Interaction of 2A proteinase of human rhinovirus genetic group A with eIF4E is required for eIF4G cleavage during infection

Virology 511 (2017) 123–134

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Interaction of 2A proteinase of human rhinovirus genetic group A with eIF4E is required for eIF4G cleavage during infection Martina Aumayr, Anna Schrempf, Öykü Üzülmez, Karin M. Olek, Tim Skern

MARK

⁎

Max F. Perutz Laboratories, Medical University of Vienna, Dept. of Medical Biochemistry, Vienna Biocenter, Dr. Bohr-Gasse 9/3, A-1030 Vienna, Austria

A R T I C L E I N F O

A BS T RAC T

Keywords: Chymotrypsin-like cysteine proteinase Host cell shut-off Initiation of protein synthesis Cap-binding protein Virus-host interactions Protein-protein interactions

In enteroviruses, the inhibition of protein synthesis from capped host cell mRNA is catalyzed by the virally encoded 2A proteinase (2Apro), which cleaves eukaryotic initiation factors (eIF) 4GI and 4GII. Despite much investigation, the exact mechanism of 2Apro cleavage remains however unclear. Here, we identify the domains responsible for the eIF4E/HRV2 2Apro interaction using molecular modelling and describe mutations that impair this interaction and delay in vitro cleavage of eIF4G isoforms. Furthermore, we produced HRV1A viruses bearing the mutation L17R, Y32A or Y86A in the 2Apro sequence. All three viruses showed reduced yield and were appreciably delayed during infection in eIF4GI cleavage. Thus, we propose for genetic group A HRVs that the eIF4E/2Apro interaction is essential for successful viral replication. In contrast, HRV4 2Apro and coxsackievirus B4 2Apro failed to form complexes with eIF4E, suggesting that the mechanism of eIF4G isoform cleavage in these and related viruses is different.

1. Introduction Viruses of the family Picornaviridae (Makela et al., 1998), including poliovirus (PV), coxsackievirus (CV), human rhinoviruses (HRV) and foot-and-mouth disease virus (FMDV), possess a single stranded RNA genome of positive sense encoding a single open reading frame (ORF). Efficient translation of the RNA genome into the polyprotein product is crucial for successful viral replication. To this end, the majority of picornaviruses shut off protein synthesis directed by host cell mRNA whilst allowing viral translation to proceed. To achieve this modified translational profile, many picornaviruses cleave the two isoforms of the cellular eukaryotic initiation factor (eIF) 4G, eIF4GI and eIF4GII (Etchison et al., 1982; Foeger et al., 2003; Lamphear et al., 1993; Liebig et al., 1993) into two distinct domains. Consequently, the N-terminal eIF4G domain that binds the cap-binding protein eIF4E is separated from the C-terminal domain that binds the eIF3, the protein that is associated with the 40S ribosomal subunit. Consequently, the host cell fails to recruit capped mRNA to the ribosome; viral mRNA translation remains unaffected as it initiates through the internal ribosome entry site (IRES) at the RNA's 5′ end (Martinez-Salas and Ryan, 2010). The cleavage of the eIF4G isoforms and the appearance of the host cell shut-off occur one to two hours post-infection and before replica-

tion of the RNA genome takes place (Penman et al., 1963). Thus, the reaction is efficiently carried out by proteinases translated from the RNA genome of the infecting virion (Bovee et al., 1998). Understanding the mechanics and dynamics of eIF4G cleavage has proved challenging. Viruses of the genus Enterovirus, including HRV, PV, CV and enterovirus 71, use a chymotrypsin-like cysteine proteinase termed 2Apro to cleave eIF4G isoforms. The three-dimensional structures of 2Apro from several viruses (HRV2 (Petersen et al., 1999), HRV C2 (Lee et al., 2014), EV71 (Cai et al., 2013; Mu et al., 2013), CVB4 (Baxter et al., 2006),) and the site of cleavage on eIF4G isoforms has been determined for 2Apro from HRV2, PV and CVB4 (Lamphear et al., 1993; Ventoso et al., 1998). However, investigation of the interaction between HRV2 2Apro, the most closely investigated 2Apro, and eIF4G isoforms has shown that the cleavage reaction is more complex than previously imagined. Thus, the cleavage of eIF4G isoforms by HRV2 2Apro is stimulated by eIF4E, the cap-binding protein, and HRV2 2Apro can form a distinct complex with eIF4E alone (Aumayr et al., 2015). Finally, the cleavage sites of HRV2 2Apro on eIF4GI and eIF4GII are not conserved, with eIF4GII being cleaved about 20 amino acids Cterminal to the site on eIF4GI (Gradi et al., 2003; Fig. S1). It is unclear, however, whether HRV2 2Apro can serve as a model for all 2Apro enzymes of the diverse enterovirus group with its twelve viral species (Knowles et al., 2012). First, HRV2 2Apro shares only 35–38%

Abbreviations: eIF, eukaryotic initiation factor; FMDV, foot-and-mouth disease virus; HRV, human rhinovirus; IRES, internal ribosome entry site; Lpro, Leader proteinase; rmsd, root mean squared deviation; wt, wildtype ⁎ Correspondence to: Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr-Gasse 9/3, A-1030 Vienna, Austria. E-mail address: [email protected] (T. Skern). http://dx.doi.org/10.1016/j.virol.2017.08.020 Received 10 July 2017; Received in revised form 11 August 2017; Accepted 14 August 2017 0042-6822/ © 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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sequence identity with PV1, CVB4 and EV71 2Apro, even though they share the same overall fold. Second, HRV2 2Apro, belonging to the HRV genetic group A, shows only 37% sequence identity with HRV14 2Apro, a member of the HRV genetic group B. Indeed, the 2A protein is the least conserved protein between genetic A and B group viruses, with an overall identity of less than 40% (Tapparel et al., 2007). In contrast, HRV2 2Apro and HRV1A 2Apro, both genetic group A viruses, have a sequence identity of 88%; the overall level of the identity of 2Apro amongst genetic A group HRVs is over 80% (Tapparel et al., 2007). Third, differences in the cleavage of eIF4GI in vitro in rabbit reticulocytes (RRLs) between HRV2, HRV4, HRV14 and CVB4 2Apro have been observed (Baxter et al., 2006; Watters and Palmenberg, 2011). Fourth, in contrast to the HRV2 enzyme, CVB4 2Apro does not form a stable complex with eIF4E; further, CVB4 2Apro is a monomer whereas HRV2 2Apro is dimeric (Aumayr et al., 2015; Liebig et al., 1993). Differences have also been observed in cell culture. HRV2 and HRV16, both genetic A group HRVs, cleave both isoforms of eIF4G simultaneously; however, PV and HRV14, a genetic group B virus, were shown to cleave eIF4GI more rapidly than eIF4GII in the infected cell (Gradi et al., 1998b). These observations strongly suggest that there may be mechanistic differences between 2Apro of genetic group A HRV on the one hand and genetic group B HRV and other enteroviruses on the other hand. Despite the observed differences, the interactions of 2Apro with eIF4E and the eIF4G/eIF4E complex represent attractive targets for anti-viral compounds. In this manuscript, we examine the importance of the interaction of 2Apro from HRV2 and HRV1A with eIF4E. Using mutational analysis and protein modelling, we first identified and characterized residues of 2Apro and eIF4E involved in forming the complex in vitro and then examined the effect of three mutations on viral replication in cell culture using HRV 1A.

