Structural and mechanistic insights into translation initiation on the enterovirus Type 1 IRES

Enteroviruses are a widespread and diverse set of pathogens responsible for over [~]1 billion human infections annually. Upon cell entry, translation of the virus genome is mediated by an internal ribosome entry site (IRES) in the 5 untranslated region (UTR). Despite early identification of the poliovirus IRES, structure determination has been hampered by the flexibility and complexity of the RNA ([~]450 nt, five structured domains), the requirement for almost all of the eukaryotic initiation factors (eIFs), and additional dependency on PCBP2 for trans-activation. As a result, there is little mechanistic information on how enterovirus Type 1 IRESs recruit and direct translational machinery. To address this, here we reconstitute human translation initiation on a model poliovirus IRES and examine 48S complexes by electron cryo-microscopy (cryo-EM). Our structures reveal a novel set of interactions between the IRES and the translation machinery. Domain IVc contacts uS19 and uS13 on the small ribosomal subunit, and the conserved apical GNRA tetraloop interacts directly with the initiator tRNA during start-codon recognition. We demonstrate that disruption of these contacts causes defects in IRES-driven translation and virus replication. Together, our results define the structural and molecular basis for translation initiation on the Type 1 IRES, and highlight the potential for RNA-RNA tertiary interactions as new targets for antiviral therapeutics.