wild-type (Table II). This variant was designated Lbpro − 11*, as it lacks 11 amino acids compared with the wild-type Lbpro; the asterisk indicates the aberrant amino acid sequence of the CTE. In addition, RNA encoding a second variant was produced by linearizing the same plasmid with HindIII, which cleaves 481 nucleotides downstream of the BamHI site; due to the position of the first stop codon encountered, the CTE in this case has 28 amino acids, 9 longer than the wild-type. Once again, they are of different sequence (Table II), so that the variant was designated Lbpro + 9*. Although the mutant sequences have a relatively high alanine content and are somewhat hydrophobic, they are not otherwise unusual. Nevertheless, examination of these variants in the RRL system (Fig.5) showed that both Lbpro − 11* and Lbpro + 9* had an impaired ability to cleave eIF4GI. With the Lbpro − 11* variant, about 90% eIF4GI cleavage is observed after 30 min of incubation, even though after 4 and 8 min similar protein concentrations to those of the wild-type experiment shown in Fig. 4 B are present. Processing with the variant Lbpro + 9* (Fig. 5, right panels) was reduced even more dramatically, as 50% eIF4GI cleavage did not occur until 30 min after cleavage, even though the RNA concentration had been adjusted to ensure that twice the amount of protein was synthesized in this experiment compared with the wild-type Lbpro in Fig.4. In the above experiments, the activity of the Lbpro C-terminal variants was monitored only on the endogenous eIF4GI present in the RRL extract. We wished to investigate whether these variants were also impaired in their ability to recognize the polyprotein cleavage site. To generate a suitable substrate, the construction ΔLbproVP was prepared (Fig. 6 A). Following linearization with BamHI, transcription generates an RNA encoding an inactive Lbpro with a 46-amino acid deletion, all 85 amino acids of VP4, and 140 amino acids of VP2. The deletion in the Lbpro part and the extension of the VP2 part make the cleavage products of LbproVP4VP2 and ΔLbproVP distinguishable from each other on PAGE. The protein produced in RRL from the pet8cΔLbproVP construction is of apparent molecular mass of 46 kDa (Fig.6 B, far right lane) and is proteolytically inactive (Fig. 6,B and C, far right lanes). The ability of ΔLbproVP to serve as substrate was examined by simultaneously translating its RNA with the pet8cLbpro RNA (Fig. 6 B, left panel). Initially, after 8 min of incubation, two products of translation were observed, the mature Lbproand the uncleaved substrate ΔLbproVP (Fig. 6 B, left panel). Subsequently, a third product of apparent molecular mass of 34 kDa becomes visible, representing the VP part of the substrate following cleavage at the junction between ΔLbpro and VP. 50% cleavage of ΔLbproVP is achieved between 15 and 30 min. Eventually, all ΔLbproVP is converted to product, as sufficient proteinase is presumably present to cleave rapidly any newly synthesized substrate so that it is no longer observed. The ΔLbpro part of the substrate has a molecular mass of 14.2 kDa; despite this, however, it is not resolved by this gel system. Cleavage of eIF4GI also takes place under these conditions. Examination of the fate of the eIF4GI in the RRLs shows that over 50% is cleaved using Lbpro before 8 min (Fig. 6 C, left panel; Table III); this is comparable with the experiments in which only one RNA was translated. The Lbproconcentration was estimated to be about 20 pg/μl by counting the band. This agrees with previous observations in which 50% cleavage of eIF4GI was achieved when the Lbpro concentration had reached 15 pg/μl (18Glaser W. Skern T. FEBS Lett. 2000; 480: 151-155Crossref PubMed Scopus (45) Google Scholar).Table IIIComparison of cleavage efficiency of wild-type Lbpro and its C-terminal deletionsTime at 50% elF4GIProteinase concn at 50% elF4GI cleavageminpg/μlLbpro<8<20LbproVP4VP2<8<25Lbpro− 188–1520–52Lbpro + 9*4575Data were calculated from Fig. 6, B and C, except for LbproVP4VP2 which was taken from Fig.7. Open table in a new tab Data were calculated from Fig. 6, B and C, except for LbproVP4VP2 which was taken from Fig.7. This experiment was then repeated using mRNAs encoding Lbpro − 18 and Lbpro + 9*. The kinetics of cleavage of ΔLbproVP by these two variants are very similar to those of the wild type (Fig. 6 B, compare themiddle and right panel with the left one; Table III), indicating that the absence of the CTE or the presence of an aberrant one do not affect the ability of the variants to process the polyprotein substrate in an intermolecular reaction. In contrast, cleavage of eIF4GI by the C-terminal variants was once again delayed (Fig. 6 C, middle and right panels; TableIII). For the