Cleavage assays were performed using purified recombinant eIF4GII551-745, murine eIF4E and an active HRV2 2Apro WT or the indicated mutant (Aumayr et al., 2015). In these reactions, 1 µg (5 µM) of substrate protein (eIF4GII551-745 or equimolar eIF4GII551-745 or eIF4E alone) were incubated with 10 ng (80 nM) of 2Apro WT or increasing concentration (100 ng/800 nM or 200 ng/1600 nM) of mutant 2Apro in assay buffer at 37 °C. At the indicated times, reactions were stopped by the addition of 5x SDS-PAGE loading buffer and heated to 95 °C for 5 min. Reactions were visualized on a 17.5% SDSPAGE gel stained with Coomassie brilliant blue. Gels were captured by a scanner (Canon) and uniform adjustments were performed using Adobe Photoshop. Densitometry was performed by the ImageJ software.

2. Materials and methods

2.4. In vitro transcription and translation

2.1. Protein expression and purification

Following linearization with BamHI for the pCITE HRV1A VP1/ 2Apro or MluI for the HRV1A full length clone and purification via the Wizard SV Gel and PCR Clean-Up System (Promega), the DNA was in vitro transcribed using T7 RNA polymerase for 120 min at 37 °C as described (Neubauer et al., 2013; Steinberger et al., 2014). To monitor self-processing of the HRV1A 2Apro, in vitro translation reactions in rabbit reticulocyte lysate (RRL, Promega) were performed as described in (Cencic et al., 2007; Steinberger et al., 2014) with a HRV1A VP1/ 2Apro RNA concentration of 14 ng/µl.

KCl, 1 mM EDTA and 5 mM DTT). Static light scattering was performed with the miniDAWN Trista light scattering instrument (Wyatt Technology, Santa Barbara, CA) and a Superdex 200 10/300 exclusion column equilibrated in assay buffer. Assessment of the molecular weight and oligomeric state of individual proteins and complexed proteins was done with the manufacturer's software ASTRA. Isothermal titration calorimetry (ITC) was performed on a MicroCal ITC microcalorimeter. Prior, proteins were dialyzed in the assay buffer lacking 5 mM DTT. Following thermal equilibration at 25 °C, an initial delay of 60 s and a single 0.5 µl titrant injection, 20 serial injections of 2 µl of the titrant was added at an interval of 180 s into the stirred sample cell (200 µl) at a stirring rate of 750 rpm at 25 °C. The protein concentration in the cell was generally 10 fold lower than protein titrant. Data analysis was performed using the Origin software package MicroCal. 2.3. In vitro cleavage assays

HRV2 2Apro, full-length murine eIF4E and human eIF4GII551-745 were expressed and purified as described in Aumayr et al. (2015). HRV1A 2Apro (HRV1A amino acids 858–998; Swiss-Prot: P23008.3) was purified using the method of Liebig et al. (1993). The plasmid HRV4 His6VP18 2Apro C110S contains six histidine residues, eight amino acids of VP1 and all of HRV4 2Apro (HRV4 amino acids 8461000; NCBI protein ID: ABF51184.1) cloned into pET3d. The His6VP18 2Apro C110S protein was expressed in soluBL21 in the presence of 100 µM ZnCl2 and purified using Nickel-agarose affinity chromatography in buffer A of Liebig et al. (1993), except that the pH was adjusted to pH 9.0. The change in pH was required to keep the HRV4 2Apro in solution, presumably reflecting the difference in the pI between HRV2 2Apro (pI 5.5) and HRV4 2Apro (pI 7.2). Following removal of the imidazole by dialysis against buffer A, pH 9.0, the protein was further purified by size-exclusion on a HiLoad 26/60 Superdex 75 column. The protein eluted at 227 ml, much later than the HRV1A and HRV2 enzymes suggesting the HRV4 enzyme is a monomer. The protein was liable to precipitate on concentration; a concentration of 0.62 mg/ml was used for the binding studies described in this paper.

2.5. Cell culture, RNA transfection and virus propagation Human cervix carcinoma HeLa cells (strain Ohio obtained from the laboratory of D. Blaas, MFPL) were cultured in minimal essential medium (MEM, Gibco) containing 10% (v/v) FCS (Gibco), 1% (v/v) 200 mM L-glutamine (Gibco), 1% (v/v) 100x penicillin/streptomycin in an incubator at 37 °C with 5% CO2. In vitro transcribed RNAs of full length HRV1A genome (a gift of D. Pavear, ViroPharma) or RNA of a full length HRV1A genome carrying a mutation in the 2Apro were used to transfect HeLa cells by DEAE-dextran. Accordingly, Hela cells were grown to a density of 50%. Transfection reactions with 1.2 µg in vitro transcribed RNA, 140 mM LiCl2, 1 mM MgCl2, 10 mM HEPES (pH 7.5) and 2 µM DEAE-dextran were prepared and incubated on ice for 30 min. The cells were then washed two times with PBS, followed by incubation with the transfection mix for 25 min at 34 °C with gentle shaking. Streptomycin was added to the cells following removal of the transfection mix comprising 2 ml of infection medium, consisting of MEM supplemented with 2% (v/v) FCS, 1% (v/v) 200 mM L-glutamine, 1% (v/v) 100x penicillin. Cells were incubated at 34 °C until lysed. HRV1A WT lysed 100% of the cells normally after about 1 day; the mutants needed 2 or 3 days until more than 50% of the cells were lysed. After harvesting the lysed cells, samples were centrifuged at 1200 rpm

2.2. Biochemical protein characterization Analytical size exclusion chromatography (SEC), static light scattering and isothermal titration calorimetry were performed as described previously (Aumayr et al., 2015). For SEC, 500 ng of pure or complexed proteins were mixed together with 1 mg aprotinin (6.5 kDa) as a standard. Analytical size-exclusion chromatography was then performed on a HiLoad® 16/60 Superdex® 200 pg (GE Healthcare), equilibrated in assay buffer (20 mM Hepes/KOH pH 7.4, 150 mM 124

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3. Results

for 5 min and the cell debris was discarded. The supernatant containing the HRV1A WT or mutant virus was cultivated by several rounds of infection of T25, T75 and T162 flasks of HeLa cells. To concentrate the virus, the supernatant was ultracentrifuged in a Beckman Coulter Optima™ L-80 XP Ultracentrifuge at 40,000 rpm for 80 min with a Ti70 rotor. After confirming with RT-PCR that the mutation was not lost during the cultivation cycles, virus titres were determined by end point dilution tests with a 50% tissue culture infective dose (TCID50) as described by Reed and Muench (1938).

