1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics

Structural insights into the recruitment of viral type 2 IRES to ribosomal preinitiation complex for protein synthesis

  1. Deepakash Das
  2. Tanweer Hussain  Is a corresponding author
  1. Department of Developmental Biology and Genetics, Indian Institute of Science, India
https://doi.org/10.7554/eLife.107788.3
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  1. Deepakash Das
  2. Tanweer Hussain
(2026)
Structural insights into the recruitment of viral type 2 IRES to ribosomal preinitiation complex for protein synthesis
eLife 14:RP107788.
https://doi.org/10.7554/eLife.107788.3
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eLife Assessment

This manuscript offers valuable structural and mechanistic insights into the assembly of the Type II internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) and the translation initiation complex, revealing a direct interaction between the IRES and the 40S ribosomal subunit. A solid experimental strategy, combining cryo-EM analysis, complementary biochemistry, and detailed structural comparisons, provides mechanistic insights into IRES-based translation initiation systems. This paper will attract researchers in cap-independent translation, host-pathogen interactions, and virology.

https://doi.org/10.7554/eLife.107788.3.sa0

Abstract

Picornaviruses employ internal ribosome entry sites (IRESs) in their genomic RNA to hijack the host’s translational machinery. The picornavirus, encephalomyocarditis virus, employs a type 2 IRES present in its 5’ untranslated region (5’UTR) and requires 43S ribosomal preinitiation complex (PIC), the central domain of eukaryotic initiation factor (eIF) 4G, eIF4A, and an essential ITAF (IRES trans-acting factor)-polypyrimidine tract binding protein 1 (PTB1) to form 48S PIC. In this study, we have used cryo-electron microscopy (cryo-EM) to determine the structure of encephalomyocarditis virus (EMCV) IRES-bound mammalian 48S PIC in a scanning-arrested closed state at the start codon. The EMCV IRES domains contact initiator tRNA (tRNAi) and 40S head at the inter-subunit interface, which reveals an altogether unique mechanism used by viruses to capture host translational machinery for its protein synthesis. The tRNAi is held away from the 40S body in contrast to canonical cap-dependent translation while the domain I apical region of EMCV IRES mimics 28S rRNA of 60S to interact with 40S ribosomal head proteins uS13 and uS19. The structural analysis accounts for numerous previously reported biochemical studies on type 2 IRES and shows how type 2 IRES interacts with 43S PIC to form 48S PIC. This study provides mechanistic insights for understanding EMCV IRES-mediated translation initiation, which could be extrapolated to other IRESs sharing similar motifs and factor requirements, including type 1 viral IRESs.

Introduction

Eukaryotic translation initiation occurs in two different modes: Cap-dependent and Cap-independent. Cap-dependent translation initiation in eukaryotes can be divided into four major steps: (i) formation of 43S ribosomal PIC (40S subunit in complex with eIF1, eIF1A, eIF3, eIF5, and ternary complex [eIF2-GTP-tRNAi]); (ii) recruitment of 43S PIC to 5’end of capped mRNA, mediated by eIF4F complex (formed by eIF4E, eIF4G, and eIF4A) yielding 48S complex (48S PIC); (iii) scanning of 5’ untranslated region (5’UTR) of mRNA and start codon recognition; and (iv) joining of large 60S ribosomal subunit to form elongation-competent 80S ribosomal complex. Among these, major rate-limiting steps include the regulation of available tRNAi as the ternary complex (eIF2.GTP.Met-tRNAi) and recruitment of mRNA on 43S PIC (Jackson et al., 2010; Brito Querido et al., 2024a). Cryo-electron microscopy (cryo-EM) studies on eukaryotic canonical 48S PICs from yeast (Hussain et al., 2014; Llácer et al., 2015) to humans (Eliseev et al., 2018; Simonetti et al., 2020; Brito Querido et al., 2024b; Brito Querido et al., 2020; Petrychenko et al., 2025) have revealed various interactions among the initiation factors, mRNA, and ribosome in scanning (POUT or open) state and scanning-arrested (PIN or closed) states. In mammals, 48S PIC formation is mediated by the interaction of eIF3 and eIF4G (Villa et al., 2013) and recent attempts to understand the mammalian canonical 48S PIC could capture interactions of eIF4 proteins (eIF4G and eIF4A) with eIF3 (Brito Querido et al., 2020; Brito Querido et al., 2024a; Brito Querido et al., 2024b).

Alternatively, several positive strand RNA viruses use internal ribosome entry sites (IRESs), which are internal cis-acting sequences present in 5’UTR of mRNA that drive the assembly of translation initiation complex without the requirement of 7-methylguanosine cap (Martinez-Salas et al., 2017; Martínez-Salas et al., 2015), initially reported in picornaviruses such as poliovirus (PV) and encephalomyocarditis virus (EMCV) RNA genome (Jang et al., 1988; Pelletier and Sonenberg, 1988; Trono et al., 1988). As of 2020, the IRESbase database reported 1328 IRESs, out of which 554 were viral IRESs from 198 viruses (Zhao et al., 2020). These RNA elements have unique secondary and tertiary structures, which allow them to hijack the host translational machinery and promote translation initiation internally by recruitment of host ribosome, canonical eIFs, and IRES trans-acting factors (ITAFs) via multiple RNA-RNA and RNA-protein interactions (Lee et al., 2017; Lozano et al., 2018). The IRESs lack sequence homology and enfold different structural organization, thus requiring different eIFs for the assembly of 48S PICs (Jackson et al., 2010). The type 1 IRES (represented by PV) and type 2 IRES (EMCV) require almost all eIFs, except eIF4E or N-terminal of eIF4G for the formation of 48S PIC (Lozano and Martínez-Salas, 2015). However, initiation on the latter does not require scanning for start codon recognition (Kaminski et al., 1994; Sweeney et al., 2014). The type 3 IRES (hepatitis C virus [HCV]) does not require eIF4 factors or scanning for 48S PIC formation (Niepmann and Gerresheim, 2020). The type 4 IRES (Cricket paralysis virus [CrPV] intergenic IRES) initiates without the requirement of any eIFs and tRNAi (Johnson et al., 2017). The HCV IRES and CrPV intergenic IRES directly interact with host 40S ribosome; however, they differ in their mechanism of attachment. Where the CrPV IRES (type 4) mimics anticodon-codon interaction via pseudoknot 1 to bind with ribosome in an elongation-competent state (Petrov et al., 2016; Fernández et al., 2014), the HCV IRES (type 3) binds to the solvent side of 40S subunit by replacing eIF3 and directly interacts with expansion segment (ES7) of 18S rRNA and ribosomal proteins near mRNA exit channel, thus placing the initiation codon at the P-site (Niepmann and Gerresheim, 2020; Brown et al., 2022). While the structures of ribosome-bound type 4 (Spahn et al., 2004; Fernández et al., 2014; Muhs et al., 2015; Murray et al., 2016; Pisareva et al., 2018; Acosta-Reyes et al., 2019; Abaeva et al., 2023; Abaeva et al., 2020) and type 3 IRES (Spahn et al., 2001; Boehringer et al., 2005; Hashem et al., 2013; Yamamoto et al., 2014; Quade et al., 2015; Yamamoto et al., 2015; Yokoyama et al., 2019; Brown et al., 2022) have been determined, there is no structural information about type 2 and type 1 IRES and their mode of recruitment to 48S PIC.

