IRES-mediated ribosome repositioning directs translation of a +1 overlapping ORF that enhances viral infection

Abstract

AbstractRNA structures can interact with the ribosome to alter translational reading frame maintenance and promote recoding that result in alternative protein products. Here, we show that the internal ribosome entry site (IRES) from the dicistrovirus Cricket paralysis virus drives translation of the 0-frame viral polyprotein and an overlapping +1 open reading frame, called ORFx, via a novel mechanism whereby a subset of ribosomes recruited to the IRES bypasses downstream to resume translation at the +1-frame 13th non-AUG codon. A mutant of CrPV containing a stop codon in the +1 frame ORFx sequence, yet synonymous in the 0-frame, is attenuated compared to wild-type virus in a Drosophila infection model, indicating the importance of +1 ORFx expression in promoting viral pathogenesis. This work demonstrates a novel programmed IRES-mediated recoding strategy to increase viral coding capacity and impact virus infection, highlighting the diversity of RNA-driven translation initiation mechanisms in eukaryotes.Significance StatementViruses use alternate mechanisms to increase the coding capacity of their viral genomes. Here, we provide biochemical evidence that ribosomes recruited to the dicistrovirus cricket paralysis virus IRES undergo a bypass event to direct translation of a downstream +1 frame overlapping open reading frame, called ORFx. Mutations that block ORFx expression inhibit +1 frame translation and infection in fruit flies. These findings highlight the diversity of RNA-driven translation initiation mechanisms in eukaryotes.