Investigating the Substrate Specificity and Oligomerisation of the Leader Protease of Foot and Mouth Disease Virus using NMR

Cencic, Regina; Mayer, Christina; Juliano, María A.; Juliano, Luiz; Konrat, Robert; Kontaxis, Georg; Skern, Tim

doi:10.1016/j.jmb.2007.08.061

Cited by 16 publications

(35 citation statements)

References 49 publications

(54 reference statements)

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“…Nevertheless, a peptide corresponding to the eIF4GI peptide (SFANLG⁎RTTL) was a poor substrate for both Lb pro and sLb pro ((Santos et al, 2009); unpublished data). However, the eIF4GI cleavage site when introduced into the background of the polyprotein substrate was efficiently cleaved by Lb pro but was still refractory to cleavage by sLb pro (Cencic et al, 2007). Examination of the state of the endogenous eIF4GI in RRLs used for the experiments showed that it was cleaved by both Lb pro and sLb pro (Cencic et al, 2007).…”

Section: Resultsmentioning

confidence: 99%

“…The constructs pCITE-1d Lb pro C51A VP4/VP2 containing the mutations at position P1 and P1′ of the Lb pro -VP4 cleavage site (VQRKLG⁎RAGQ, VQRKLK⁎RAGQ, VQRKLG⁎AAGQ) were created by site-directed PCR mutagenesis of pCITE-1d Lb pro C51A VP4/VP2. The construct pCITE-1d Lb pro C51A VP4/VP2 containing the eIF4GI sequence SFANLG⁎RTTL at the Lb pro -VP4 cleavage site, termed pCITE-1d Lb pro C51A VP4/VP2 SFANLG⁎RTTL (FMDV residues 29–195 of Lb pro , residues 669–678 of eIF4GI, residue 5–85 of VP4 and 78 residues of VP2), has been described (Cencic et al, 2007). The construct pCITE-1d Lb pro C51A VP4/VP2 containing residues 599–678 of eIF4GI, termed pCITE-1d Lb pro C51A eIF4GI 599-668 VP4/VP2 SFANLG⁎RTTL, (FMDV residues 29–195 of Lb pro , residues 599–678 of eIF4GI, residue 5–85 of VP4 and 78 residues of VP2) was created by PCR amplification of residues 599–678 of eIF4GI using the plasmid pKS eIF4GI 400–739 as template and cloning of this fragment into pCITE-1d Lb pro C51A VP4/VP2 via the restriction sites Bpu10 I and Sac I.…”

Section: Methodsmentioning

confidence: 99%

“…In vitro translation reactions were performed as described (Cencic et al, 2007) with the following modification. To translate substrate proteins and proteinases, RNA was added to the reaction at concentrations of 14 ng/µl.…”

Section: Methodsmentioning

confidence: 99%

“…1 h)(Mayer et al, 2008). A separate function for sLb pro has not been identified; however, one report suggested that Lb pro and sLb pro may differ in their cleavage efficiencies in intermolecular cleavage of the polyprotein substrate (Cencic et al, 2007). …”

Section: Introductionmentioning

confidence: 99%

“…sLb pro cannot form homodimers in this way because it lacks the six most C-terminal residues. The Lb pro homodimer has been observed by X-ray crystallography and NMR (Cencic et al, 2007; Guarn é et al, 1998, 2000; Steinberger et al, 2013) with the K D being estimated from NMR analyses to be in the millimolar range (Cencic et al, 2007). Therefore, formation of the homodimer at concentrations of Lb pro achieved when it is synthesised in the infected cell seems unlikely unless there is a high local concentration.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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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 Lpro (Labpro and Lbpro). 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, Lbpro removes six residues from its own C-terminus, generating sLbpro. We present the structure of sLbpro bound to the inhibitor E64-R-P-NH2, illustrating how sLbpro can cleave between Lys/Gly and Gly/Arg pairs. In intermolecular cleavage on polyprotein substrates, Lbpro was unaffected by P1 or P1′ substitutions and processed a substrate containing nine eIF4GI cleavage site residues whereas sLbpro failed to cleave the eIF4GI containing substrate and cleaved appreciably more slowly on mutated substrates. Introduction of 70 eIF4GI residues bearing the Lbpro binding site restored cleavage. These data imply that Lbpro and sLbpro may have different functions in infected cells.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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NMR analysis of the interaction of picornaviral proteinases Lb and 2A with their substrate eukaryotic initiation factor 4GII

et al. 2015

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Messenger RNA is recruited to the eukaryotic ribosome by a complex including the eukaryotic initiation factor (eIF) 4E (the cap‐binding protein), the scaffold protein eIF4G and the RNA helicase eIF4A. To shut‐off host–cell protein synthesis, eIF4G is cleaved during picornaviral infection by a virally encoded proteinase; the structural basis of this reaction and its stimulation by eIF4E is unclear. We have structurally and biochemically investigated the interaction of purified foot‐and‐mouth disease virus (FMDV) leader proteinase (Lbpro), human rhinovirus 2 (HRV2) 2A proteinase (2Apro) and coxsackievirus B4 (CVB4) 2Apro with purified eIF4GII, eIF4E and the eIF4GII/eIF4E complex. Using nuclear magnetic resonance (NMR), we completed 13C/15N sequential backbone assignment of human eIF4GII residues 551–745 and examined their binding to murine eIF4E. eIF4GII551–745 is intrinsically unstructured and remains so when bound to eIF4E. NMR and biophysical techniques for determining stoichiometry and binding constants revealed that the papain‐like Lbpro only forms a stable complex with eIF4GII551–745 in the presence of eIF4E, with K D values in the low nanomolar range; Lbpro contacts both eIF4GII and eIF4E. Furthermore, the unrelated chymotrypsin‐like 2Apro from HRV2 and CVB4 also build a stable complex with eIF4GII/eIF4E, but with K D values in the low micromolar range. The HRV2 enzyme also forms a stable complex with eIF4E; however, none of the proteinases tested complex stably with eIF4GII alone. Thus, these three picornaviral proteinases have independently evolved to establish distinct triangular heterotrimeric protein complexes that may actively target ribosomes involved in mRNA recruitment to ensure efficient host cell shut‐off.

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2018

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Investigating the Substrate Specificity and Oligomerisation of the Leader Protease of Foot and Mouth Disease Virus using NMR

Cited by 16 publications

References 49 publications

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

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

NMR analysis of the interaction of picornaviral proteinases Lb and 2A with their substrate eukaryotic initiation factor 4GII

Exploring Fundamentals

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