Investigating the quaternary structure of the FMDV Leader protease

Das Maul-und Klauenseuchevirus (MKSV) stellt ein Mitglied der Picornavirus-Familie dar und ist ein kleines, nicht-umhülltes Virus mit einem einzelsträngigen RNA-Genom mit positiver Polarität. Das RNA-Genom wird direkt in ein Polyprotein translatiert, welches anschließend von viralen Proteasen prozessiert wird. Das erste Protein dieses Polyproteins ist die Leader-Protease, die in Abhängigkeit von der Translationsinitiation an zwei verschiedenen Startkodons in den beiden Formen Labpro und Lbpro vorkommen kann. Lbpro ist eine papain-ähnliche Cystein-Protease, die sich durch einen Schnitt zwischen dem eigenen C-Terminus und dem N-Terminus des nachfolgenden Proteins VP4 vom Polyprotein abspaltet. Lbpro ist eine sehr spezifische Protease, die nur zwei zelluläre Substrate schneidet, nämlich die beiden Homologe des eukaryotischen Initiationsfaktors 4G (eIF4G), eIF4GI and eIF4GII. eIF4G spielt eine wichtige Rolle bei der eukaryotischen Translationinitiation, da es als Gerüstprotein dient und dadurch die „gecappte“ mRNA und das Ribosom zusammenführt. Der Begriff „Host cell shut off“ beschreibt den Vorgang während einer MKSV Infektion, bei dem eIF4G von der Leader Protease geschnitten wird und dadurch die zelluläre cap-abhängige Translationsinitiation verhindert wird. Dabei bleibt die Translation der viralen RNA allerdings unbeeinträchtigt, da die Translation durch eine IRES (internal ribosome entry site) eingeleitet wird. Das letztendliche Ziel dieser Studie ist die Verhinderung der Selbst-Prozessierung von Lbpro. Infolgedessen würde Lbpro mit dem viralen Capsidprotein VP4 verbunden bleiben. Daher wäre VP4 nicht mehr in der Lage sich korrekt in die virale Capsidstruktur einzugliedern, weshalb die Bildung von lebensfähigen Viruspartikeln verhindert wäre. Die Selbstprozessierung von Lbpro kann entweder inter- oder intramolekular ablaufen; allerdings wurde gezeigt, dass die cis-Spaltungsreaktion bevorzugt wird. Aus diesem Grund stellt die intramolekulare Selbst-Prozessierung von Lbpro einen wichtigen Angriffspunkt für die Entwickung von antiviralen Medikamenten dar. Aufgrund dieser Fakten haben wir uns in dieser Arbeit darauf konzentriert die intramolekulare Selbst-Prozessierung von Lbpro auf molekularer Ebene zu untersuchen. Es wurde beobachtet, dass Lbpro durch Insertion des C-terminalen Fortsatzes in das aktive Zentrum des benachbarten Moleküls stabile Dimere in Lösung bildet. Daher haben wir versucht das Dimer durch zielgerichtete Mutagenese zu trennen um in der Lage zu sein die Selbst-Prozessierung in cis untersuchen zu können. Zwei Regionen der Protease wurden mutiert: die Grenzflächen-Region zwischen den dimeren Lbpro Molekülen und der CTE, der an das aktive Zentrum des benachbarten Moleküls bindet. Da angenommen wurde, dass Trp 105 und Thr 117 zu den intermolekularen Interaktionen in der Grenzflächen-Region zwischen dimeren Lbpro Molekülen beitragen, wurden diese Aminosäuren entweder durch Ala ersetzt um potenzielle anziehende Interaktionen zu entfernen oder durch Arg um eine Abstoßung hervorzurufen. Die Mutationen W105A, T117A, W105A T117A und W105R in 98 der Grenzflächen-Region konnten weder die enzymatische Aktivität von Lbpro beeinflussen, noch konnten sie die Bildung von Dimeren verhindern. Aufgrund dieser Tatsachen, scheinen die Aminosäuren Trp 105 und Thr 117 keinen maßgeblichen Beitrag zur Dimer-Stabilität zu leisten. Interessanterweise war eine einzelne Mutation der C-terminalen Aminosäure Leu 200 zu Phe ausreichend um das Dimer zu trennen. Obwohl das monomere Lbpro L200F eine verzögerte Selbst- Prozessierung aufweist, konnte eine transiente Binding des CTE an das aktive Zentrum festgestellt werden. Daher liefern diese Untersuchungen interessante Einblicke im Bezug auf die intramolekulare Selbst-Prozessierung. Die Daten zeigen, dass die letzten sieben Aminosäuren des CTE in einer ähnlichen Weise an das aktive Zentrum gebunden sind wie es im Dimer der Fall ist. Allerdings, war es nicht möglich Signale für die letzten 12 Aminosäuren des CTE zu detektieren. Dies wird wahrscheinlich durch die Frequenz der transienten Interaktion zwischen CTE und aktivem Zentrum bedingt, die mit Kernresonanzspektrometrie schwierig zu detektieren ist. Die zusätzliche Mutation L143A stellt die Aktivität in der Selbst-Prozessierung wie auch die dimere Struktur von Lbpro L200F wieder her, obwohl das Lbpro L143A L200F Dimer destabilisiert erscheint. Weitere Untersuchungen betrafen die nukleäre Lokalisation von Lbpro. Es wurde angenommen, dass Lbpro durch rezeptor-vermittelten Transport in den Zellkern eindringt. Aus diesem