variant lacking the CTE, Lbpro − 18, 50% cleavage was achieved between 8 and 15 min at a proteinase concentration between 20 and 52 pg/μl; with the variant with the aberrant C terminus, Lbpro + 9*, 50% cleavage was obtained after about 45 min at a concentration of 75 pg/μl. In the experiment shown in Fig. 6, Lbpro can only be cleaving ΔLbproVP and eIF4GI intermolecularly. It has been a long-standing question, however, as to whether the initial cleavage of the Lbpro from the growing polyprotein (i.e. from LbproVP4VP2) is also an intra- or an intermolecular event (20Guarné A. Tormo J. Kirchweger K. Pfistermueller D. Fita I. Skern T. EMBO J. 1998; 17: 7469-7479Crossref PubMed Scopus (117) Google Scholar, 21Medina M. Domingo E. Brangwyn J.K. Belsham G.J. Virology. 1993; 194: 355-359Crossref PubMed Scopus (126) Google Scholar, 24Belsham G.J. Brangwyn J.K. Ryan M.D. Abrams C.C. King A.M.Q. Virology. 1990; 176: 524-530Crossref PubMed Scopus (24) Google Scholar). To investigate this, we translated the ΔLbproVP RNA together with that of LbproVP4VP2 to see whether the cleavage kinetics of ΔLbproVP or eIF4GI were changed (Fig.7). This can be achieved because, as stated above, all the cleavage products from LbproVP4VP2 and ΔLbproVP can be separated from one another by SDS-PAGE. The result is shown in Fig. 7 A. Four products are visible during the time course. These are the uncleaved ΔLbproVP (46 kDa) and the VP cleavage products (34 kDa) (the 14.2-kDa ΔLbpro cleavage product is once again not resolved) as well as the mature Lbpro (19.8 kDa) and the VP4VP2 (15 kDa) cleavage product. However, the uncleaved protein LbproVP4VP2 is not a major product of the reaction; the uncleaved precursor is essentially only visible with the inactive leader proteinase mutant LbproC51A VP4VP2 (Fig. 7 A, far right lane). Therefore, the processing of the Lbpro from the growing LbproVP4VP2 polypeptide chain must be extremely efficient, strongly implying that this reaction is an intramolecular one. The processing of the endogenous eIF4GI by Lbpro expressed as LbproVP4VP2 is comparable with that observed when the Lbpro is translated as a mature protein (compare Fig.6 C with Fig. 7 B). The cleavage of ΔLbproVP with Lbpro expressed from LbproVP4VP2 is also comparable to that with Lbpro (compare Fig. 6 B with Fig. 7 A) and is therefore not delayed by self-processing. Furthermore, the self-processing of LbproVP4VP2 occurs much more efficiently than the cleavage of ΔLbproVP, even though the sequence of the cleavage site is the same. This strongly implies that Lbpro frees itself from the growing polypeptide chain by an intramolecular cleavage. In summary, the reactions in order of efficiency catalyzed by the Lbpro are the intramolecular processing at the Lbpro VP4 junction and the cleavage of eIF4GI as part of the eIF4F complex followed by the much less efficient intermolecular cleavage at the Lbpro VP4 junction. The experiments described here examine the relationship of the self-processing reaction of the FMDV Lbpro to the cleavage of the cellular substrate eIF4GI. By using mutagenesis to vary the cleavage site at the Lbpro VP4 junction and to produce C-terminal variants, factors affecting the rate of both reactions could be ascertained. As the polyprotein and eIF4GI substrates examined are those that are cleaved during replication of the virus in vivo, these findings are directly relevant to the biological activity of the Lbpro. We began by investigating whether the inhibition of self-processing affected the rate of eIF4GI cleavage. For this, we mutated the Lbpro to prevent self-processing without affecting either the catalytic residues or the structure of the enzyme. Initially, mutations were introduced at the P1 position (Table I). The substitution of the P1 lysine with either glycine or glutamine impaired self-processing but did not inhibit it completely. Replacement of the P1′ glycine with arginine (thus mimicking the P1′ residue in the eIF4GI cleavage site) in the mutant protein containing glycine at P1 restored self-processing to wild-type levels, thus confirming the unusual requirement of this enzyme for a basic residue at either the P1 or P1′ positions. Complete inhibition of the self-processing reaction was obtained by the introduction of a second mutation, namely leucine to serine at P2, in the P1 lysine to glutamine mutant protein. This stresses the importance of the presence of a hydrophobic residue at P2, a property that Lbpro shares with most other papain-like enzymes (30Rawlings N.D. Barrett A.J. Methods Enzymol. 