The 5’UTR of EMCV, genus Cardiovirus in the Picornaviridae family (Gorbalenya et al., 2020), folds into various stem-loops numbered D-L, where domains H-L, followed by the initiation codon at the 834th residue (~450 nucleotides in length), make a functional IRES moiety (Carocci and Bakkali-Kassimi, 2012). It requires the core of 43S PIC, the central domain of eIF4G, eIF4A, and an essential ITAF-polypyrimidine tract binding protein 1 (PTB1), and the presence of eIF4B enhances 48S formation (Hellen and Wimmer, 1995; Martinez-Salas et al., 2017; Sweeney et al., 2014). Functional characterization of these domains showed H, and I interact with 40S ribosomal subunit (Chamond et al., 2014), and domain J-K recruits eIF4G, enhanced by eIF4A (Kolupaeva et al., 1998; Pestova et al., 1996; Lomakin et al., 2000). The structure of domain J-K-eIF4G1(HEAT1)-eIF4A revealed that positively charged patches on the eIF4G1-HEAT1 domain interact with two separated negatively charged clefts (in domains J and St) without perturbing its innate function of recruiting eIF4A (Imai et al., 2016; Imai et al., 2023). The binding sites for PTB1 were revealed to be dispersed, encompassing domains H to L (Kafasla et al., 2009). These domains function as a single entity to form 48S PIC, and any mutation in conserved IRES motifs can drastically affect the translation rates (Fernández-Miragall et al., 2009; Fernández et al., 2011). For example, the RAAA and GNRA loop in the domain I of type 2 IRES are crucial for IRES activity (Robertson et al., 1999), and the GNRA loop is also found in other IRES families such as type 1 (PV) and type 5 (Aichi virus A) picornaviruses (Abdullah et al., 2023). The inherent flexibility within the IRES domains provides a challenge for structural studies of full-length IRES and to capture IRES in the context of 48S PIC. EMCV IRES can independently interact with 40S subunit without any eIFs (Chamond et al., 2014) and does not require scanning to recognise the start codon (Pestova et al., 1996), unlike type 1 IRESs, which scan for the start codon (Sweeney et al., 2014). Besides, EMCV IRES can form 48S PIC with HEAT1-eIF4G without the requirement of eIF4G residues 1015–1104 that are known to interact with eIF3 (Lomakin et al., 2000). These residues are indispensable in case of canonical initiation on capped mRNAs and for type 1 IRESs (Villa et al., 2013; Sweeney et al., 2014). However, how EMCV IRES interacts with 40S subunit, or what molecular interactions are essential to form the EMCV IRES-48S PIC, remains a question.

In this study, we have used pull-down assay to isolate EMCV IRES-bound 48S PIC from rabbit reticulocyte lysate (RRL) and subjected the complex to cryo-EM analysis. The cryo-EM map of EMCV IRES-bound 48S PIC, henceforth mentioned as EMCV IRES-48S PIC, shows densities corresponding to EMCV IRES domains that reveal how EMCV IRES contacts the ternary complex and 40S head at the inter-subunit interface. The structural details presented here account for numerous biochemical studies that have been reported earlier on type 2 IRES (Pestova et al., 1996; Roberts and Belsham, 1997; López de Quinto and Martínez-Salas, 1997; Robertson et al., 1999; Fernández-Miragall and Martínez-Salas, 2003; Welnowska et al., 2011; Chamond et al., 2014; Kwon et al., 2017; Lozano et al., 2018; Maloney and Joseph, 2024). Furthermore, the structural analysis suggests how type 2 IRES would interact with 43S PIC to form 48S PIC. Importantly, the study reveals a unique strategy used by viral IRES to capture the host translational apparatus for making viral polypeptide.

Results

Overview and features of EMCV IRES-48S PIC

To isolate 48S PIC on EMCV IRES, we used a Talon affinity-based pull-down from nuclease-treated RRL. EMCV IRES RNA used harboured residues from 280 to 905 with AUG (start codon) at 834th position. PTB1 was recombinantly overexpressed with an N-terminal 6X His tag, followed by a 3C protease cleavage site. PTB1 was incubated with the IRES, followed by RRL addition, and 48S PIC was stalled using GMP-PnP. The complex was eluted from the Talon matrix employing 3C protease cleavage and pelleted (Figure 1—figure supplement 1A). The EMCV IRES-48S PIC was subsequently analysed by cryo-EM. The processed data yielded three major classes: (i) 40S without any factors (Map A), (ii) 40S-IRES-tRNAi (Map B), and (iii) 40S-IRES-ternary complex (Map B1), namely, EMCV IRES-48S PIC (Figure 1—figure supplement 1B; Table 1). Map B and Map B1 have an overall resolution of 4.6 Å and 5.0 Å, respectively (Figure 1—figure supplement 1B). The core of 40S is at around 4.0 Å, and the local resolution across the maps was largely in the range of 4.0–8 Å (Figure 1—figure supplement 2A and B). Only the extreme tip of beak of 40S in Map B1 (Figure 1—figure supplement 2A) and ends of IRES and eIF2γ are around 12 Å in Map B2 (Figure 1—figure supplement 2B).

Table 1
Cryo-electron microscopy (cryo-EM) data and model statistics.
Data collectionMap A – 40S without factorsMap B – 40S-tRNAi-EMCV IRESMap B1 – 40S-tRNAi-EMCV IRES-eIF2α-eIF2γ
MicroscopeTalos Arctica
CameraGatan K2 Summit Direct Detector
Magnification×36,000
Voltage (kV)200
Electron dose (e-/Å2)50–55
Defocus range–2 to –0.5
Pixel size (Å)1.17
Number of micrographs22549
Cryo-EM reconstruction
Final number of particles125,50355,23128,439
Point group symmetryC1C1C1
FSC threshold0.1430.1430.143
Map overall resolution (Å)4.514.555.01
Resolution metricGold-standard FSCGold-standard FSCGold-standard FSC
Sharpening B factor(Å2)–153.5–117.9–122.3
Atomic model refinement   
Resolution (0.5) (Å)5.05.46.0
D FSC model (0/0.143/0.5)4.4/4.5/5.04.4/4.5/5.44.9/4.9/6.0
Initial models used6YAN6YAN; 8OZ06YAN; 8OZ0
CC overall   
CC(mask)/(box)/(peaks)/(volume)0.73/0.83/0.72/0.660.77/0.84/0.71/0.770.78/0.84/0.72/0.78
Molprobity score2.32.282.23
Clash score19.0819.0918.64
No. of atoms/No. of residues   
Chains394139
Total atoms (Hydrogens:0)764558005783717
No. of residues – proteins/nucleotides4916/17444918/19125660/1912
Bond (RMSD) Lengths (Å)0.0020.0030.003
Bond (RMSD) Angles (⁰)0.6580.6500.659
Ramachandran plot (%)   
Outliers0.210.270.20
Allowed8.948.217.09
Favoured90.8691.5292.72
Rotamer outliers (%)0.020.020.00
Cβ outliers (%)0.000.000.00
CaBLAM outliers (%)6.095.825.47

The cryo-EM densities in EMCV IRES-48S PIC correspond to 40S, tRNAi, eIF2α, eIF2γ, and RNA in the mRNA channel, along with an extra density connecting 40S ribosomal head to tRNAi (Figure 1A and B). However, it lacks distinct density for PTB1, eIF4G, eIF4A, and eIF3, and hence these factors are not modelled. Since nuclease-treated RRL lacked endogenous RNA, the presence of density for mRNA in the channel indicates trapping of EMCV IRES in 48S PIC. Also, the density connecting 40S head to tRNAi in Maps B and B1 could be assigned to a double-stranded RNA structure found in EMCV IRES (Figure 1A and B).