3.1. Mapping the HRV2 2Apro binding site for eIF4E We first wished to map the HRV2 2Apro binding site for eIF4E. To this end, we first attempted to generate diffracting crystals of the HRV2 2Apro/eIF4E complex; however, at present all attempts in this direction have remained unsuccessful. Our previous data showed that the formation of the HRV2 2Apro/eIF4E complex required dissociation of the HRV2 2Apro homodimer (Aumayr et al., 2015). Therefore, to locate the eIF4E binding site on HRV2 2Apro, we decided to investigate whether there was overlap between the eIF4E binding site and the dimerization interface of HRV2 2Apro. To obtain information on the dimerization interface, we used the crystal structure of the HRV2 2Apro (PDB ID: 2HRV) (Petersen et al., 1999) and the PISA server (Krissinel and Henrick, 2007; Velankar et al., 2012). The PISA server computes all assemblies that may be potentially formed in a given crystal packing and assesses the physiological relevance of crystallographic interfaces. For the HRV2 2Apro structure, PISA computes two possible dimer interfaces (Fig. 1 and Table 1). Importantly, the complexation significant scores (CSS) differ markedly; dimer 1 has a score of 0.1, whereas dimer 2 scored 1.0 (Table 1). This indicates that the dimer 2 is the physiologically most probable one. Close examination of the two dimer interfaces calculated by PISA shows that dimer interface 1 is that described in the asymmetric unit of the crystal structure whereas dimer interface 2 is formed with a symmetry mate generated by the software PyMOL from the 2HRV PDB file. Dimer interface 2 comprises a disulphide bridge, hydrogen bonds between β-strands eI2 of the two monomers and van der Waals contacts between their side-chains (Table 1, Fig. 1). In contrast, dimer interface 1 shows minimal interactions between the monomers. We attempted to confirm the PISA postulate by generating the molecular envelope of the HRV2 2Apro solution structure using smallangle X-ray scattering (SAXS) (Fig. S2). The molecular envelope from the SAXS data confirmed that the volume of the HRV2 2A molecule corresponds to that of a dimer; however, it was not possible to determine which of the two predicted models would better fit into the envelope. Indeed, the radii of gyration and the Chi-square values are nearly identical for both computed dimers (Fig. S2). This difficulty arises because the HRV2 2Apro monomer is almost spherical; thus, the two possible dimer arrangements, when superimposed on top of each other, reveal a similar shape (Fig. S2). Despite our inability to confirm the dimerization interface with

2.6. Cleavage of eIF4GI during HRV1A infection of HeLa cells HeLa cells were infected with an MOI of 1 with wt or mutant HRV1A. For the HRV1A WT and the Y32A mutant, extracellular virus and eIF4G-specific cleavage activity from HeLa cells were monitored at the following time-points: 0 h, 4 h, 6 h, 9 h and 11 h. The HRV1A mutants L17R and Y86A were monitored at 9 h as the difference between the HRV1A wt and the HRV1A Y32A was most pronounced at this time. At each time point, the supernatant was taken and virus titres were determined by end point dilution test TCID50 to determine the amount of extracellular virus. After taking the supernatant, cells were washed with 1xPBS before 1 ml 1x PBS was added and cells were scraped from the plate. To collect the cells, the sample was then centrifuged at 14,000 g for 5 min and the cell pellet was resuspended in 300 µl of preheated 2x sample buffer. The samples were then heated up at 95 °C for 5 min and passed several times through a hypodermic G25 needle. Cell debris was then centrifuged at 14,000 k for 20 min at 4 °C. Proteins in the supernatant were separated on a 6% SDS PAGE gel. Western blot analysis of eIF4GI was carried out by electrotransferring the proteins on a polyvinylidene difluoride (PVDF) membrane, followed by blocking with i-block solution and incubation with a rabbit serum recognizing the N-terminal part of eIF4GI (1:8000, a gift from R. Rhoads, Shreveport, Louisiana, USA (Liebig et al., 1993)). After washing with 1x PBST, blots were incubated with peroxidase-coupled secondary antibodies and subjected to enhanced chemiluminescence (SuperSignal™ West Pico kit (Pierce)). Subsequently, the membrane was exposed to a CL-X Posure™ film (Thermo Scientific). 2.7. SAXS investigation of the molecular envelope of HRV2 2Apro SAXS analyses were performed at the 0.99 Å wavelength ESRF at BioSAXS beamline BM29 coupled to the Superdex 200 10/300 exclusion column (Grenoble, France) and equipped with PILATUS 1M.

Fig. 1. Identification of the HRV2 2Apro dimer interface. Drawing (using PyMOL and the PDB coordinates 2HRV) of three monomers of the HRV2 2Apro. The central monomer in cyan and yellow makes one dimer interface (dimer interface 1) with the monomer in grey as found in the PDB co-ordinates. The central monomer makes a second dimer interface (dimer interface 2), predicted by PISA, with the monomer in yellow and dark blue. Residues of the predicted dimer interface that were mutated in this study are shown as pink sticks and are labelled. The yellow domains of the monomers comprising dimer interface 2 are predicted to form an eight stranded anti-parallel β-sheet.

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eIF4GII551-745) (Aumayr et al., 2015). We employed this fragment of eIF4GII because we have not been able to express and purify suitable quantities of the corresponding fragment from eIF4GI. The eIF4GII fragment comprises the eIF4E binding site (Gradi et al., 1998a; Mader et al., 1995) as well as the binding and cleavage site of the HRV 2Apro. It can be cleaved by HRV2 2Apro even though eIF4GII551-745 and HRV2 2Apro do not form a stable complex that can be detected by SEC (Aumayr et al., 2015). However, ITC measurements did reveal an interaction between eIF4GII551-745 and HRV2 2Apro C106S with KD of 75 µM (Table 3; an example raw ITC data is shown in Fig. S3). ITC also detected an interaction between eIF4GII551-745 and the mutant protein HRV2 2Apro C106S C138S; the KD was 2.5 µM, 30 fold lower than the 75 µM of the wild-type (Table 3). In contrast, HRV2 2Apro Y32A C106S interacted with eIF4G551-745 with a much higher KD of 242 µM. An interaction of HRV2 2Apro L17R C106S with eIF4G551-745 could not be measured by ITC. We also investigated the ability of the mutant proteins to interact with the purified eIF4GII551-745/eIF4E complex. HRV2 2Apro C106S interacts with the eIF4GII551-745/eIF4E complex with a KD of 5 µM (Table 3). The mutants HRV2 2Apro C106S C138S and HRV2 2Apro Y32A C106S showed values of 4.6 µM and 72 µM, respectively. An interaction between the HRV2 2Apro L17R C106S mutant and the eIF4GII551-745/eIF4E complex was measurable this time; however, the KD of 278 µM is very high, about 60 fold higher than the wild-type.

Table 1 PISA analysis (Krissinel and Henrick, 2007) of the HRV2 2Apro (PDB 2HRV) structure. NHB, the number of potential hydrogen bonds across the interface, NSB, the number of potential salt bridges across the interface and NDS, the number of potential disulphide bonds across the interface, CSS, complexation significant score. Dimer interface

Interface area [Å]

ΔiG [kcal/ mol]