1994; 244: 461-486Crossref PubMed Scopus (336) Google Scholar). In all of the above self-processing mutant proteins, however, a reduction in the rate or extent of eIF4GI cleavage was not observed. Thus, cleavage at the Lbpro VP4 junction is not an essential prerequisite for cleavage of eIF4GI. This implies that the enzyme is active as part of the growing polypeptide chain and that it may be active while the polypeptide chain is still bound to the ribosome. Why is the cleavage of eIF4GI so efficient, despite the lack of self-processing? To begin, it can be assumed that the substrate eIF4GI is in the optimal conformation, as the binding of eIF4E to eIF4GI stimulates Lbpro cleavage of eIF4GI (31Ohlmann T. Pain V.M. Wood W. Rau M. Morley S.J. EMBO J. 1997; 16: 844-855Crossref PubMed Scopus (51) Google Scholar), and most of the eIF4GI in RRLs is complexed to eIF4E (29Rau M. Ohlmann T. Morley S.J. Pain V.M. J. Biol. Chem. 1996; 271: 8983-8990Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). For the enzyme, the C-terminal deletion experiments indicate a role for the C terminus. Thus, the mutant protein lacking the entire CTE cleaved eIF4GI at a significantly reduced rate; however, a much more drastic reduction in the reaction rate could be seen with the variants possessing an aberrant CTE. Nevertheless, all C-terminal variants recognized the Lbpro VP4 cleavage site in the intermolecular cleavage at the same rate. Thus, the CTE is required for efficient intermolecular cleavage of eIF4GI but not for that on the polyprotein substrate. Three explanations for this observation appear plausible. First, the C terminus may be involved in a direct interaction with eIF4GI, perhaps fitting specifically into a pocket, or even eIF4E; removal of the CTE would prevent this interaction. However, it is not clear from this theory why the presence of aberrant CTEs would have a further effect on eIF4GI processing than the variants lacking a CTE. Second, the CTE could stabilize the Lbpro in a conformation that favors eIF4GI cleavage but that is not required for intermolecular polyprotein processing. The lack of the CTE would obviate the stabilizing effect; however, the presence of the aberrant CTE might destabilize this conformation or even prevent it from being adopted. The third possibility would require that binding of the Lbpro CTE to eIF4GI induces a conformational change in this protein that exposes the cleavage site. In this case, proteolysis would be the rate-limiting step; in the CTE deletion mutants, the rate-limiting step would be the exposure of the active site in the absence of the CTE. It is interesting to note that, compared with papain, the Lbpro achieves its active conformation without a pro-domain and without activation at low pH (32Turk B. Turk V. Turk D. Biol. Chem. Hoppe-Seyler. 1997; 378: 141-150PubMed Google Scholar). Indeed the experiments with the CTE variants appear to suggest a much more important role for the C terminus, a region of Lbpro that has no equivalent in papain (20Guarné A. Tormo J. Kirchweger K. Pfistermueller D. Fita I. Skern T. EMBO J. 1998; 17: 7469-7479Crossref PubMed Scopus (117) Google Scholar). Finally, cleavage reactions of the Lbpro on intra- and intermolecular polyprotein substrates were examined simultaneously. Thus, RNAs were translated so that the rate at which both intra- and intermolecular self-processing occurred could be measured (Fig. 7). These experiments were compared with one in which intramolecular self-processing was not required because the Lbprocontained a stop codon at its C terminus (Fig. 6). No difference in the rates of cleavage of eIF4GI or the polyprotein intermolecular substrate were observed. In contrast, the kinetics of cleavage of the Lbpro VP4 junction were different, depending on whether the enzyme and substrate were part of the same polypeptide chain or not. The cleavage of the substrate when part of the same chain was much more rapid, suggesting that this reaction is an intramolecular one, with the C terminus of Lbpro folding into the active site of the same molecule, as proposed by modeling studies based on the crystal structure (20Guarné A. Tormo J. Kirchweger K. Pfistermueller D. Fita I. Skern T. EMBO J. 1998; 17: 7469-7479Crossref PubMed Scopus (117) Google Scholar). This also implies that the ΔLbproVP substrate adopts a conformation competent for the intramolecular reaction and must unfold so that it can be processed intrans. In summary, we have shown that self-processing is not a prerequisite for cleavage of eIFGI by Lbpro and have established the hierarchy of the Lbpro cleavage reactions. The first two are the extremely efficient intramolecular self-processing and the intermolecular cleavage of eIF4GI followed by the much less efficient intermolecular cleavage of the polyprotein sequence. We thank Dieter Blaas and Joachim Seipelt for critical reading of the manuscript." @default.
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