Figure 1 with 2 supplements see all
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Features of EMCV IRES-48S PIC.

(A) Different views of encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-48S preinitiation complex (PIC) (Map B1) by 45° rotation along one axis. The map shows densities assigned to 40S ribosome, RNA (in channel), ternary complex, and IRES density at the inter-subunit region of head. (B) View of Map B and zoomed view of densities corresponding to IRES and initiator tRNA (tRNAi).

Besides, an extra density is evident at the mRNA entry site, contacting 40S ribosomal proteins uS3, eS10, and uS5, as well as h16 (helix 16) of 18S rRNA (Figure 1—figure supplement 2C). In the human 48S PIC, this region positions eIF3g-RRM (RNA recognition motif) and downstream ORF (Figure 1—figure supplement 2D; Brito Querido et al., 2020; Brito Querido et al., 2024b). We anticipate that this extra density in EMCV IRES-48S PIC could be contributed by downstream ORF; however, it is difficult to assign it to eIF3g-RRM due to the lack of eIF3 (core/peripheral) density. Furthermore, our recent study on the interaction of yeast eIF4B with 40S ribosome suggested occupancy of eIF4B (N-terminal-RRM) in the same region (Datey et al., 2025), which opens up the possibility of mammalian eIF4B-NT-RRM to bind to this region in the absence of eIF3, although in the human 48S PIC eIF4B was tentatively positioned slightly away from this location (Brito Querido et al., 2024b). An interesting possibility could be positioning one of the RRM domains of PTB1 bound to UCUUU sequence of 18S rRNA (PTB1 can bind to UCUUU sequence; Maris et al., 2020) present at the tip of h16 (528–532 nucleotides) of 18S rRNA. An extra density in the same position is also present in Map A (40S without EMCV IRES or factors; Figure 1—figure supplement 2E) as well, and PTB1 was used as a bait for the pull-down; therefore, this density may be contributed by PTB1-RRM interacting with h16 of 18S rRNA.

The EMCV IRES-48S PIC is trapped in a closed conformation

In EMCV IRES, A-834-U-835-G-836 (AUG-834) forms the start codon (Kaminski et al., 1994; Hellen and Wimmer, 1995; Pestova et al., 1996). Previous experiments based on toeprints of EMCV IRES-48S PIC assembly suggested A-826-U-827-G-828 (AUG-826) as the codon where 48S PIC can assemble and AUG-834 as the start codon (Pestova et al., 1996). Furthermore, the intensity of the toeprint at AUG-834 was much higher than at AUG-826 (Pestova et al., 1996; Sweeney et al., 2014). Also, AUG-834 is present in a Kozak context (CRCCaugG; R is a purine) (Kozak, 1989), where the –3 position is A-831 and +4 is G-837 (Figure 2—figure supplement 1D), whereas AUG-826 is present in a poor Kozak context. In this work, the EMCV IRES construct used has both AUG-826 and AUG-834. Based on the above-mentioned reports, poor and strong Kozak context of AUG-826 and AUG-834, respectively, and placement of AUG-834 at the P-site in EMCV IRES-40S binary complex (Chamond et al., 2014), we reason AUG-834 to base-pair with the anticodon in EMCV IRES-48S PIC (Figure 2A), and the flanking nucleotide residues were added as per the sequence (Figure 2—figure supplement 1D). The recognition of the start codon at the P-site by tRNAi leads to accommodation of ternary complex, and 48S PIC adopts a PIN state in contrast to POUT state observed during scanning (Llácer et al., 2015; Llácer et al., 2021; Yi et al., 2022; Petrychenko et al., 2025). A distinct difference of ~7.0 Å could be seen by comparing the tRNAi position of EMCV IRES-48S PIC with that in the open state (Figure 2B), depicting a PIN state of tRNAi. The entrapment of the start codon in the P-site also evokes closure of the mRNA latch formed by 18S rRNA helices – h34 of 40S head and h18 of body (Hussain et al., 2014; Llácer et al., 2015; Hinnebusch, 2017). The h34 of 18S rRNA of EMCV IRES-48S PIC is shifted by 9 Å as compared to the human canonical open state 48S PIC (PDB Id – 7QP6, Yi et al., 2022). This conformation of 18S rRNA correlates well with that of closed 48S PIC (PDB Id – 7QP7, Yi et al., 2022), suggesting EMCV IRES-48S PIC was captured in a closed state (Figure 2C). This closed conformation locks the mRNA in the channel formed within 40S head and body. On the other hand, the conformation of 18S rRNA in Map A (40S ribosome without initiation factors) shows an open state (Figure 2—figure supplement 1E). The transition from open (PDB Id – 7QP6) to closed states (PDB Id – 7QP7) structures is also accompanied by an upward shift of N-terminal domain of eS17 (connecting the head to body) by ~10 Å (Yi et al., 2022). The EMCV IRES-48S PIC structure correlates with the conformation of eS17 in the human 48S-closed PIC, i.e., eS17 N-terminal helix associated with the ribosomal head shifts upward by ~10 Å, keeping C-terminal domain position constant (Figure 2D; Figure 2—figure supplement 1F).

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Closed conformation of EMCV IRES-48S PIC.

(A) Fitting of initiator tRNA (tRNAi)-base paired to start codon AUG (left). Zoomed view of codon-anticodon interaction (middle), and B-factor for codon-anticodon interaction (right). (B) The tRNAi in encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-48S preinitiation complex (PIC) is in PIN state similar to that in human 48S PIC PIN state (PDB Id – 7QP7) and its anticodon shows ~7 Å shift as compared to human 48S PIC open state (PDB Id – 7QP6). (C) Comparison of ribosomal conformation (18S rRNA) of EMCV IRES 48S PIC with human open and closed state (PDB Id – 7QP6 and 7QP7, respectively). Focusing on the entry site and the helices governing latch conformation, helix 34 moves toward helix 18 by 9 Å. (D) Movement of eS17 in open and closed states of ribosome. Zoomed view of comparison between eS17 in EMCV IRES 48S PIC and human 48S PIC open state, showing an upward shift of the N-terminal domain by ~10 Å.