ΔiG Pvalue

NHB

NSB

NDS

CSS

1 2 3 4 5

730.3 617.0 248.7 200.8 134.0

− 6.2 − 10.1 − 1.0 − 0.1 0.0

0.475 0.128 0.487 0.677 0.666

9 10 2 2 1

0 4 1 2 0

0 1 0 0 0

0.100 1.000 0.000 0.000 0.000

SAXS, we decided to examine whether mutations in dimer interface 2 would affect the ability of HRV2 2Apro to bind eIF4E. Fig. 1A shows that, alongside residue C138, which forms the disulphide bond, residues L17 and Y32 are close to the proposed interface. Indeed, substitution of L17 with arginine was previously shown to delay HRV2 2Apro cleavage of eIF4GI in rabbit reticulocyte lysates (RRLs); further, the mutant protein failed to bind to eIF4GI fragments, in contrast to the wild-type (Foeger et al., 2003). We did not, however, at the time consider that the phenotype might be due to an inability to interact with eIF4E. To examine this idea, we therefore introduced the single mutations L17R, Y32A and C138S into the catalytically inactive HRV2 2Apro C106S variant, expressed and purified the respective mutant proteins and examined their interaction with eIF4E. Differences in the formation of the eIF4E/HRV2 2Apro heterodimer were immediately observed with all three mutant proteins. The stable complex between HRV2 2Apro and eIF4E is only obtained after overnight incubation ((Aumayr et al., 2015) Table 2, Fig. 2A); however, the introduction of the mutation C138S led to a more rapid formation of the 1:1 heterodimeric complex, with eIF4E/HRV2 2Apro C106S C138S being already formed after 10 min (Table 2). In contrast, the HRV2 2Apro mutants L17R and Y32A failed to stably bind eIF4E; a complex was not observed on SEC (Table 2, Fig. 2). ITC measurements confirmed the lack of interaction of the L17R and Y32A mutants with eIF4E (Table 3). However, they also indicated that the C138S mutant had a slightly higher affinity for eIF4E than the wild-type (6.3 µM compared with 12 µM). The higher affinity may also be a reason for the more rapid formation of the complex with this mutant than with the wild-type.

3.3. Effects of L17R and Y32A on the 2Apro cleavage of eIF4G To investigate the effect of the interaction of eIF4E and HRV2 2Apro on eIF4G cleavage, we chose to further investigate the two mutations that abrogated complex formation, namely L17R and Y32A. To this end, we first employed a recently described activity assay with purified components (Aumayr et al., 2015). In this activity assay, 10 ng of HRV2 2Apro (80 nM) are sufficient to detect cleavage products from 1 µg (5 µM) of eIF4GII551-745 in the absence of eIF4E after 60–90 min; in the presence of 1 µg (5 µM) eIF4E, cleavage products of eIF4GII551745 become visible after 10 min (Fig. 3A). The introduction of either the L17R or the Y32A mutation into HRV2 2Apro delayed cleavage of eIF4GII551-745, regardless of whether eIF4E was added or not. Thus, both in the absence or presence of eIF4E, eIF4GII551-745 was not cleaved by 10 ng of protease (Fig. 3B and C) in marked contrast to wildtype HRV2 2Apro (Fig. 3A). Instead, 200 ng (1.6 µM) or 100 ng (800 nM) of the mutant proteins (HRV2 2Apro L17R or Y32A, respectively) were required to obtain a similar cleavage efficiency to that of HRV2 2Apro (Figs. S4A and S4B). Next, we investigated the question whether the mutations L17R and Y32A affect the ability of the 2Apro to perform self-processing and cleavage of full-length eIF4GI in RRLs. Foeger et al. (2003) showed previously that the intramolecular (cis) cleavage efficiency of the HRV2 2Apro L17R was not affected whereas endogenous eIF4GI cleavage was

3.2. Mutations in the C138 interface also affect the interaction of HRV2 2Apro with eIF4G and the eIF4E/eIF4G complex We next wished to examine the effect of the mutations L17R, Y32A and C138S on the interaction with eIF4G. To this end, we used a fragment of human eIF4GII containing residues 551-745 (termed Table 2 Complex formation and molecular mass of protein complexes determined by SEC-MALLS. Proteins

eIF4E/ eIF4E/ eIF4E/ eIF4E/ eIF4E/

HRV2 HRV2 HRV2 HRV2 HRV2

2Apro 2Apro 2Apro 2Apro 2Apro

C106S C106S C106S C106S C106S

(overnight) L17R Y32A Y86A C138S (10 min)

eIF4GII551-745/eIF4E/HRV2 eIF4GII551-745/eIF4E/HRV2 eIF4GII551-745/eIF4E/HRV2 eIF4GII551-745/eIF4E/HRV2 eIF4GII551-745/eIF4E/HRV2

2Apro 2Apro 2Apro 2Apro 2Apro

C106S C106S C106S C106S C106S

L17R Y32A Y86A C138S

Complex

Apparent molecular mass (SEC-MALLS, kDa)

Stoichiometry

+ − − − +

38–40 – – – 38–40

1:1 – – – 1:1

+ − − + +

104–106 – – 77 118–120

? – – 1:1:1 ?

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Fig. 2. HRV2 2Apro C106S mutants L17R and Y32A fail to form a stable heterodimer with eIF4E. Complex formation was analysed via SEC on a HiLoad 16/60 Superdex 200 prep grade (A–D) or HiLoad 16/60 Superdex 75 prep grade (E) column together with aprotinin (6.5 kDa) as an internal standard. 0.5 mg of the individual or complexed proteins were analysed together with 1 mg of aprotinin. Complex formation was performed by incubation at 4 °C overnight. eIF4E was incubated with: (A) HRV2 2Apro C106S (taken from Aumayr et al. (2015)) (B) HRV2 2Apro C106S C138S (C) HRV2 2Apro C106S L17R. (D) HRV2 2Apro C106S Y32A on HiLoad 16/60 Superdex 200 prep grade. (E) HRV2 2Apro C106S Y32A on HiLoad 16/60 Superdex 75 prep grade. All proteins are catalytically inactive due to the presence of the C106S mutation. Heterodimeric complexes under SEC conditions were observed between eIF4E and HRV2 2Apro C106S or between HRV2 2Apro C106S C138S (A and B); in contrast, no complexes were observed between eIF4E and HRV2 2Apro C106S L17R (C) or HRV2 2Apro C106S Y32A (D and E). 127

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Table 3 Binding constants (KD, µM) for the interactions of HRV2 2Apro WT or mutants with eIF4GII551-745, eIF4E or the eIF4GII551-745/eIF4E complex as determined by ITC. The average and standard deviation for the HRV2 2Apro WT were calculated from three experiments. Cell

Ligand

eIF4E eIF4E eIF4E eIF4E eIF4E

HRV2 HRV2 HRV2 HRV2 HRV2

2Apro 2Apro 2Apro 2Apro 2Apro

C106S C106S C106S C106S C106S

C138S L17R Y32A Y86A

eIF4GII551-745 eIF4GII551-745 eIF4GII551-745 eIF4GII551-745 eIF4GII551-745

HRV2 HRV2 HRV2 HRV2 HRV2

2Apro 2Apro 2Apro 2Apro 2Apro

C106S C106S C106S C106S C106S

C138S L17R Y32A Y86A

eIF4GII551-745/eIF4E eIF4GII551-745/eIF4E eIF4GII551-745/eIF4E eIF4GII551-745/eIF4E eIF4GII551-745/eIF4E

HRV2 HRV2 HRV2 HRV2 HRV2

2Apro 2Apro 2Apro 2Apro 2Apro

C106S C106S C106S C106S C106S

C138S L17R Y32A Y86A

ΔS° [cal mol−1 deg−1]

ΔG [kcal mol−1]

KD [µm]