Grund wurde enzymatisch inaktives Lbpro in humanen Zellen exprimiert. Allerdings war es nicht möglich Lbpro in nennenswerten Mengen im Zellkern nachzuweisen. Aufgrund dieser Ergebnisse, konnte eine nukleäre Lokalisation und ein rezeptor-vermittelter Transport von Lbpro in den Zellkern nicht bestätigt werden.Foot-and-mouth disease virus (FMDV), being a member of the picornavirus family, is a small, non-enveloped virus with a single-stranded RNA genome of positive polarity. The RNA genome is directly translated into a long polyprotein which is subsequently processed by viral proteases. The first protein encoded on this polyprotein is the Leader protease that can exist in two different forms, Labpro and Lbpro, dependent on the translation initiation at two different start codons. Lbpro is a papain-like cysteine protease that frees itself from the polyprotein by cleavage between its own C-terminus and the N-terminus of the subsequent protein VP4. Lbpro is a very specific protease cleaving only two cellular substrates, the two homologues of the eukaryotic translation initiation factor 4G (eIF4G), eIF4GI and eIF4GII. eIF4G plays an important role in eukaryotic translation initiation as it acts as a scaffold protein that brinds together the capped mRNA and the ribosome. The term ‚host cell shut off’ describes the process during FMDV infection, at which eIF4G is cleaved by the Leader protease resulting in the inhibition of cellular capdependent translation initiation. However, the translation of the viral RNA remains unaffected as translation is initiated via an IRES (internal ribosome entry site). The overall goal of this study is to inhibit the Lbpro self-processing step. Consequently, Lbpro would remain connected with the capsid protein VP4. As a result, VP4 would not be able to fit correctly into the viral capsid structure, thus inhibiting the formation of viable virus particles. Lbpro self-processing can occur either inter- or intramolecularly; however, the cis cleavage reaction was shown to be preferred. Therefore, intramolecular self-processing of Lbpro is an important target for the development of anti-virals. Due to these facts, in this work we focused on the investigation of the intramolecular selfprocessing reaction of Lbpro at the molecular level. It was observed that Lbpro forms stable dimers in solution by inserting the C-terminal extension (CTE) of one molecule into the active site of the neighbouring molecule. Therefore, we tried to separate the dimer by site-directed mutagenesis in order to be able to investigate self-processing in cis. Mutations were introduced at two regions of the protease: the interface region between dimeric Lbpro molecules and the CTE which binds to the active site of the neighbouring molecule. As Trp 105 and Thr 117 were thought to contribute to the intermolecular interactions in the interface region between dimeric Lbpro molecules, these residues were substituted either by Ala to remove potential attractive interactions or by Arg to provoke repulsion. However, the mutations W105A, T117A, W105A T117A and W105R in the interface region neither affected the enzymatic activity of Lbpro nor could they inhibit dimer formation. Therefore, residues Trp 105 and Thr 117 do not appear to make crucial contributions to the stability of the dimer. Interestingly, the single mutation of the C-terminal residue Leu 200 to Phe was sufficient to disrupt the dimer. Although monomeric Lbpro L200F appeared delayed in self-processing, a transient binding of the CTE to the active site could be determined. Therefore, these findings 96 provide interesting insights concerning intramolecular self-processing. The data indicate that the last seven amino acids of the CTE are bound to the active site in a similar way as present in the dimer. However, it was not possible to detect signals for the last 12 residues of the CTE. This is probably caused by the rate of transient interaction between the CTE and the active site, which is difficult to detect by NMR. The additional mutation L143A restores the self-processing activity as well as the dimeric structure of Lbpro L200F, although the Lbpro L143A L200F dimer appears rather destabilised. Further investigations concerned the nuclear localisation of Lbpro. It was considered that Lbpro enters the nucleus via receptor-mediated transport. Therefore, enzymatically inactive Lbpro was expressed in human cells. However, it was not possible to detect Lbpro in the nucleus in appreciable amounts. Due to these findings, nuclear localisation and receptor-mediated nuclear transport of Lbpro into the nucleus could not be confirmed