The IRES density connecting 40S head to tRNAi is contributed by the domain I apical part

In EMCV IRES-48S PIC complex, we observed a density, likely for double-stranded RNA, connecting 40S head to the tRNAi elbow region (Figure 3A). The extra density interacts with the elbow region and acceptor stem of tRNAi and ribosomal proteins: uS19 (RPS15) and uS13 (RPS18) (nomenclature; Ban et al., 2014). The EMCV IRES contains four major stem-loops (H-L) in the functional IRES region (Figure 3B; Hellen and Wimmer, 1995). Among these, domains H and I have been shown to interact with 40S (Chamond et al., 2014), and mutations of conserved residues in these domains severely compromise translation on EMCV IRES (López de Quinto and Martínez-Salas, 1997; Hellen and Wimmer, 1995). Moreover, incubation of foot and mouth disease virus (FMDV) IRES, an EMCV-like type 2 IRES, with 40S ribosomes has shown a decrease in SHAPE reactivity in its domain 3 apex (Lozano et al., 2018), which corresponds to EMCV IRES domain I apex. We reasoned that domains H and I may contribute to the double-stranded RNA density emanating from 40S head. The density architecture (Figure 3C) could be interpreted as a long main stem (S1) extending away from 40S where the base is anchored to the ribosome by two branches (B1 and B2) and to tRNAi by one branch (B3), further divided into two sub-branches (B3a and B3b). Visualising the IRES RNAfold-determined secondary structure (Figure 3D, which correlates with the experimental structure proposed in Duke et al., 1992), this architecture could be contributed by the apical part of domain I.

Figure 3 with 2 supplements see all
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Deciphering the IRES density.

(A) Extra density connecting the head of 40S to initiator tRNA (tRNAi) elbow region is contributed by encephalomyocarditis virus (EMCV) internal ribosome entry sites (IRES) RNA in Map B1. Rotated views of RNA density showing its connection to the 40S head via uS13 and uS19 and to tRNAi via its elbow region. (B) Organisation of EMCV IRES domains from D to L, where H-L makes the functional IRES moiety (Hellen and Wimmer, 1995). (C) Deciphering the architecture of the obtained IRES density. The density could be interpreted as a long main stem (S1) extending away from the ribosome, where the base is anchored to the ribosome by two branches (B1 and B2) and to tRNAi by one branch (B3), further divided into two sub-branches (B3a and B3b). (D) Secondary structure of apical region of domain I (made using RNAfold) marking the stem and branches, along with imported loops. (E) Fitting of domain I apex in the density (rotated views). The sub-domains are coloured as proposed in C,D.

To determine the tertiary structure of the domain I apex, the IRES region from nucleotides 507–619 was modelled using AlphaFold3 (Figure 3—figure supplement 1A). The model was decomposed and reconstructed based on the best fit in the obtained density for the IRES using Chimera and Coot (Figure 3—figure supplement 1A). The final model was generated after multiple rounds of geometry correction and real space refinements (Figure 3E). The final model holds a correlation coefficient of 0.8 with respect to the map (Figure 3—figure supplement 1B), where B1 is AAG loop, B2 is CAAA loop, B3a – GCGA loop, B3b – C-rich loop1, and S1 is the major double-stranded stem of domain I. The B-factor of the modelled IRES largely ranges from 124 to 200 (Figure 3—figure supplement 1C). To check the possibility of other IRES domains that might contribute to the extra density, AlphaFold3 was used to predict the tertiary structure of isolated EMCV IRES domains (Figure 3—figure supplement 2A–J), using sequences as shown in Supplementary file 1. The predicted tertiary structure of domain H or experimental models of domains D to F did not fit in the observed IRES density (Figure 3—figure supplement 2K–L). The domain J-K adopts a Y-shaped structure, and placement of its cryo-EM (PDB Id – 8HUJ) or NMR (PDB Id – 2NBX) structure in the density would clash with 40S (Figure 3—figure supplement 2M). Moreover, in the EMCV IRES-48S PIC, domain J-K binds eIF4G, and the location of eIF4G has been mapped close to ES6 of 18S rRNA, located near the left foot 40S ribosome (Yu et al., 2011). The domain I apex model in EMCV IRES-48S PIC shows the RAAA and AAG motif contacts uS19 and uS13, and the GNRA loop with tRNAi (Figure 4A). In addition, incubation of EMCV IRES with RRL protected domain I apex regions, including the CAAA loop in the SHAPE reactivity profile (Maloney and Joseph, 2024). These interactions with 40S head and tRNAi could be facilitated by the long length and flexible nature of domain I.

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EMCV IRES domain I apex contacts uS13, uS19 and tRNAi.

(A) Model showing connections of domain I apex with uS13, uS19, and initiator tRNA (tRNAi). (B) uS13 interacts with B3 branch or sub-domain of internal ribosome entry site (IRES) via its alpha helix (100–117 residues). (C) Multiple points of contacts between uS19 and domain I motifs: RAAA and AAG. The electrostatic potential map of uS19 suggests that encephalomyocarditis virus (EMCV) IRES interacts via ionic interactions with its phosphate backbone. (D) Sequence alignment of h38 with domain I apex of EMCV IRES showing sequence identity. Overlapping of uS19 from 80S (PDB Id – 4UG0) and EMCV IRES-48S preinitiation complex (PIC) show that the interaction of uS19 with EMCV IRES is similar to interaction with that of h38. (E) The fit of GNRA or GCGA stem and its contact with tRNAi at the elbow region and acceptor stem.

The interaction of domain I of EMCV IRES with ribosomal proteins and initiator tRNA

The domain I is the longest domain in EMCV IRES, which harbours important motifs such as GNRA, RAAA, and C rich, crucial for IRES activity (Roberts and Belsham, 1997; Fernández-Miragall and Martínez-Salas, 2003) and conserved across all cardioviruses (Hellen and Wimmer, 1995). We mutated the GNRA loop and RAAA loop in EMCV IRES and checked for luciferase activity using a firefly luciferase reporter downstream of the wild-type and mutant IRESs. We found a drastic reduction in the luciferase activity in the mutants as compared to that of the wild-type (Figure 4—figure supplement 1A), which correlates with previous studies that showed the importance of these motifs in regulating IRES activity (Roberts and Belsham, 1997; López de Quinto and Martínez-Salas, 1997; Robertson et al., 1999; Fernández-Miragall and Martínez-Salas, 2003). Among these, the CAAA and AAG motifs share potential contact sites with uS13 and uS19. The alpha helix (100–117 residues) of uS13 contacts the IRES element at the B3 stem (connecting the GNRA loop to RAAA loop) (Figure 4B). uS19 contacts the IRES majorly at its CAAA motif through multiple sites involving N-terminal residues, residues 67–75, and C-terminal 102–124 (Figure 4C). These regions of uS13 and uS19 are rich in basic residues, which might interact with the negatively charged backbone of the IRES element (Figure 4B and C). The role of uS13 and uS19 also involves the formation of inter-subunit bridges during 60S joining to form elongation-competent 80S complexes. uS13 interacts with uL5 (RPL11) and uS19 with helix 38 or h38 (1748–1778, in humans) in 28S rRNA to form inter-subunit bridges B1b/c and B1a, respectively (Figure 4—figure supplement 1B; Ben-Shem et al., 2011; Bowen et al., 2015; Khatter et al., 2015). These interactions are dynamic, owing to ribosomal subunit rotation and swivelling during 80S ribosomal translocation states (Khatter et al., 2015).

On superimposition of 80S ribosomal structure to EMCV IRES-48S PIC model, the IRES density clashes with the position of uL5 and h38 of 28S rRNA (Figure 4—figure supplement 1C), suggesting repositioning of IRES domain from 40S head during 48S to 80S transition. Interestingly, the h38 residues interacting with uS19 share considerable similarity in sequence to domain I apex in the EMCV IRES (Figure 4D), suggesting the domain I apex of EMCV IRES could mimic h38 (60S)-40S interaction (Figure 4D). The similarity of h38 with the domain I residues provides additional support for annotation of domain I apex in the density.

