The dicistrovirus intergenic region internal ribosome entry site (IRES) utilizes a unique mechanism, involving P-site tRNA mimicry, to directly assemble 80S ribosomes and initiate translation at a specific non-AUG codon in the ribosomal A site. A subgroup of dicistrovirus genomes contains an additional stem-loop 5′-adjacent to the IRES and a short open reading frame (ORFx) that overlaps the viral structural polyprotein ORF (ORF2) in the þ1 reading frame. Using mass spectrometry and extensive mutagenesis, we show that, besides directing ORF2 translation, the Israeli acute paralysis dicistrovirus IRES also directs ORFx translation. The latter is mediated by a U∶G base pair adjacent to the P-site tRNA-mimicking domain. An ORFx peptide was detected in virus-infected honey bees by multiple reaction monitoring mass spectrometry. Finally, the 5′ stem-loop increases IRES activity and may couple translation of the two major ORFs of the virus. This study reveals a novel viral strategy in which a tRNA-like IRES directs precise, initiator MettRNA-independent translation of two overlapping ORFs.frameshifting | Israeli acute paralysis virus | protein synthesis | pseudoknot | genetic recoding

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The dicistrovirus intergenic internal ribosome entry site (IGR IRES) directly recruits the ribosome and initiates translation using a non-AUG codon. A subset of IGR IRESs initiates translation in either of two overlapping open reading frames (ORFs), resulting in expression of the 0 frame viral structural polyprotein and an overlapping +1 frame ORFx. A U–G base pair adjacent to the anticodon-like pseudoknot of the IRES directs +1 frame translation. Here, we show that the U-G base pair is not absolutely required for +1 frame translation. Extensive mutagenesis demonstrates that 0 and +1 frame translation can be uncoupled. Ribonucleic acid (RNA) structural probing analyses reveal that the mutant IRESs adopt distinct conformations. Toeprinting analysis suggests that the reading frame is selected at a step downstream of ribosome assembly. We propose a model whereby the IRES adopts conformations to occlude the 0 frame aminoacyl-tRNA thereby allowing delivery of the +1 frame aminoacyl-tRNA to the A site to initiate translation of ORFx. This study provides a new paradigm for programmed recoding mechanisms that increase the coding capacity of a viral genome.

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The ''RNA world'' hypothesis rests on the assumption that RNA polymerase ribozymes can replicate RNA without the use of protein. In the laboratory, in vitro selection has been used to create primitive versions of such polymerases. The best variant to date is a ribozyme called B6.61 that can extend a RNA primer template by 20 nucleotides (nt). This polymerase has two domains: the recently crystallized Class I ligase core, responsible for phosphodiester bond formation, and the poorly characterized accessory domain that makes polymerization possible. Here we find that the accessory domain is specified by a 37-nt bulged stem-loop structure. The accessory domain is positioned by a tertiary interaction between the terminal AL4 loop of the accessory and the J3/4 triloop found within the ligase core. This docking interaction is associated with an unwinding of the A3 and A4 helixes that appear to facilitate the correct positioning of an essential 8-nt purine bulge found between the two helices. This, together with other constraints inferred from tethering the accessory domain to a range of sites on the ligase core, indicates that the accessory domain is draped over the vertex of the ligase core tripod structure. This geometry suggests how the purine bulge in the polymerase replaces the P2 helix in the Class I ligase with a new structure that may facilitate the stabilization of incoming nucleotide triphosphates.