− 46 ± 1.9 − 19 – – − 419

− 13 ± 9.2 − 12 – – − 121

12 ± 5.1 6.3 – – 8.9

− 12 ± 13 −4 –

− 6.8 ± 4.8 − 8.9 –

5

−5

75 ± 12 2.5 – 242 34

− − − − −

5.1 ± 1.8 4.6 278 72 61

− 1 − − −

12 ± 2.9 6340 49 4

20 ± 8.7 7 2053 14 9

delayed. Cleavage of eIF4GI by wt HRV2 2Apro or HRV2 2Apro L17I was complete within 60 min, cleavage by HRV2 2Apro L17R was however only 50% complete after 300 min. To investigate the effect of Y32A, we employed an HRV1A system (Neubauer et al., 2013) with which we can investigate 2Apro selfprocessing and eIF4GI cleavage in RRLs as with the HRV2 2Apro enzyme. With HRV1A, however, we can also introduce the mutations into a full-length cDNA clone and transfect in vitro transcribed RNA and generate mutant viruses. HRV2 RNA cannot be used as the inherent transfection efficiency is too low (Neubauer et al., 2013). To emphasise the suitability of HRV1A 2Apro, we first confirmed that the residues L17, Y32 and C138 are conserved between the 2Apro of HRV1A and HRV2 (Fig. S5); indeed, the overall identity of the HRV1A and HRV2 2Apro is 88%. Second, we purified the HRV1A 2Apro C106S and showed that it forms a heterodimeric complex with eIF4E (Figs. S6A and S6B). To demonstrate that the binding of 2Apro to eIF4E is a property of genetic group A rhinoviruses, we also expressed and purified the catalytically inactive HRV4 2Apro C110S containing an Nterminal His-tag, eight residues of VP1 and all 148 residues of HRV4 2Apro. We chose the HRV4 2Apro because it had been shown to be expressed in a soluble form that could be purified (Watters and Palmenberg, 2011). However, we were unable to purify the protein using the published procedure; thus, we developed our own protocol (see Section 2 and Fig. S6C–E), the key component being the use of pH 9 to maintain solubility. The purified HRV4 2Apro C110S failed to bind to eIF4E (Fig. S6F), thus indicating that the ability of 2Apro to bind eIF4E is confined to those from genetic group A viruses. To confirm that the HRV4 2Apro is functional, we also expressed and purified the wild-type protein; proteolysis of eIF4GI in a HeLa cell extract was readily observed (Fig. S6G). To assay HRV1A 2Apro activity in RRLs, in vitro transcribed RNA encoding HRV1A VP1/2Apro is translated in the presence of [35S]-Met. The 2Apro cleaves itself off the precursor protein, generating the cleavage products VP1 and 2Apro. Fig. 4 (upper panel) shows that wild-type HRV1A 2Apro and the Y32A mutant cleave 50% of the precursor protein within 20–30 min (Fig. 4A and B, upper panel). Two points about this assay should be noted. First, detection of the HRV1A 2Apro is impaired by the low number of methionine residues. In addition, it also co-migrates with the bulk of the globin. Consequently, only a faint band is detected at a molecular mass corresponding to HRV1A 2Apro. Second, the HRV2 and HRV1A VP1 cleavage product is unstable in the RRLs as we have documented previously (Neubauer et al., 2013; Sousa et al., 2006). eIF4GI is present as a natural component of the RRLs. Thus, its

Fig. 3. In vitro cleavage of eIF4GII551-745 by HRV2 2Apro in the absence or presence of eIF4E. (A) In vitro cleavage of 1 µg (5 µM) eIF4GII551-745 in the absence or presence of 1 µg (5 µM) eIF4E by (A) 10 ng (80 nM) HRV2 2Apro wt, (B) 10 ng (80 nM) HRV2 2Apro L17R and (C) 10 ng (80 nM) HRV2 2Apro Y32A. The molar ratio of HRV2 2Apro to eIF4GII551-745 was 1:60 in each case. Cleavage products were analysed by SDS-PAGE on gels containing 17.5% acrylamide.

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Fig. 4. HRV1A 2Apro Y32A is impaired in eIF4GI cleavage but not self-processing. In vitro translation assays of self-processing and eIF4GI cleavage of (A) HRV1A VP1/2Apro WT or (B) HRV1A VP1/2Apro Y32A. Rabbit reticulocyte lysates (RRL) were incubated with in vitro transcribed mRNA (14 ng/µl) of HRV1A VP1/2Apro or HRV1A VP1/2Apro Y32A and incubated at 30 °C. Translated proteins were labelled with 35S-methionine and 10 µl aliquots were taken at the indicated time points. The reactions were stopped by adding an excess of unlabelled methionine and 2x Laemmli sample buffer. The intramolecular cleavage of HRV1A VP1/2Apro wt or 2Apro Y32A mutant was analysed on gels containing 17.5% polyacrylamide, followed by fluorography (upper pictures). Uncleaved precursor HRV1A VP1/2Apro and the cleavage products VP1 and 2Apro are indicated. To observe cleavage of endogenous eIF4GI in the RRLs, 10 µl aliquots were analysed on gels containing 6% polyacrylamide followed by Western blotting using serum to detect the N-terminal part of the eIF4GI protein (lower pictures). Uncleaved eIF4GI and cleavage products (CPN) are indicated. Negative controls without any RNA are also included at the right of each gel. Protein standards are shown on the left.

cleavage by the newly synthesised 2Apro can be observed in the same sample. eIF4GI migrates as a series of bands on SDS-PAGE with a molecular mass of 220 kDa (Etchison et al., 1982). HRV1A 2Apro cleavage of eIF4GI thus generates a series of N-terminal cleavage products upon challenge by an N-terminal antibody. Fig. 4 (lower panels) show that the HRV1A 2Apro cleaves 50% of eIF4GI within 30– 60 min of incubation (Fig. 4A, lower panel) whereas the HRV1A 2Apro Y32A fails to cleave eIF4GI within 300 min (Fig. 4B, lower panel).

(Fig. 5C). Further, cells infected with the WT were observed in the microscope to detach from the surface between 9 h and 11 h post infection; in contrast, cells infected with the HRV1A 2Apro Y32A remain attached at 11 h post-infection (Fig. 5D). Examination of the mutant HRV1A 2Apro L17R was more challenging. We performed four unsuccessful transfection experiments with RNA from the mutant until we obtained any virus at all. The presence of the mutation L17R was confirmed by DNA sequencing of the appropriate RT-PCR product. We were however only able to generate sufficient virus stock for one time point. Given the results with HRV1A 2Apro Y32A, we therefore chose a time point of 9 h to examine viral replication of the mutant virus bearing 2Apro L17R. eIF4GI cleavage was severely delayed compared to that seen in the HRV1A WT (Fig. 5B). Indeed, it was even less efficient than the HRV1A Y32A (Figs. 5A and B). Examination of the extracellular virus revealed that the virus absorbed to the cells as the WT, but that no extracellular virus had been produced at this 9 h time point (data not shown).