The GCGA (GNRA motif, where N is any nucleotide and R is a purine) is known for long-range RNA-RNA interactions, widespread in ribosomal RNA and in some catalytic RNAs. It forms a characteristic U-turn structure (Fiore and Nesbitt, 2013) and interacts with minor grooves of helical RNA elements (Figure 4—figure supplement 1D; Reiter et al., 2010). A single-point mutation within this tetraloop (GCGA to GCGC) severely reduced the IRES activity, suggesting it is essential for IRES activity (Roberts and Belsham, 1997; Fernández-Miragall and Martínez-Salas, 2003). The density extending from the elbow region of tRNAi could fit in the characteristic U-turn, adopted by conventional GNRA motifs. In EMCV IRES, the GNRA motif is represented by GCGA loop, preceded by a C-rich region and in close contact with the tRNAi elbow and acceptor stem (Figure 4E). Thus, we infer that EMCV IRES interacts with tRNAi by virtue of its GCGA loop.

The position of eIF2-ternary complex is shifted towards 40S head in EMCV IRES-48S PIC in contrast to canonical 48S PIC

We could fit eIF2α and eIF2γ in their respective densities in Map B1 (Figure 5A). Focussed classification or refinement did not yield any distinct density corresponding to the position of eIF2β, probably due to the flexibility associated with repositioning of eIF2β during transition from open to closed complexes (Llácer et al., 2015; Llácer et al., 2021; Yi et al., 2022; Petrychenko et al., 2025). Previous reports on EMCV IRES suggested its direct interaction with eIF2 (Scheper et al., 1991; Scheper et al., 1994) and inactivation of eIF2 compromises EMCV IRES-mediated translation (Welnowska et al., 2011; Kwon et al., 2017), indicating EMCV IRES’s dependence on the canonical ternary complex. Here, we observe a direct interaction of EMCV IRES with ternary complex via tRNAi, a feature not observed in previously determined HCV (Yamamoto et al., 2014; Yamamoto et al., 2015; Brown et al., 2022) and CrPV IRES-bound ribosomal structures (Acosta-Reyes et al., 2019; Pisareva et al., 2018).

Figure 5
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State of ternary complex in EMCV IRES-48S PIC.

(A) Inter-subunit view of encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) 48S preinitiation complex (PIC) showing position for ternary complex on map. Fitting of eIF2α and eIF2γ in its corresponding density in Map B1. (B) Overlapping EMCV IRES 48S PIC on mammalian late-stage 48S PIC (PDB Id – 6YAN) indicates a shift in position of eIF2γ towards 40S head. (C) Zoomed view showing the positions of eIF2γ, initiator tRNA (tRNAi), and eIF2α in the EMCV IRES 48S PIC relative to those in the mammalian late-stage 48S PIC (PDB ID 6YAN). The black arrow indicates the shift in position. (D) Superimposition of the EMCV IRES-48S PIC on the human late-stage 48S PIC (before 60S joining; PDB Id – 8PJ5), showing the conformation of tRNAi in association with eIF2 and IRES and tRNAi with eIF5B in the canonical context, respectively. (Right) Zoomed view of tRNAi conformation in both complexes.

As found in canonical 48S PICs (Brito Querido et al., 2024b; Petrychenko et al., 2025), eIF2α-domain 1 is in close contact with ribosomal protein uS7, and domain 2 with tRNAi elbow and domain 3 with eIF2γ in EMCV IRES-48S PIC. The position of eIF2γ and eIF2α-domain 3 (D3) is distinct from mammalian 48S-closed PICs (PDB Id – 6YAN and 7QP7) as we could observe a shift by ~10 Å towards the head of 40S on superimposing the 18S rRNA from EMCV IRES 48S PIC and mammalian late-stage 48S PIC (Figure 5B). The ternary complex is flexible, and it moves away from ribosomal head towards the body on recognition of authentic start codon as studied in yeast (Villamayor-Belinchón et al., 2024) and human (Petrychenko et al., 2025). This opposite directional shift of the ternary complex in the EMCV IRES-48S PIC is evident in tRNAi acceptor stem as well (Figure 5C). This shift could be due to the rigid stem B3 (consisting of G-C base pairs), connecting 40S head to GNRA loop, which interacts with the tRNAi at its elbow and acceptor arm, and the association of eIF2α-D3 and eIF2γ with the acceptor arm of tRNAi orchestrated with the conformational change.

During the transition of 48S PIC to 80S elongation-competent complex, there are major changes in the conformation of tRNAi due to the joining of eIF5B, and release of eIF2 (Petrychenko et al., 2025). This joining event of eIF5B positions the tRNAi elbow and acceptor stem towards the 40S body to aid 60S ribosomal subunit joining (Petrychenko et al., 2025). However, in the context of EMCV IRES-48S PIC, we have seen the positioning of tRNAi elbow and acceptor stem towards the 40S head, away from the body (Figure 5C). On superimposing the human 48S PIC structure (before 60S joining), 48S-5 (PDB Id – 8PJ5; Petrychenko et al., 2025), we could see that tRNAi in EMCV IRES-48S PIC is away from the canonical tRNAi position (in contact with eIF5B) (Figure 5D). Therefore, we anticipate a change in tRNAi conformation during eIF5B joining and eIF2 release. Furthermore, the IRES (domain I) interacting with the tRNAi elbow needs to be displaced from the position to facilitate the interaction of tRNAi with eIF5B, and this rearrangement would also aid in 60S joining and avoid steric clash with the IRES domain I.

