A complex IRES at the 5'-UTR of a viral mRNA assembles a functional 48S complex via an uAUG intermediate.

RNA viruses are pervasive entities in the biosphere with significant impact in human health and economically important livestock. As strict cellular parasites, RNA viruses abuse host resources, redirecting them towards viral replication needs. Taking control of the cellular apparatus for protein production is a requirement for virus progression and diverse strategies of cellular mimicry and/or ribosome hijacking evolved to ensure this control. Especially in complex eukaryotes, translation is a sophisticated process, with multiple mechanisms acting on ribosomes and mRNAs. The initiation stage of translation is specially regulated, involving multiple steps and the engagement of numerous initiation factors some of them of high complexity. The use of structured RNA sequences, called Internal Ribosomal Entry Sites (IRES), in viral RNAs is a widespread strategy for the exploitation of eukaryotic initiation. Using a combination of electron cryo-microscopy (cryo-EM) and reconstituted translation initiation assays with native components, we characterized how a novel IRES at the 5-UTR of a viral RNA assembles a functional translation initiation complex via an uAUG intermediate, redirecting the cellular machinery for protein production towards viral messengers. The IRES features a novel extended, multi-domain architecture, circling the 40S head, leveraging ribosomal sites not previously described to be exploited by any IRES. The structures and accompanying functional data, illustrate the importance of 5-UTR regions in translation regulation and underline the relevance of the untapped diversity of viral IRESs. Given the large number of new viruses metagenomic studies have uncovered, the quantity and diversity of mechanisms for translation hijacking encrypted in viral sequences may be seriously underestimated. Exploring this diversity could reveal novel avenues in the fight against these molecular pathogens.