Subash Chapagain, UBC Jan Lab

Subash Chapagain (Department of Biochemistry and Molecular Biology, UBC) Xu Zhong (Department of Earth, Ocean and Atmospheric Sciences, UBC) Kevin Low (Department of Biochemistry and Molecular Biology, UBC) Eric Jan (Department of Biochemistry and Molecular Biology, UBC)

Expressing & Multiplying

RNA viruses have evolved strategies to expand their limited coding capacity through translational recoding mechanisms such as frameshifting and stop-codon readthrough. We have recently revealed a subset of internal ribosome entry sites (IRES) from Dicistroviruses that can direct translation in alternative 1 open reading frame (ORFx). The evolutionary origin and diversity of this unique IRES-mediated alternative frame translation has not been investigated. In this study, using a database of dicistrovirus IRES-containing genomes (~4000), we found that ~30% genomes contain a potential 1 frame ORFx downstream of the IRES (>15 amino acids in length). We also identified 105 genomes that contained a potential 2 frame ORFy. Furthermore, 54 genomes contained predicted overlapping triple ORFs - the 0 frame ORF, 1 ORFx and 2 ORFy that may be IRES driven. Using bicistronic RNA reporter-based assays, we functionally screened a subset of these IRESs that can drive alternative reading frame translation. One IRES (6b-2) from a viral genome identified from Alaska wetland fen soil sample can drive translation in all three frames in rabbit reticulocyte and insect cell-free lysates and Drosophila S2 cells. Systematic mutational analysis showed that alternative frame translation was IRES-dependent and dependent on 0-frame initiation. Domain swapping with an IRES that cannot direct alternative reading frame translation revealed the tRNA-like anticodon:codon mimicry domain of the 6b-2 IRES is sufficient to drive ORFx/y translation. Together, these findings reveal a novel viral strategy whereby an IRES drives translation in all three frames, uncovering hidden viral ORFs that may modulate viral host interactions.