Discussion

The mode of binding of IRES recruitment on 40S ribosomal PIC varies among different types of IRESs. CrPV intergenic IRES binds to 40S by mimicking tRNA-mRNA interaction with the help of pseudoknot 1 (Petrov et al., 2016; Fernández et al., 2014; Murray et al., 2016; Acosta-Reyes et al., 2019), whereas HCV IRESs associate with the solvent side of 40S body by replacing eIF3 with its domain 3 (Spahn et al., 2001; Hashem et al., 2013; Niepmann and Gerresheim, 2020; Brown et al., 2022). In this study, we capture EMCV IRES in 48S PIC context where the domain I of EMCV IRES interacts with 40S head and tRNAi elbow stem (Figure 6A; Figure 6—video 1). The sequence similarity of h38 of 28S rRNA with domain I and its ability to interact with uS19-N terminal via RAAA motif (Khatter et al., 2015) suggests molecular mimicry by EMCV IRES for its recruitment to the ribosome. Moreover, the conservation of domain I apex sequence and motifs (RAAA, AAG loop, C-rich, and GNRA) across cardioviruses, including Theiler’s murine encephalitis virus (TMEV), Vilyuisk human encephalomyelitis virus (VHEV), Theiler’s-like rat virus (TRV), and Saffold viruses 1 and 2 (SAFV-1 and SAFV-2), as well as apthoviruses (AAG loop is replaced by ACG loop) such as FMDV (Hellen and Wimmer, 1995; Liang et al., 2008), suggests that these IRESs might adopt similar strategies for 48S formation (Figure 6B and C). Like EMCV IRES, the type 1 IRES (PV, coxsackievirus, etc.) also harbours the GNRA loop, preceded by a C-rich loop at its longest domain, known for long-range RNA-RNA interactions. The segment harbouring GNRA loop is highly conserved across the type 1 family of IRESs (Kim et al., 2015). The domain I of EMCV IRES is similar to domain IV of polioviral IRES or other type 1 IRESs in terms of length, secondary structure, and conserved motifs (GNRA, C-rich) positioning (Figure 6C). Therefore, we anticipate a similar interaction of domain IV (in type 1 IRES class) with tRNAi. Also, this interaction of IRES with tRNAi could be a strategy by which these IRESs can sequester the tRNAi pool in the cell, rendering them unavailable for capped cellular mRNAs. During the revision of this work, a preprint reported a structure of polioviral IRES-48S PIC (Velazquez et al., 2025), which shows that domain IV apex (similar to domain I apex in EMCV IRES) interacts with uS13 and uS19, and the GNRA loop directly interacts with tRNAi during start codon recognition (Velazquez et al., 2025) as observed in EMCV IRES-48S PIC. Similarly, the Aichi virus IRES (type 5; Abdullah et al., 2023) harbours a GNRA loop in its longest domain, which is domain J. Deletion of the GNRA loop compromises the IRES activity; however, substitution mutations in this region either elevate the IRES activity or it remains unaltered (Yu et al., 2011). We hypothesise that Aichi virus IRES might use this motif to mediate long-range interactions with tRNAi, similar to type 1 and type 2 IRESs, as all these IRESs require eIF2-ternary complex for the formation of 48S PIC.

Figure 6 with 1 supplement see all
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Summarising the known interactions of EMCV IRES domains in context to 48S PIC.

(A) Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) secondary structure depicting the role of binding partners for each domain in context to translation initiation. The known tertiary structure of EMCV IRES and its binding partners are depicted. The domains responsible for binding PTB1 are boxed. (B) Conservation of domain I apex sequence and secondary structure across type 2 IRES family. (C) Comparison of secondary structure of EMCV and foot and mouth disease virus (FMDV) IRES to that of polioviral IRES (type 1).

The EMCV IRES does not require scanning, and the start codon (A-834) is directly placed in the P-site, which would eventually place the domain L at the mRNA exit site, preceded by domain J-K that interacts with eIF4G-eIF4A (Figure 6A). Earlier, biochemical studies suggested eIF4G to be positioned close to ES6 of 18S rRNA in EMCV IRES-bound 48S PIC (Yu et al., 2011). The human 48S PIC with a 5’ capped mRNA showed a similar location for eIF4G, that is at the mRNA exit site contacting eIF3 (Brito Querido et al., 2020; Brito Querido et al., 2024b). Locating eIF4F has been challenging due to the inherent flexibility associated with the eIF4F complex on mRNA and requires association with eIF3 in canonical 48S context (Brito Querido et al., 2020; Brito Querido et al., 2024b). However, the canonical eIF3-eIF4G interaction (Villa et al., 2013) is dispensable for EMCV IRES-48S PIC formation (Lomakin et al., 2000; Sweeney et al., 2014), and no density for eIF3 was observed even after focused classification. However, after the initial submission of this work, a preprint reported a structure of reconstituted EMCV IRES-48S PIC where eIF3 is present at the canonical position (Bhattacharjee et al., 2026). This position of eIF3 suggests the possibility that eIF4G-eIF4A proteins could be placed similarly to the canonical eIF3-eIF4G-eIF4A position (Brito Querido et al., 2024b) in context to EMCV IRES-48S PIC, thus placing eIF4G-domain J-K close to ES6 of 40S ribosome. This plausible placement of eIF4G in EMCV IRES-48S PIC corroborates well with the previous hydroxyl radical cleavage assay experiment that traced the location of eIF4G in context to EMCV IRES-48S PIC (Yu et al., 2011). In addition to initiation factors, the ITAF-PTB1 serves as an essential ITAF for 48S PIC formation on EMCV IRES, but the obtained map shows no distinct density to PTB1. PTB1 binds to the base of domains H and I, and domain K loop (Kafasla et al., 2009; Dorn et al., 2023), and the flexibility associated with these domains might have hindered capturing of PTB1 in the reported 48S complex (Figure 6A). However, the unassigned extra density at the mRNA entry site could be contributed by PTB1-RRM interacting with 18S rRNA, as discussed previously.

The structural studies on type 2 IRESs have been limited due to their flexible nature. In the cryo-EM map and structural analysis presented here, we could capture a part of EMCV IRES in 48S context (Figure 6A) and fetch a significant understanding of ribosomal recruitment by EMCV IRES and 48S PIC formation. As mentioned above, the cryo-EM map of reconstituted EMCV IRES-48S PIC (Bhattacharjee et al., 2026) shows similar findings, where the density for the apical portion of domain I of the IRES is only observed and it interacts with uS13 and uS19 on 40S ribosome, and tRNAi. A higher resolution of the reconstituted EMCV IRES-48S PIC (3.2 Å) helped the authors to identify interactions of individual IRES nucleotides with their binding partners (Bhattacharjee et al., 2026). In the future, the entrapment of additional factors would significantly provide more insights into EMCV IRES 48S PIC. The conservation of secondary structures and motifs such as GNRA within the picornaviruses (type 1, type 2, and Aichi virus IRESs) suggests common strategies of interaction in the context of 48S PICs in the Picornaviridae family.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Homo sapiens)PTB1Nucleotide databaseNM_002819.53C protease site (LEVLFQGP) was inserted at the N-terminal and inserted into pET28a in between BamHI and HindIII restriction sites, retaining the N-terminal 6X Histidine tag
Gene (Human Rhinovirus)HRV_3C proteaseProtein database1CQQ_A
Gene (Encephalomyocarditis virus)EMCV IRESNucleotide databaseNC_001479.1280–905 nucleotides
Gene (Photinus pyralis)LUCIFERASESnapgenepGL3-basicFirefly luciferase
Recombinant DNA reagentpcDNA3.1 (Plasmid)SnapgenepcDNA3.1Vector
Recombinant DNA reagentpET28a (Plasmid)SnapgenepET28aVector
Strain, strain background (Escherichia coli)BL21 (DE3)Recombinant protein expression strainMaintained in the lab
Commercial assay, kitRiboMAX Large Scale RNA Production SystemsPromegaP1280In vitro transcription kit
Commercial assay, kitRabbit Reticulocyte Lysate, Nuclease-TreatedPromegaL4960Lysate used for pull-down
Commercial assay, kitLuciferase substrate- Steady-Glo Luciferase Assay SystemPromegaE2510

Plasmid constructs and molecular cloning

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EMCV IRES 905 was obtained from EMCV-L plasmid- EMCV IRES (nt 280–905) into pCR2.1 and inserted in pcDNA3.1 using BamHI and XbaI restriction sites. PTB1 was cloned from HEK293 cDNA, and 3C protease site (LEVLFQGP) was inserted at the N-terminal and inserted into pET28a in between BamHI and HindIII restriction sites, retaining the N-terminal 6X Histidine tag.

