Mechanistic insights into the recruitment of viral type 2 IRES to ribosomal pre-initiation complex and its interaction with eIF4 factors and PTB1

Abstract

Viruses often employ internal ribosome entry sites (IRESs) in their genomic RNA to hijack the host’s translational machinery. The IRES elements are characterized by unique secondary and tertiary RNA structures capable of recruiting initiation factors or ribosomal subunits to initiate translation. Based on their secondary structure and requirement of initiation factors, the viral IRESs have been classified into several types. The picornavirus, Encephalomyocarditis virus (EMCV) employs a type 2 viral IRES which requires the core of the 43S complex, the central domain of eIF4G, eIF4A, and an essential ITAF-polypyrimidine tract binding protein 1 (PTB1) to form the 48S preinitiation complex (48S PIC), major rate regulatory step in translation initiation. However, owing to the flexibility associated with the EMCV IRES, there is a dearth in mechanistic understanding of the assembly of translation initiation complex on the IRES such as how the 48S PIC is assembled, how PTB1 acts as a chaperone, and whether this role affects the interaction of IRES with eIF4 proteins.

To understand the mechanism of recruitment of EMCV IRES on the pre-initiation complex, we have used Cryo-EM to analyse the EMCV IRES-PTB1-48S PIC, isolated from the Rabbit Reticulocyte Lysate (RRL) using a pull-down approach. The cryo-EM map of EMCV IRES-bound 48S PIC shows densities corresponding to EMCV IRES domains, which reveal how the IRES contacts the ternary complex and 40S head at the inter-subunit interface. We have identified a possible mimicry mechanism in the domain I apical sequence that enables the IRES to interact with 40S ribosomal head proteins- uS13 and uS19. The structural details account for numerous biochemical studies on Type 2 IRES and suggest how Type 2 IRES would interact with 43S PIC to form 48S PIC. Thus, uncovering a unique mechanism of IRES recruitment to translation initiation complexes.

Besides, the EMCV IRES serves as a landing pad for proteins-eIF4G and eIF4B, and PTB1. However, how PTB1 stabilizes the IRES structure as a whole remains unknown. We have reconstituted the EMCV IRES-PTB1 complex and obtained a low resolution Cryo-EM map. Interestingly, the PTB1 binding sites on the IRES is in close proximity to eIF4G-binding site and the addition of PTB1, increased the affinity of EMCV IRES for eIF4G. Corollary, we used Alphafold3 to predict the structure of PTB1-IRES domains, which contributed to propose how PTB1 enhances the affinity of the IRES for eIF4G. Perhaps highlighting the necessity of PTB1 in 48S PIC formation on EMCV IRES.

Another eIF4 factor which stimulates the initiation event on EMCV IRES is eIF4B, which could be divided into 3 major regions in the human orthologue- RNA recognition motif (RRM), DRYG motif, and arginine-rich motif (ARM). Although, eIF4B has been characterized to interact non-specifically with RNAs via its ARM region, previous cross-linking experiments on Foot and Mouth disease virus (FMDV) IRES (type 2) have suggested the binding of eIF4B is sequence-dependent for this IRES. Therefore, we sought to check how eIF4B interacts with the IRES domains. Our studies on recombinant eIF4B showed a sequence-independent binding of eIF4B to the IRES via its ARM domain stimulated by the presence of C terminal tail and DRYG motifs. Moreover, human eIF4B can exist as a dimer, and this property is achieved by DRYG repeats. We found the N-terminal tail-RRM domain of eIF4B might disrupt the self-assembly in the absence of C C-terminal tail.

Overall, we have uncovered avenues related to type 2 IRESs with regard to its recruitment on the translation initiation complex, its interaction with eIF4G and PTB1; and explored the dynamics associated with human eIF4B with regard to its RNA-binding and self-association. The studies provide the structural foundation for understanding the mechanism of EMCV IRES-mediated translation initiation, which could be extrapolated to other IRESs sharing similar motifs and factor requirements, such as type 1 viral IRESs.