Foot-and-mouth disease virus leader proteinase: Structural insights into the mechanism of intermolecular cleavage

Translation of foot-and-mouth disease virus RNA initiates at one of two start codons leading to the synthesis of two forms of leader proteinase L(pr)o (Lab(pro) and Lb(pro)). These forms free themselves from the viral polyprotein by intra- and intermolecular self-processing and subsequently cleave the cellular eukaryotic initiation factor (eIF) 4G. During infection, Lb(pro) removes six residues from its own C-terminus, generating sLb(pro). We present the structure of sLb(pro) bound to the inhibitor E64-R-P-NH2, illustrating how sLb(pro) can cleave between Lys/Gly and Gly/Arg pairs. in intermolecular cleavage on polyprotein substrates, Lb(pro) was unaffected by P1 or P1' substitutions and processed a substrate containing nine eIF4GI cleavage site residues whereas sLb(pro) failed to cleave the eIF4GI containing substrate and cleaved appreciably more slowly on mutated substrates. Introduction of 70 eIF4GI residues bearing the Lb(pro) binding site restored cleavage. These data imply that Lb(pro) and sLb(pro) may have different functions in infected cells. (C) 2014 the Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).Austrian Science FoundationFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Med Univ Vienna, Max F Perutz Labs, A-1030 Vienna, AustriaUniv Vienna, Dept Struct & Computat Biol, Max F Perutz Labs, A-1030 Vienna, AustriaUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-0404420 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-0404420 São Paulo, BrazilAustrian Science Foundation: P20889Austrian Science Foundation: P24038FAPESP: 12/50191-4RCNPq: 471340/2011-1CNPq: 470388/2010-2Web of Scienc

Investigating the structural basis of the intra- and intermolecular substrate specificity of the foot-and-mouth disease virus leader protease