EMCV IRES-Luciferase constructs: The firefly luciferase gene was inserted downstream of EMCV IRES in-frame with A-834 residue of IRES to generate the wild-type EMCV IRES-Luciferase construct in pcDNA3.1 (WT-Luc). The CAAAA (RAAA) loop and GCGA (GNRA) loop were mutated to GCTGA and TACG, as per functional assay reports for FMDV IRES and EMCV IRES to generate RAAAmut-Luc and GNRAmut-Luc, respectively (López de Quinto and Martínez-Salas, 1997; Fernández-Miragall and Martínez-Salas, 2003). The sequence of oligos or primers used is listed in Supplementary file 1.

Protein overexpression and purification

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PTB1 (Histidine tag-3Cprotease site PTB1 gene) was overexpressed using 0.5 mM IPTG in E. coli BL21 cells at 30°C, 120 rpm for 4 hr in Luria broth. 2 l harvested culture was lysed using Buffer N1 (20 mM HEPES pH 7.4, 300 mM KCl, 2 mM MgCl2, 10% glycerol, 10 mM imidazole, 5 mM β-mercaptoethanol, 0.05% Triton X-100, 2 mM PMSF) and sonicated at 18% amplitude, 10 s ON and 20 s OFF pulses, 30 cycles and centrifuged at 20,000 rpm for 20 min, and the supernatant was loaded on Ni-NTA column and eluted using a gradient of N1 to N2 buffer (20 mM HEPES pH 7.4, 300 mM KCl, 2 mM MgCl2, 10% glycerol, 500 mM imidazole, 5 mM β-mercaptoethanol). PTB1 was eluted at 250 mM imidazole concentration. The eluant fractions (diluted to 100 mM KCl) were further loaded on Heparin column and eluted by applying a gradient of 100 mM to 1000 mM KCl. The protein was eluted at 250 mM KCl concentration. The eluant fractions were stored at –80°C after size exclusion chromatography in Buffer S (20 mM HEPES pH 7.4, 200 mM KCl, 2 mM MgCl2, 5% glycerol, 1 mM DTT) at 2.5 mg/ml.

3C protease was overexpressed using 0.5 mM IPTG in E. coli BL21 cells at 30°C, 120 rpm in Luria broth. 2 l harvested culture was lysed using Buffer N11 (20 mM HEPES pH 7.4, 200 mM KCl, 2 mM MgCl2, 10% glycerol, 10 mM imidazole, 5 mM β-mercaptoethanol, 0.05% Triton X-100, 2 mM PMSF) and sonicated at 18% amplitude, 10 s ON and 20 s OFF pulses, 30 cycles and centrifuged at 20,000 rpm for 20 min, and the supernatant was loaded on Ni-NTA column and eluted using a gradient of N11 to N22 buffer (20 mM HEPES pH 7.4, 200 mM KCl, 2 mM MgCl2, 10% glycerol, 500 mM imidazole, 5 mM β-mercaptoethanol). 3C protease was eluted at 250 mM imidazole concentration. The eluant fractions were subjected to size exclusion chromatography in Buffer S (20 mM HEPES pH 7.4, 100 mM KOAc, 2 mM MgCl2, 10% glycerol, 1 mM DTT), and peak fractions were concentrated and stored at 2.5 mg/ml.

In vitro transcription of EMCV IRES

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EMCV IRES 905-pcDNA3.1 was linearised using XbaI restriction enzyme and transcribed using Promega-RiboMAX Large Scale RNA Production Systems as per the manufacturer’s protocol. 1 µg of linearised plasmid yielded 87 µg of RNA after DNase treatment and RNA cleanup (RNA clean up kit, NEB). WT-Luc, GNRAmut-Luc, and RAAAmut-Luc were linearised using XhoI and transcribed in vitro using the same strategy.

Assembly of EMCV IRES 48S PIC using Talon affinity chromatography

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12 µg of IRES was heated at 95°C to dissolve any secondary structures acquired and refolded using Buffer R (20 mM HEPES pH 7.4, 150 mM KOAc, 2 mM MgCl2, 2 mM β-mercaptoethanol, 0.25 mM spermidine) at 37°C for 5 min, and PTB1 (1:2=IRES:PTB1) was added and further incubated at 30°C for 5 min. Simultaneously, RRL (Promega) was incubated at 30°C with ATP, amino acid mix minus leucine, murine RNase inhibitor for 5 min, and then mixed with the IRES-PTB1 vial with instant addition of 6 mM GMP-PnP per reaction (100 µl) and incubated at 30°C for 8 min, followed by ice incubation. The reaction was loaded onto Talon beads equilibrated with Buffer A (20 mM HEPES, pH 7.4, 150 mM KOAc, 2 mM MgCl2, 4% glycerol, 5 mM imidazole, 2 mM β-mercaptoethanol). After recommended passes, reloading, and incubation, the flowthrough was collected and the beads were washed with Buffer A until A260 attains a baseline value (~0), following which 3C protease in Buffer A was added to the beads and left for overnight incubation. Fractions were eluted using Buffer A (400 µl each in 10 vials) and subjected to analysis such as A260 measurements and agarose gel electrophoresis. Samples having the RNA bands were pelleted using a 1 ml sucrose cushion (20 mM HEPES pH 7.4, 150 mM KOAc, 2 mM MgCl2, 30% sucrose, 1 mM DTT) in SW60 tubes and centrifuged at 50,000 rpm for 10 hr at 4°C. The pellet was resuspended in 20 µl Buffer R and used for cryo-EM grid preparation (no crosslinker was used to avoid artifacts).

Negative stain analysis and mass spectrometry

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The final sample was diluted 10 times using Buffer R and applied on 400-mesh Cu TEM grids, which were freshly glow-discharged (negative polarity) for 30  s in GloQube glow-discharge system, stained using 1% uranyl acetate solution, and analysed using Talos L120C transmission electron microscope at ×57,000 magnification. Furthermore, the sample was digested by trypsin and subjected to NanoOrbitrap analysis for the identification of proteins in the complex.

Cryo-EM sample preparation

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3 µl of resuspended pellet (2.96 A260) was applied on glow-discharged Quantifoil R 1.2/1.3 300 mesh 2 nm carbon-coated grid and blotted using 8 s and 8.5 s blot time, zero blot force at 16°C, and 100% humidity and plunged into liquid ethane. Cryo-EM data were collected on Talos Arctica transmission electron microscope equipped with an FEG at 200 kV (Thermo Fisher Scientific). All data were collected using a Gatan K2 Summit Direct Detector at a nominal magnification of ×36,000, and a pixel size of 1.17 Å with a total electron dose of 55 e-/Å2 fractionated over 20 frame movies with a dose rate of ~2.5 e-/Å2/frame.

