The structure and mechanism of action of a distinct class of dicistrovirus intergenic region IRESs

Internal ribosomal entry sites (IRESs) engage with the eukaryotic translation apparatus to promote end-independent initiation. We identified a conserved class of ∼150 nt long intergenic region (IGR) IRESs in dicistrovirus genomes derived from members of the phyla Arthropoda, Bryozoa, Cnidaria, Echinodermata, Entoprocta, Mollusca and Porifera. These IRESs, exemplified by Wenling picorna-like virus 2, resemble the canonical cricket paralysis virus (CrPV) IGR IRES in comprising two nested pseudoknots (PKII/PKIII) and a 3′-terminal pseudoknot (PKI) that mimics a tRNA anticodon stem–loop base-paired to mRNA. However, they are ∼50 nt shorter than CrPV-like IRESs, and PKIII is an H-type pseudoknot that lacks the SLIV and SLV stem–loops that are primarily responsible for the affinity of CrPV-like IRESs for the 40S ribosomal subunit and that restrict initial binding of PKI to its aminoacyl (A) site. Wenling-class IRESs bound strongly to 80S ribosomes but only weakly to 40S subunits. Whereas CrPV-like IRESs must be translocated from the A site to the peptidyl (P) site by elongation factor 2 for elongation to commence, Wenling-class IRESs bound directly to the P site of 80S ribosomes, and decoding begins without a prior translocation step. A chimeric CrPV clone containing a Wenling-class IRES was infectious, confirming that the IRES functioned in cells.