A divergent mechanism of initiation mediated by Nedicistrovirus and related intergenic region (IGR) IRESs

Abstract

The canonical mechanism of translation initiation is coordinated by >10 initiation factors and involves 5’-end ribosomal attachment. Many viral mRNAs use alternative initiation mechanisms that involve 5’ end-independent binding of ribosomal complexes to an internal ribosome entry site (IRES). The simplest example occurs on the ~200 nt-long intergenic region (IGR) in the genomes of dicistroviruses, e.g. Cricket Paralysis virus (CrPV). CrPV-like IGR IRESs adopt a triple pseudoknot (PK) structure that mediates factor-independent initiation at a non-AUG start codon. Stem loops (SL) IV and V in PKIII interact with the proteins eS25 and uS7 of the 40S ribosomal subunit, and the conserved L1.1 loop in PKII interacts with the L1 stalk of the 60S subunit. PKI is inserted in the decoding center of the 40S subunit, mimicking an authentic tRNA-mRNA interaction. PKI’s placement prevents eukaryotic elongation factor (eEF) 1A•GTP/aminoacyl-tRNA (aa-tRNA) from binding to the ribosomal A site, which occurs only after a “pseudotranslocation” step mediated by eEF2. Recent advances in metagenomics have led to the identification of novel dicistrovirus-like viruses with shorter, structurally distinct IGRs. Nedicistrovirus (NediV) and Antarctic picorna-like virus 1 (APLV-1) IGRs are ~160 nt long and lack key motifs found in canonical IGR IRESs. Importantly, the IGRs of both viruses are shorter than all known IGR IRESs, but do resemble CrPV-like IRESs structurally albeit with some significant differences. In vitro reconstitution experiments demonstrated that these novel NediV-like IGRs form elongation-competent 80S ribosomal complexes in the absence of factors and/or tRNA and initiate from a non-AUG codon. These NediV-like IRESs bind directly to the P site of 80S ribosomal complexes and not 40S subunits alone, leaving the A site accessible. The NediV IRES promotes translation in mammalian cell-free extracts only after preincubation with purified ribosomal subunits and functions at both low and high [Mg2+]. NediV IRES-mediated translation is enhanced during experimentally induced stress conditions. Bioinformatic approaches were used to generate structural models of the NediV-like IRESs, which were generally validated by mutagenesis and chemical/enzymatic probing experiments. Domain exchange experiments demonstrated that the NediV-like IRESs have a modular nature with discrete ribosome-binding and tRNA mimicry domains, and that structural differences between the NediV-like and CrPV-like IRESs directly contribute to mechanistic differences between the two IGR IRES groups. These NediV-like IGR IRESs bypass the eEF2-mediated “pseudotranslocation” step and thus initiate translation by a mechanism even simpler than that used by CrPV-like IGR IRESs. The unique differences in both structure and mechanism and subsequent identification of candidate IRESs support the conclusion that NediV and APLV-1 are members of a novel IGR IRES class.