Data processing

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Micrographs were collected and processed using CryoSPARC v3.3 (Punjani et al., 2017). The micrographs were patch motion-corrected and CTF was estimated. Using a blob picker, particles with an average diameter of 300 Å were picked and extracted. The extracted particles were subjected to multiple rounds of 2D classification. Final 2D classes showing promising ribosome 2D features were selected for ab initio reconstruction. The junk was discarded, and the 237,054 good particles were classified into two classes using a mask around tRNAi. We obtained two classes: Empty 40S (1.25L particles – Class 1 or Map A) and 40S with tRNA bound (1.11L particles – Class 2). Class 2 was further classified into two classes using a 3D mask around the IRES density and subjected to non-uniform refinement with global CTF refinement to yield Map B, having tRNAi and IRES density (55k particles). This was subjected to non-uniform refinement (Punjani et al., 2020) with global CTF refinement. Map B class was further classified using a mask around eIF2α and eIF2γ to yield two classes: 40S-tRNAi-IRES-eIF2 (28k particles – Map B1) and 40S-tRNAi-IRES (26k particles). The obtained maps were then subjected to model building and refinement. The local resolution for the obtained maps was estimated using PHENIX (Liebschner et al., 2019).

Map analysis and model building

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To the obtained maps, 40S ribosome (PDB Id – 6YAN) was fitted using UCSF Chimera. The 40S head (head proteins+18S rRNA [1197–1688]+eS17) and 40S body (body proteins+18S rRNA [1–1196; 1689–1870]) was fitted to the obtained maps separately and subjected to rigid body fit and real space refinement using PHENIX (Liebschner et al., 2019). The models were merged using Coot (Emsley et al., 2010) and subjected to real space refinement. tRNAi and mRNA models were taken from PDB Id – 8OZ0 and fitted to the maps B and B1 in Chimera. eIF2α and eIF2γ from PDB Id – 8OZ0 were rigid body fitted to Map B1 and then mutated as per Rabbit eIF2 protein sequence (NCBI Reference Sequence XP_002719561.1; XP_051683593.1) and subjected to a final real space refinement. The IRES domain I apex model was predicted from AlphaFold3 (Abramson et al., 2024) and the helical sub-domains were dismantled and fitted according to best fit using Chimera (Pettersen et al., 2004), followed by chain joining in Coot with manual real space refinement (Afonine et al., 2018). The geometry was corrected using Geometry minimisation tool in PHENIX with rounds of real space refinement. Other domain tertiary structures were predicted using AlphaFold3. The final model yielded 40S-EMCV IRES-tRNAi (Map B) and 40S-EMCV IRES-tRNAi-eIF2αγ (Map B1). All the figures were made using ChimeraX (Pettersen et al., 2021). The models were real space refined using PHENIX, and the Fourier shell correlation for ‘map to model’ for each was determined at 0.5 FSC (Figure 1—figure supplement 2A–C; Table 1).

Luciferase assay

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WT-Luc, GNRAmut-Luc, and RAAAmut-Luc RNA were subjected to polyadenylation using E. coli Poly(A) Polymerase and ATP supplied in New England Biolabs kit using the manufacturer’s protocol. Post polyadenylation, the RNA was extracted using RNA cleanup kit (NEB). The RNA was denatured at 95°C and refolded using Buffer R at 37°C for 5 min. To 1 µg of RNA, 10 µl RRL was added with 0.25 µM amino acid mix minus leucine, and amino acid mix minus methionine, 1 mM ATP, 0.5 mM GTP, and the remaining volume was adjusted using Buffer R to a final volume of 30 µl. Each reaction was divided into three sets: 10 µl each and incubated at 30°C for 3 hr. The luciferase activity in each reaction was measured by adding 10 µl of Steady-Glo Luciferase reagent (Promega) and quantified using a Tecan plate reader. The graphs were plotted using GraphPad prism.

Secondary structure determination and multiple sequence alignments

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The secondary structure for domain I apex was obtained from RNAfold (Gruber et al., 2008). Multiple sequence alignments were performed using Clustal Omega (Sievers and Higgins, 2021). Sequence accession number for various sequence used: EMCV (NC_001479.1), FMDV (NC_039210.1), TMEV (DQ401688.1), TRV (AB090161.1), VHEV (M80888.1), SAFV (FM207487.1), poliovirus (NC_002058.3).

Data availability

Maps and atomic coordinates of the 40S ribosome (Map A), 40S ribosome-EMCV IRES-tRNAi (Map B) and 40S ribosome-EMCV IRES-ternary complex (Map B1) have been deposited in the EMDB database with accession codes EMD-64646, EMD-64644, and EMD-64645, respectively and in the PDB database with accession codes 9UZM, 9UZK, and 9UZL, respectively.

The following data sets were generated
    1. Das D
    2. Hussain T
    (2026) EMDataResource
    ID EMD-64646. 40S ribosome without initiation factors.
    https://www.emdataresource.org/EMD-64646
    1. Das D
    2. Hussain T
    (2026) EMDataResource
    ID EMD-64644. EMCV IRES captured on mammalian 40S with initiator tRNA.
    https://www.emdataresource.org/EMD-64644
    1. Das D
    2. Hussain T
    (2026) EMDataResource
    ID EMD-64645. EMCV IRES captured on mammalian 40S ribosome with initiator tRNA and eIF2.
    https://www.emdataresource.org/EMD-64645
    1. Das D
    2. Hussain T
    (2026) Worldwide Protein Data Bank
    EMCV IRES captured on mammalian 40S with initiator tRNA.
    https://doi.org/10.2210/pdb9UZK/pdb
    1. Das D
    2. Hussain T
    (2026) Worldwide Protein Data Bank
    EMCV IRES captured on mammalian 40S ribosome with initiator tRNA and eIF2.
    https://doi.org/10.2210/pdb9UZL/pdb

References

    1. Emsley P
    2. Lohkamp B
    3. Scott WG
    4. Cowtan K
    (2010) Features and development of Coot
    Acta Crystallographica. Section D, Biological Crystallography 66:486–501.
    https://doi.org/10.1107/S0907444910007493
    1. Johnson AG
    2. Grosely R
    3. Petrov AN
    4. Puglisi JD
    (2017) Dynamics of IRES-mediated translation
    Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 372:20160177.
    https://doi.org/10.1098/rstb.2016.0177

Article and author information

Author details

  1. Deepakash Das

    Department of Developmental Biology and Genetics, Indian Institute of Science, Bangalore, India
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0009-0000-3138-4033

  2. Tanweer Hussain

    Department of Developmental Biology and Genetics, Indian Institute of Science, Bangalore, India
    Contribution
    Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing
    For correspondence
    hussain@iisc.ac.in
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4735-2380

Funding

Wellcome Trust/DBT India Alliance (IA/I/17/2/503313)

  • Tanweer Hussain

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission.

Acknowledgements

EMCV IRES (nt 280–905) into pCR2.1 was a kind gift from Dr. Bruno Sargueil, CNRS UMR8015, Université Paris Descartes, France, and 3C protease-pET24 was a kind gift from Prof. Raghavan Varadarajan, Indian Institute of Science (IISc), India. We thank various central facilities, namely Cryo-EM, Mass Spectrometry, and Computational Cluster at the Division of Biological Sciences, IISc, for support. DD acknowledges the DBT-JRF Programme for fellowship. The authors acknowledge the DST-FIST support to the department. This work was supported by the Intermediate Fellowship from DBT-Welcome Trust India Alliance to TH (IA/I/17/2/503313).

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© 2025, Das and Hussain

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  1. Deepakash Das
  2. Tanweer Hussain
(2026)
Structural insights into the recruitment of viral type 2 IRES to ribosomal preinitiation complex for protein synthesis
eLife 14:RP107788.
https://doi.org/10.7554/eLife.107788.3

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