3.4. eIF4GI cleavage in HeLa cells is delayed by HRV1A bearing mutations 2Apro L17R or 2Apro Y32A To determine whether the mutated residues that inhibit eIF4E/ 2Apro heterodimer complex formation in vitro also have an impact on full length eIF4GI cleavage during viral replication in HeLa cells, we introduced the 2Apro mutations L17R, Y32A or L17R and Y32A into cDNA of the full length HRV1A genome and transfected HeLa cells with the appropriate in vitro transcribed RNAs. Viruses bearing the double mutation L17R Y32A were not viable. Viruses carrying either the L17R or the Y32A mutations were viable; the presence of the mutations in the virus stocks was confirmed by RT-PCR amplification and DNA sequencing. We first compared HRV1A wt and HRV1A 2Apro Y32A virus. A time-course of infection in HeLa cells at an MOI of 1 was performed at the time points indicated in Fig. 5. Cleavage of eIF4GI was examined by immunoblotting. HRV1A wt initiates cleavage of eIF4GI at about 4 h post infection; in contrast, the HRV1A 2Apro Y32A does not initiate cleavage of eIF4GI until about 6 h post infection (Fig. 5A). HRV1A wt shows 100% cleavage after 9 h, whereas with HRV1A 2Apro Y32A, only about 50% of eIF4GI is cleaved at this time (Fig. 5A). This supports the notion that the eIF4E interactions with the 2Apro are crucial for the cleavage of full length eIF4GI during HRV1A replication in HeLa cells. Extracellular virus production was also monitored by performing TCID50 measurements of each of the supernatants at the indicated time points (Fig. 5C). In the first hours, extracellular virus decreases, as virus is absorbed to the cells (Fig. 5C). After about 6 h post infection, progeny virus particles from the WT appear; in contrast, an increase of HRV1A 2Apro Y32A virus is only slightly noticeable after about 11 h

3.5. Docking model of the trimeric complex eIF4E/eIF4G/HRV2 2Apro In the absence of diffracting crystals of the eIF4E/HRV2 2Apro complex, we tried to generate a suitable docking model for the eIF4E/ HRV2 2Apro complex using ClusPro 2.0 protein-protein docking service (Comeau et al., 2004a, 2004b; Kozakov et al., 2013). Constraints for the decision which docking models would be the most likely were as follows. First, as the HRV2 2Apro homodimer is disassembled to form a 1:1 complex with eIF4E, we propose that eIF4E binds to the HRV2 2Apro dimer interface. Second, residues in the dimer interface that were shown to influence the eIF4E/HRV2 2Apro C106S complex formation (L17, Y32 and C138) should be involved in the interaction. Third, we predict that the HRV2 2Apro would not bind to a site of eIF4E that is normally occupied by the m7GDP cap structure. To help satisfy these constraints, we chose for the docking a crystal structure (PDB: 1EJH) of murine eIF4E bound to m7GDP and a 16 residue oligopeptide of eIF4GI containing the eIF4E binding site. Using molecules A of the HRV2 2Apro structure and 1EJH, we selected one such docking model from the ten proposed by ClusPro of the heterotrimeric complex eIF4E/eIF4GI/HRV2 2Apro C106S that satisfied all three constraints 129

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Fig. 5. eIF4GI cleavage in HeLa cells is delayed in HRV1A mutants deficient in binding eIF4E. After transfection of HeLa cells with in vitro transcribed RNA of full length HRV1A wt or HRV1A carrying the Y32A or L17R mutation in 2Apro, virus was cultivated and concentrated. Five 6-wells were then infected with a MOI of 1 of wt or mutant virus. At the following time points (0 h, 4 h, 6 h, 9 h and 11 h), cells were monitored under the light microscope, extracellular virus in the supernatant was harvested and eIF4GI cleavage was monitored. For the HRV1A 2Apro L17R, only enough virus for 1 time point (9 h) could be cultivated. (A, B) Cleavage of eIF4GI by HRV1A wt, HRV1A 2Apro Y32A or HRV1A 2Apro L17R was monitored by scratching the cells from the surface and breaking up the cells as described in materials and methods, followed by Western blot with anti-eIF4GI antibody. (C) The amount of extracellular virus present in the supernatant was measured by 50% tissue culture infective dose (TCID50). The average and standard deviation of three experiments was calculated. TCID50 points were connected by lines (black for HRV1A wt and blue for HRV1A 2Apro Y32A). (D) Cells were visualized under the light microscope (10x magnification) at the indicated time points.

Fig. 6. Model of the formation of the heterotrimeric complex eIF4E/eIF4GI/HRV2 2Apro. The model was created with ClusPro 2.0 protein-protein docking service with the PDB identifier 2HRV and 1EJH. Restraints for the docking model are described in the text. eIF4E is in grey, eIF4GII622-637 in red, HRV2 2Apro in dark blue. Residues mutated at the predicted dimer interface are shown. eIF4E residues which affected heterodimeric complex formation are shown in green (S83, R123 and D127) whereas residues, which did not affect formation are shown in yellow (D71 and H78). Drawings were visualized in PyMOL (DeLano, 2002).

indicates that L17 is not directly involved in the interaction with eIF4E, but rather orients the Y32 residue for interaction with the eIF4E protein by turning it in the direction of the eIF4E residues S83 and D127. However, the residue C138 is closer to the eIF4GI helix, perhaps explaining why this mutation did not prevent eIF4E/HRV2 2Apro interaction. The model thus provides plausible explanations why substitutions at L17 and Y32 but not at C138 interrupted eIF4E/ HRV2 2Apro complex formation. In addition, the model also suggested that a third residue of HRV2 2Apro, namely Y86, would interact with residue R123 of eIF4E (Fig. 6). No other side-chain interaction could be predicted with confidence from the model. To investigate the importance of Y86, we generated the mutant

(Fig. 6). After these models were generated, the structure of human eIF4E complexed to a fragment of eIF4GI and m7GTP was reported; as the mouse and human structures superpose with an rmsd of 0.6 Å, very similar models with ClusPro are obtained using 5T46. In this docking model, HRV2 2Apro contacts the so-called dorsal face of eIF4E on the other side of the molecule to the cap-binding region (Gruner et al., 2016; Peter et al., 2015). The dorsal face is also contacted by eIF4GI; however, the 2Apro binding site does not overlap that of eIF4GI. Instead, the 2Apro is positioned so that the examined residues L17 and Y32 can contact eIF4E at the top of the dorsal face. Thus, in detail, residue Y32 of the HRV2 2Apro interacts with eIF4E residues S83 and D127 in helix α2. Further analysis of the model

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Fig. 7. Y86 is also involved in the interaction with eIF4E and delays eIF4G cleavage. (A) No stable heterodimeric complex between eIF4E and HRV2 2Apro Y32A C106S is formed. Complex formation was analysed via SEC on a HiLoad 16/60 Superdex 200 prep grad as described in Fig. 2. (B) In vitro translation assay of the self-processing (upper picture) and eIF4GI cleavage of HRV1A VP1/2Apro Y86A (lower picture). The intramolecular cleavage efficiency is not affected by the Y86A mutation; however, eIF4GI cleavage is impaired (compare to Fig. 3A). (C) In vitro cleavage of 5 µM eIF4GII551-745 by 1600 nM HRV2 2Apro Y32A in the absence or presence of 5 µM eIF4E. (D) Cleavage of eIF4GI by HRV1A 2Apro Y86A at 9 h post infection was monitored as described in Fig. 5D in materials and methods, followed by Western blotting with anti-eIF4GI antibody.