Die Leader Protease (Lpro) des Maul- und Klauenseuche Virus ist ein vielversprechendes Zielobjekt zur Entwicklung von antiviralen Medikamenten. Lpro ist das erste Protein, welches im viralen RNA Genom kodiert ist. Die Translation kann an zwei alternativen Startkodons initiiert werden, die zur Bildung von Labpro und Lbpro führen, welche sich durch 28 Aminosäuren am N-Terminus unterscheiden. Während einer viralen Infektion entsteht durch die Entfernung von sechs bis sieben Aminosäuren vom C-Terminus eine dritte Form, die sLbpro genannt wird. Lpro schneidet sich selbst durch einen intra- (cis) oder intermolekularen (trans) Prozess vom viralen Polyprotein, wobei gezeigt wurde, dass die cis Reaktion bevorzugt wird. Die Hemmung von Lpro verhindert seine Abtrennung vom Kapsidprotein VP4, welches dadurch nicht länger in der Lage ist sich korrekt in das virale Kapsid einzugliedern. Um potente und spezifische Wirkstoffe gegen Lpro entwickeln zu können, ist es entscheidend die Substratspezifität und den Mechanismus der Selbstprozessierung der Protease genau zu verstehen. Diese Arbeit beschreibt daher eine Vielzahl an Experimenten zu diesem Zweck. Über die Struktur der "Prime"-Seite der Substratbindungsregion von Lpro gibt es nur spärliche Informationen. Aus diesem Grund haben wir die Kristallstruktur von sLbpro im Komplex mit dem Epoxid-basierten Inhibitor E64-R-P-NH2 bestimmt. Die Struktur zeigt die Interaktion von Arg und Pro des Inhibitors mit den S1' und S2' Bindungsstellen von sLbpro. Des Weiteren gibt die Struktur eine Erklärung wie Lbpro durch die einzigartige Anordnung von den drei sauren Aminosäuren Asp 49, Glu 96 und Glu 147 in der Substratbindungsstelle basische Aminosäuren an der P1 und der P1' Position akzeptieren kann. Des Weiteren wurde die unterschiedliche Enzymaktivität von Lbpro und sLbpro gegenüber Polyprotein-Substraten untersucht. Die Spalteffizienz von Lbpro wurde weder durch Mutationen an der P1 und P1' Stelle noch durch die Substitution mit der eIF4GI-Spaltstelle beeinflusst. Im Gegensatz dazu sank die Effizienz von sLbpro mutierte Polyprotein-Spaltstellen zu schneiden deutlich und das Substrat mit der eIF4GI-Spaltstelle konnte überhaupt nicht geschnitten werden. Durch die Einfügung von weiteren 70 Aminosäuren von eIF4GI, die die zuvor charakterisierte Bindungsstelle für Lbpro enthielt, konnte die Effizienz von sLbpro die eIF4GI-Spaltstelle zu schneiden allerdings wieder hergestellt werden. Demnach wird für die effiziente Spaltung von eIF4GI und dem Polyprotein-Substrat die stabilisierende Interaktion von der eIF4GI-Bindungsstelle oder der letzten sechs Cterminalen Aminosäuren benötigt. Die Tatsache, dass Lbpro und sLbpro gleichzeitig in der infizierten Zelle vorkommen könnte auf einen Mechanismus hindeuten um die Enzymeigenschaften während der viralen Infektion zu modifizieren. Darüber hinaus nahmen wir uns vor die Struktur und Dynamik der intramolekularen Selbstprozessierung von Lbpro mittels NMR zu untersuchen. Zu diesem Zweck wurden Lbpro-Varianten untersucht, deren C-Terminus um fünf Aminosäuren verlängert war. Es konnten Änderungen an der chemischen Verschiebung von bestimmten Aminosäuren der Substratbindungskluft festgestellt werden, die auf eine intramolekulare Interaktion zwischen dem C-Terminus und der Substratbindungsstelle hindeuten. Allerdings, konnten keine Signale für C-terminale Aminosäuren detektiert werden, die mit der Substratbindungskluft interagierten. Im Gegensatz dazu, konnten für C-terminale Aminosäuren, die ungebunden waren und nicht interagierten, in der Tat Signale beobachtet werden. Zusammenfassend kann gesagt werden, dass durch die innewohnende Eigenschaft des C-Terminus nur flüchtig zu interagieren die direkte Untersuchung des C-Terminus während der intramolekuaren Prozessierung schwierig ist.The leader protease (Lpro) of the foot-and-mouth disease virus represents a promising target for the development of antiviral drugs. Lpro is the first protein encoded on the viral RNA genome and its translation can be initiated at two alternative start codons giving rise to Labpro and Lbpro which differ by 28 residues at their N-terminus. A third form arises during infection when Lbpro removes six to seven residues from its C-terminus to create sLbpro. Lpro cleaves itself off the viral polyprotein either inter- (trans) or intramolecularly (cis); however, the cis reaction was shown to be preferred. The inhibition of Lpro prevents its cleavage from the capsid protein VP4 which is no longer able to integrate into the viral capsid correctly. In order to design potent and specific inhibitors, thorough understanding of the substrate specificity and the self-processing mechanism of Lpro is crucial. Thus, this thesis covers a number of experiments to provide this knowledge. Structural information on the prime side of the substrate binding region of Lpro is sparse. To this end, we solved the crystal structure of sLbpro in complex with the epoxide-based inhibitor E64-R-P-NH2 which shows the interaction of Arg and Pro of the inhibitor with the S1' and S2' subsite of sLbpro. Furthermore, the structure explains how Lbpro can accept a basic residue at the P1 and P1' position using a unique alignment of the three acidic residues Asp 49, Glu 96 and Glu 147 in the active site. Furthermore, the differences in the cleavage activities of Lbpro and sLbpro were observed. The cleavage efficiency of Lbpro on a polyprotein substrate was neither affected by mutations at the P1 and P1' position nor by the substitution with the eIF4GI cleavage site. In contrast, sLbpro cleavage of mutated polyprotein cleavage sites was significantly delayed and cleavage of the eIF4GI cleavage site completely failed. However, the introduction of further 70 residues of eIF4GI containing the previously characterized binding site for Lbpro restored the cleavage efficiency of sLbpro on the eIF4GI cleavage site. Thus, for efficient cleavage of eIF4GI and the polyprotein substrate stabilizing interactions either via the eIF4GI binding site or via the final six C-terminal residues are required. The fact that both Lbpro and sLbpro are present in the infected cell might suggest a mechanism to modify the properties of the enzyme during viral infection. Moreover, we attempted to investigate the structure and dynamics of intramolecular selfprocessing of Lbpro by NMR. To this end, Lbpro variants were investigated that were extended at the C-terminus by five amino acids. Amide shift changes for residues of the substrate binding cleft were observed indicating intramolecular interactions between the C-terminus and the substrate binding cleft. However, no signals could be detected for interacting Cterminal residues. In contrast, for C-terminal residues that were unbound and not interacting intramolecularly signals could indeed be observed. Concluding, due to the intrinsic nature of the C-terminus to interact transiently, the direct investigation of the C-terminus during intramolecular processing remains elusive.submitted by Jutta SteinbergerAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheAuch erschienen in Virology 443: 271-277 (2013) und Virology 468-470C: 397-408, 2014Wien, Med. Univ., Diss., 2014OeBB(VLID)171441

