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

Abstract

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.

Dissecting distinct proteolytic activities of FMDV Lpro implicates cleavage and degradation of RLR signaling proteins, not its deISGylase/DUB activity, in type I interferon suppression

“…Interestingly, the introduced mutations in L pro had different effects on the cleavage or degradation of RLR signaling proteins. Upon infection with EMCV-L pro C133S, we observed a~2 hr delay in eIF4G cleavage, consistent with a previous report [48]. This mutation did not affect the cleavage/degradation of the various RLR signaling proteins.…”

“…In addition, we introduced the mutations L143A and C133S. Mutation C133S was reported to reduce the affinity of L pro for eIF4G [48] whereas mutation of to L143 to alanine with its shorter side-chain rescued polyprotein processing in the context of the additional mutation L200F [49]. Based on the structure of L pro , mutation L143A has been predicted to open up the catalytic pocket.…”

Irreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategies

“…The Lb pro vector ( 23 ) was expressed and purified according to ref. 33 . For the ISG15 CTD -∆C probe, ISG15 (amino acids 79–154) was cloned in frame into the intein/chitin binding domain pTXB1 vector.…”

Structures and Corresponding Functions of Five Types of Picornaviral 2A Proteins

“…It has been shown that RV 2A pro can cleave the eIF4G-eIF4E complex more efficiently than eIF4G alone ( Haghighat et al, 1996 ). Moreover, 2A pro from RV-A2 and CV-B4 can form a stable complex with eIF4GII/eIF4E, but not with eIF4GII alone ( Aumayr et al, 2015 ). These findings suggest that the eIF4F complex may be a preferred substrate for this cleavage.…”