2Apro Y86A and examined its properties. First, we investigated whether this mutant is able to bind eIF4E. The introduction of Y86A into the HRV2 2Apro C106S clearly inhibits heterodimeric complex formation with eIF4E (Fig. 7A), as no stable heterodimeric complex between eIF4E and HRV2 2Apro Y86A C106S could be observed on SEC and SEC-MALLS (Table 3); however, ITC did show a dissociation constant similar to the HRV2 2Apro C106S (Table 3), indicating that the mutation affects the koff rate for the interaction. The interaction between eIF4GII551-745 and HRV2 2Apro Y86A C106S, as measured by ITC, was similar to that seen with the wild-type (Table 3). However, a heterotrimeric complex eIF4GII551-745/eIF4E/HRV2 2Apro Y86A C106S could again be observed using SEC and SEC-MALLS (Table 2); surprisingly, this heterotrimeric complex has a molecular mass of 77 kDa, suggesting that the stoichiometry of the eIF4GII551pro Y86A C106S is 1:1:1 (Table 3). This is in 745/eIF4E/HRV2 2A contrast to the complexes formed with HRV2 2Apro wt and HRV2 2Apro C138S, for which the stoichiometry has remained unclear (Aumayr et al., 2015). The mutation Y86A in the HRV1A VP1/2Apro did not affect the intramolecular cleavage efficiency, with 50% of cleavage of the precursor protein VP1/2A between 20 and 30 min (Fig. 7B, upper panel). However, the cleavage of endogenous eIF4GI present in the RRL was again impaired (Fig. 7B, lower panel) compared to the HRV1A 2Apro wt (Fig. 3B, lower panel). HRV1A 2Apro Y86A could cleave 50% of eIF4GI after about 300 min (Fig. 7B, lower panel) whereas HRV1A 2Apro wt reaches 50% cleavage between 30 and 60 min (Fig. 3B, lower panel). Consistent with these results, no cleavage of eIF4GII551-745 with 10 ng

of HRV2 2Apro Y86A in the absence or the presence of eIF4E could be observed (Fig. S4C). In contrast to the HRV2 2Apro wt (Fig. 4A), 100 ng of HRV2 2Apro Y86A were required to obtain cleavage fragments in a similar time-frame (Fig. 7C). Finally, HRV1A virus carrying the Y86A mutation was prepared. As with the HRV1A 2Apro L17R mutant, we were again unable to generate sufficient virus stock for more than one time-point; therefore, we again chose the time-point of nine hours to examine viral replication of the HRV1A 2Apro Y86A mutant. With this mutant, eIF4GI cleavage in HeLa cells is again delayed compared to the wt (Figs. 7D, 5A). Nevertheless, the amount of eIF4GI cleaved at nine hours is higher than that observed with HRV1A 2Apro L17R or Y32A. 3.6. Role of eIF4E residues S83, R123 and D127 in the interaction with HRV2 2Apro Next, we aimed to investigate whether residues of eIF4E identified by the docking model influenced the interaction with the HRV2 2Apro. In the docking model, residues S83 and D127 of eIF4E interact with Y32A whereas residue R123 interacts with Y86A. We therefore mutated these residues in eIF4E to alanine and investigated their influence on the interaction with HRV2 2Apro C106S. In addition, D71 and H78 were also mutated to alanine as negative controls as these residues should, according to the model, not affect eIF4E binding to HRV2 2Apro C106S. All mutant proteins except for eIF4E S83A could be expressed and purified, even though the expression of eIF4E D127A was very poor. 131

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precluded formation of the heterodimeric complex eIF4E/HRV2 2Apro C106S as shown by SEC (Figs. 8B and C). 4. Discussion The host cell shut-off of eukaryotic protein synthesis resulting from the cleavage of eIF4G isoforms by 2Apro is essential for the successful replication of enteroviruses (Chase and Semler, 2012; Whitton et al., 2005). However, the exact molecular mechanism of this cleavage is still not fully understood. Several lines of evidence suggest that there are differences in the mechanism of eIF4G cleavage between genetic group A rhinoviruses on the one hand and genetic group B viruses and other enteroviruses on the other. First, during infection with the genetic A group viruses, HRV2 and HRV16, eIF4GI and eIF4GII are cleaved simultaneously (Seipelt et al., 2000); in contrast, HRV14, a genetic group B virus and PV cleave eIF4GII only after eIF4GI has been cleaved (Gradi et al., 1998b, 2003). Differences were also seen in vitro in RRLs; HRV1A and HRV2 2Apro cleave within 60 min whereas CV 2Apro and HRV14 2Apro do not complete cleavage within five hours of incubation (Baxter et al., 2006; Schmid, 2002). Second, the 2Apro of enteroviruses have been documented to differ substantially in their primary sequences (Fig. S5). These differences are reflected when the tertiary and quaternary structures are compared. Thus, the rmsd for Cα between the HRV2 (2HRV) and enterovirus 71 2Apro (PDB: 3W95) is relatively high at 1.28 Å over 134 residues; HRV2 and HRV1A 2Apro are homodimers whereas HRV4, CVB4 and EV71 2Apro are monomeric (Baxter et al., 2006; Falah et al., 2012; Wang et al., 1998). Third, eIF4E is known to stimulate HRV2 2Apro cleavage of eIF4G isoforms (Aumayr et al., 2015; Haghighat et al., 1996), presumably through a direct interaction of HRV2 2Apro with eIF4E to form a stable heterodimeric complex. In this paper, we characterized the eIF4E/HRV2 2Apro heterodimer and showed that it is necessary for efficient replication of a closely related genetic A group virus, HRV1A. Given that the formation of the eIF4E/HRV2 2Apro binary complex requires the dissociation of the HRV2 2Apro homodimer, we postulated that identification of the dimerization interface of HRV2 2Apro would indicate residues that would affect interaction between eIF4E and HRV2 2Apro. This was indeed the case, as the PISA algorithm proposed the region of HRV2 2Apro around residue C138 as the dimerization interface. All three mutations tested from this region (L17R, Y32A and C138S) affected the interaction of HRV2 2Apro with eIF4E but in different ways. Two of the mutations (L17R, Y32A) abrogated the interaction completely. In contrast, the C138S 2Apro was not only still able to form a complex with eIF4E but the formation of the 1:1 complex was complete after 10 min of incubation rather than an overnight incubation required by the wild-type. This suggests that the absence of a disulphide bridge between the HRV2 2Apro monomers might facilitate the formation of the complex eIF4E. Indeed, it is can be envisaged that the breakage of this bond is the rate-limiting factor in the formation of the eIF4E complex with the wt HRV2 2Apro. In addition, the C138S 2Apro showed a 30 fold tighter binding to eIF4GII551-745 as measured by ITC (Table 3), implying that the disulphide bond also hampered the interaction with the substrate protein. We then used the ClusPro protein-protein docking service (Comeau et al., 2004a, 2004b; Kozakov et al., 2013) to provide information on the interaction of eIF4E with HRV2 2Apro. This identified a further residue of HRV2 2Apro, Y86, as well as residues R123 and D127 of eIF4E as being important for this interaction. Indeed, mutation of all of these three residues also abrogated eIF4E/HRV2 2Apro complex formation. The mutant Y86A HRV2 2Apro mutant had an unusual property, as it was nevertheless still able to form a ternary complex with eIF4GII551-745 and eIF4E. Furthermore, the stoichiometry of the complex was 1:1:1 (Table 3); for the wild-type and the HRV2 2Apro C106S C138S, we have as yet been unable to determine the stoichiometry of the respective heterotrimeric complexes (Aumayr et al., 2015).