Translation Directed by Hepatitis A Virus IRES in the Absence of Active eIF4F Complex and eIF2

Translation directed by several picornavirus IRES elements can usually take place after cleavage of eIF4G by picornavirus proteases 2Apro or Lpro. The hepatitis A virus (HAV) IRES is thought to be an exception to this rule because it requires intact eIF4F complex for translation. In line with previous results we report that poliovirus (PV) 2Apro strongly blocks protein synthesis directed by HAV IRES. However, in contrast to previous findings we now demonstrate that eIF4G cleavage by foot-and-mouth disease virus (FMDV) Lpro strongly stimulates HAV IRES-driven translation. Thus, this is the first observation that 2Apro and Lpro exhibit opposite effects to what was previously thought to be the case in HAV IRES. This effect has been observed both in hamster BHK and human hepatoma Huh7 cells. In addition, this stimulation of translation is also observed in cell free systems after addition of purified Lpro. Notably, in presence of this FMDV protease, translation directed by HAV IRES takes place when eIF2α has been inactivated by phosphorylation. Our present findings clearly demonstrate that protein synthesis directed by HAV IRES can occur when eIF4G has been cleaved and after inactivation of eIF2. Therefore, translation directed by HAV IRES without intact eIF4G and active eIF2 is similar to that observed with other picornavirus IRESs.Dirección General de Investigación Científica y Técnica; Fundación Ramón Areces; FWF Austrian Science FundPeer Reviewe

Business trends (emerging business models)

Coming from public transit and combustion engine driven individual transportation, new technologies will foster future business opportunities in urban mobility. This work elaborates on these business models, paying special attention to customer value propositions as the customers' acceptance is probably the most critical success factor. Covering the big topics mobility needs, communication technologies as well as environment and energy, business trends like personal rapid transit, car sharing and e-mobility are closely examined. From this analysis it is concluded that the demand for individual and flexible means of transportation that compromise neither with environmental pollution nor with convenience rises

HAV IRES translation in BHK cells after cleavage of eIF4G.

<p>A) BHK-T7 cells were transfected or co-transfected for 2 h with 1 µg plasmid encoding HAV(IRES)-luc alone or in presence of 1 µg pTM1-2A or pFMDV-L, respectively. After 2 hpt, cells were treated with 200 µM Ars for 15 min and then metabolically labeled with 0.2 µCi per well [³⁵S]Met/Cys in presence (+) or absence (−) of Ars for 45 min. Finally, cells were processed by SDS-PAGE, fluorography and autoradiography. B) The same samples were used to analyze eIF4GI, eIF2α phosphorylation and total eIF2α by western blot using specific antibodies as detailed in Materials and Methods. C) BHK-T7 cells were transfected under the conditions described above. Cells were then collected and processed to assay for luc activity as described in Materials and Methods. The bars represent the luc activity in presence (+) or absence (−) of Ars. The RLUs values obtained were as follows: pHAV-luc in absence (−) or presence (+) of Ars were 3.9×10⁵ and 1.8×10⁵, respectively. pHAV-luc co-transfected with pTM1-2A (−) or (+) Ars were 0.2×10⁵ and 0.1×10⁵, respectively, and finally pHAV-luc co-transfected with pFMDV-L (−) or (+) Ars were 25×10⁵ and 17×10⁵, respectively. Error bars indicate standard deviation (SD). D) BHK-T7 cells were transfected with cap-luc, HAV(IRES)-luc or PV(IRES)-luc mRNAs. At 2 hpt cells were collected and luc activity was measured. The RLUs values obtained were as follows: cap-luc: 1.13×10⁶; HAV(IRES)-luc:1.46×10⁶ and PV(IRES)-luc: 0.44×10⁶.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

In vitro translation of HAV(IRES)-luc mRNA in presence of purified L^pro.

