Abstract
Taking control of the cellular apparatus for protein production is a requirement for virus progression. To ensure this control, diverse strategies of cellular mimicry and/or ribosome hijacking have evolved. The initiation stage of translation is especially targeted as it involves multiple steps and the engagement of numerous initiation factors. 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 initiation complex via an uAUG intermediate. The IRES features a novel extended, multi-domain architecture, that circles the 40S head. 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.
Highlights
Metagenomic studies of environmental samples have uncovered a great diversity of viruses that have a pervasive presence in the biosphere (Zhang et al, 2019; Zhang et al, 2018; Greninger, 2018)
In contrast to the well-characterized type IV family of Internal Ribosomal Entry Sites (IRES) found in the InterGenic Region (IGR-IRES) of these viruses, the 5’ UnTranslated Regions (5’-UTRs) of Dicistroviruses seem to harbor divergent sequences, making structural modelling based on sequence conservation difficult (Kieft, 2009)
In order to address this gap in knowledge, we produced a truncated version of the 5’-UTR region of the genomic RNA of the Cricket Paralysis Virus (CrPV) that contains the IRES in order to obtain structural information about its 40S-bound conformation by electron cryo-microscopy
Summary
Metagenomic studies of environmental samples have uncovered a great diversity of viruses that have a pervasive presence in the biosphere (Zhang et al, 2019; Zhang et al, 2018; Greninger, 2018). Viruses exploit the complexity of eukaryotic initiation to gain access to the host machinery for protein production (Jaafar and Kieft, 2019) Strategies such as mimicking the cap structure or transferring caps from cellular mRNAs (‘cap-snatching’) allow viral mRNAs to hijack host ribosomes, redirecting them towards the production of viral proteins (Jan et al, 2016; Jaafar and Kieft, 2019). A more prominent viral strategy for ribosome hijacking is the use of structured RNA sequences in viral mRNAs (Yamamoto et al, 2017) These sequences are called Internal Ribosomal Entry Sites (IRES), and a tentative classification based on their degree of RNA structure and dependency on canonical initiation factors divided them in four main types (Filbin and Kieft, 2009; Johnson et al, 2017). Two high-resolution cryo-EM reconstructions of 40S–5’-UTR-IRES–eIF3 complexes, combined with biochemical analysis, allowed us to characterize how this IRES uses an extended structure with a modular, multi-domain architecture to bind to and manipulate the 40S
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