Fig. 8. eIF4E R123A and D127A mutant proteins that abrogate heterodimeric complex formation. Complex formation was analysed via SEC on a HiLoad 16/60 Superdex 200 prep grade column together with aprotinin (6.5 kDa) as an internal standard. 0.5 mg of the individual or complexed proteins were analysed together with 1 mg of aprotinin. Complex formation of two or more proteins was performed by incubation at 4 °C for 10 min. (A) eIF4E H78A and HRV2 2Apro C106S. (B) eIF4E R123A and HRV2 2Apro C106S. (C) eIF4E D127A and HRV2 2Apro C106S. Stable complexes (under conditions of SEC) were observed between the eIF4E H78 and HRV2 2Apro wt ((A), identical to wt, see Fig. 2A), but not between eIF4E R123A and HRV2 2Apro C106S and eIF4E D127A and HRV2 2Apro C106S.

All eIF4E mutants are still able to form stable complexes with eIF4GII551-745 (data not shown), indicating that they are correctly folded. As predicted, the mutations D71A or H78A in eIF4E did not affect complex formation with HRV2 2Apro C106S, independent of whether eIF4GII551-745 was present or not (Fig. 8A and data not shown). However, substitution of either R123 or D127 with alanine

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the eIF4GII, thereby stabilizing the ternary complex and localizing the protease to the required region of eIF4G. The above postulate that the binding of the genetic group A 2Apro to eIF4E affects the conformation of eIF4G is supported by previous data. First, it has been known for many years from the structure of the cocrystal of eIF4E and eIF4G that the binding of eIF4G to eIF4E affects the conformation of eIF4G (Marcotrigiano et al., 1997). We recently confirmed this observation with NMR studies (Aumayr et al., 2015). Second, experiments with the Leader proteinase (Lpro) of foot-andmouth virus (FMDV), an unrelated picornaviral proteinase with a papain-like fold, showed that the removal of eIF4E from RRLs could prevent Lpro cleavage of eIF4GI (Ohlmann et al., 1997). This suggested an effect on the conformation of eIF4GI. Aumayr et al. (2015) could show that, in vitro at least, eIF4E was required for Lbpro to form a ternary complex with eIF4E and eIF4G. In contrast to HRV2 2Apro, however, Lbpro only interacted with the C-terminus of eIF4E and failed to form a binary complex with eIF4E. Taken together, these results strongly imply that eIF4E is required for eIF4G to attain a conformation that promotes picornaviral cleavage. In summary, the above experiments indicate that the interaction between the genetic A group rhinoviruses HRV1A or HRV2 2Apro and the eIF4E is important for an efficient host-cell shut off in vivo. However, this mechanism is most certainly not applicable to HRV4 2Apro, CVB4 2Apro and probably all other picornaviral 2Apro proteases outside of the genetic A group rhinoviruses. These 2Apro do not interact with eIF4E (Fig. S6F and Aumayr et al. (2015)) and must therefore have a different mechanism to ensure efficient cleavage of eIF4G isoforms in vivo; experiments to illuminate this mechanism are currently being performed. It is tempting to speculate that the difference in the interaction of 2Apro with eIF4E might explain why HRV2 and HRV16 2Apro cleave both eIF4G isoforms at the same rate in infected HeLa cells whereas HRV14 and PV1 cleave eIF4GI more rapidly in contrast to eIF4GII (Gradi et al., 1998b; Seipelt et al., 2000; Svitkin et al., 1999). Further structural and biochemical experiments will be required to elucidate how HRV4 2Apro, CVB4 2Apro and related 2Apro that do not directly bind eIF4E perform efficient cleavage of eIF4G isoforms during viral replication.

The structures and assembly of these complexes will require further investigation, as will the interactions that stabilize the HRV2 2Apro dimer and how they are broken during formation of the eIF4E/HRV2 2Apro heterodimer. It should be noted that the HRV2 2Apro still behaved as a homodimer in the presence of the mutations discussed above, implying that the hydrogen bonds of the β-strands between the monomers may provide the greatest stabilisation of the dimer (Fig. 1). We then examined how these differences in binding to eIF4E were reflected in the cleavage of eIF4G. First, all mutants that failed to bind eIF4E required a higher concentration than the wt to efficiently cleave the eIF4GII551-745 fragment in vitro. When expressed in RRLs, all 2Apro mutants showed wild-type levels of self-processing; however, HRV1A 2Apro bearing mutations L17R, Y32A and Y86A were appreciably delayed in cleavage of full length eIF4GI. This was also the case when HRV1A viruses bearing these mutations were used to infect HeLa cells. In both assays, the mutations at L17 or Y32 had a more pronounced effect than that at Y86, presumably reflecting the fact that HRV2 2Apro could still form a heterotrimeric complex but not a heterodimeric one. These experiments illustrate that the stimulation of eIF4E on the picornaviral cleavage of eIF4G is not only observed in in vitro studies with recombinant proteins, but is also of importance in virus infected cells. There is one caveat to the investigation of the eIF4GI cleavage by the mutant 2Apro during HeLa cell infection, namely that we have not measured the amount of 2Apro synthesised. It is therefore possible that some of the effect observed may reflect a reduced synthesis of the mutant 2Apro compared to that of the wildtype. The roles of Y86 and L19 in 2Apro have been investigated previously without any clear conclusion. Y86 and its neighbour Y85, which together form the so-called dityrosine flap, were thought initially to participate in substrate recognition as these residues are oriented within a putative substrate binding groove (Sommergruber et al., 1992, 1997). Sommergruber et al. (1997) showed that mutating Y86 to phenylalanine or threonine did not interfere with the intramolecular cleavage reaction, but that it had a reduced cleavage efficiency on the trans eIF4G cleavage on HeLa cell extracts. Conversely, substitutions of the corresponding residues in PV 2Apro affected neither self-processing nor eIF4G cleavage activity. This differential effect of the mutations on eIF4G cleavage might again indicate a difference in the mechanisms of enteroviral 2Apro (Yu and Lloyd, 1992). The substitution of L17 with arginine, the residue found at the equivalent position (R20) in CVB4 2Apro, was shown by Foeger et al. (2003) to impair eIF4GI cleavage by HRV2 2Apro but not selfprocessing. However, it was suggested that these residues were crucial for binding directly to eIF4GI; at this time, a role for eIF4E was not considered. Pertinently, though, Baxter et al. (2006) showed that substitution of R20 with leucine improved cleavage of eIF4GI in RRLs. In the light of the results presented here, it will be of interest to investigate whether CVB4 2Apro R20L has an increased affinity for eIF4E. How can the cleavage of HRV2 2Apro be enhanced by the binding to eIF4E? To answer this question, we took advantage the very recently determined crystal structure of human eIF4E bound to residues 592– 653 of eIF4GI (PDB ID: 5T46; the cleavage site of HRV2 2Apro is at 681 so that it is not present in the structure). Superimposition of this structure onto our model (rmsd of 0.779 Å for the eIF4E residues) in Fig. 6 which has only 16 amino acid residues of eIF4GII revealed that the additional residues of eIF4GI do not compromise our model (Fig. S7). In fact, the C-terminal residues of the eIF4G fragments are shown in both the model and the superimposition to be directly below the heterodimeric interface, in close proximity to the HRV2 2Apro (Figs. 6 and S7). The intrinsically unstructured nature of eIF4G would allow the residues up to the cleavage site of eIF4GI or eIF4GII to be easily wrapped around the heterodimer interface to come close to the active site of the HRV2 2Apro and thus facilitate cleavage by HRV2 2Apro. During this interaction, eIF4E acts as a bridge to anchor the 2Apro to

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