<p>HAV IRES was tested in RRL in presence of purified protease FMDV L^pro. First, two different concentrations of protease were added, 10 µg/ml or 40 µg/ml, for 20 min at 30°C. Lysates were then incubated with 50 ng poly(I:C) at the same temperature and,finally, HAV(IRES)-luc mRNA was added and incubated for 1 h at 30°C. Then, aliquots of these samples were processed to measure luc activity (A) and to analyze eIF4GI cleavage (B).</p

HAV IRES translation in presence of cleaved eIF4G in Huh7-T7 cells.

<p>A) Huh7-T7 cells were transfected or co-transfected for 3 h with 1 µg plasmid encoding HAV(IRES)-luc alone or in presence of 1 µg pTM1-2A or pFMDV-L, respectively. After 2 hpt, cells were treated with 200 µM Ars for 15 min and then metabolically labeled with 0.2 µCi per well[³⁵S]Met/Cys in presence (+) or absence (−) of Ars for 45 min. Finally, cells were processed by SDS-PAGE, fluorography and autoradiography. B) The same samples were used to analyze eIF4GI, eIF2α phosphorylation and total eIF2α by western blot. C) Huh7-T7 cells were transfected under the conditions described above. Cells were then recovered and processed to assay for luc activity as described in Materials and Methods. The bars represent the luc activity in presence (+) or absence (−) of Ars. The RLUs values obtained were as follows: pHAV-luc in absence (−) or presence (+) of Ars were 4.3×10⁵ and 1.8×10⁵, respectively. pHAV-luc co-transfected with pTM1-2A (−) or (+) Ars were 0.3×10⁵ and 0.2×10⁵, respectively, and finally pHAV-luc co-transfected with pFMDV-L (−) or (+) Ars were 23.4×10⁵ and 17.3×10⁵, respectively. Error bars indicate SD. D) Huh7-T7 cells were transfected with 1 µg plasmid pHAV-luc alone or with increasing concentrations of plasmid pTM1-2A for 3 h. After 3 hpt, cells were recovered and processed to measure luc activity. Values obtained are represented in the graph (upper panel). The same samples were used to analyze eIF4GI cleavage (lower panel).</p

HAV(IRES)-luc mRNA translation in cell free systems.

<p>A) RRL were incubated with increasing concentrations of poly(I:C) for 30 min at 30°C. After, cap-luc mRNA was added and incubated for 1 h at the same temperature. Then luc activity was measured. The values obtained are represented in the graph. B) RRL were incubated at 30°C for different time periods with 50 ng poly(I:C). In addition, to analyse the effects of mRNA or L^pro on eIF2α phosphorylation, RRL were incubated with the same concentration of poly(I:C) and 100 ng HAV(IRES)-luc mRNA alone or in presence of different amounts of purified L^pro for 30 min at the same temperature. Then, eIF2α phosphorylation was analyzed by western blot. C) Plasmids encoding HAV(IRES)-luc, EMC(IRES)-L and EMC(IRES)-2C were linearized and transcribed <i>in vitro</i>. The translation reaction was then carried out in RRL at 30°C. First, different concentrations of EMC(IRES)-L mRNA was added for 1 h to ensure eIF4G cleavage. Then, the mixture was incubated with 50 ng poly(I:C) during 30 min and finally 100 ng HAV(IRES)-luc mRNA was added and incubated for 1 h at 30°C. As control, EMC(IRES)-2C mRNA was used. In this case, samples were incubated first with different concentrations of EMC(IRES)-2C mRNA. Then, the mixture was incubated with 50 ng poly(I:C) during 30 min and finally, as above, 100 ng HAV(IRES)-luc mRNA was added and incubated for 1 h at 30°C. The graph represents the RLUs from HAV(IRES)-luc mRNA translation in presence of increasing concentrations of EMC(IRES)-L mRNA (left panel) or EMC(IRES)-2C mRNA (right panel). D) Bars represent the percentage of luc synthesis when eIF2α is phosphorylated in the presence of EMC(IRES)-L mRNA or EMC(IRES)-2C mRNA with respect to values without inhibitor, which are taken as 100%.</p