The IRES(5′UTR) of the dicistrovirus cricket paralysis virus is a type III IRES containing an essential pseudoknot structure

Cricket paralysis virus (CrPV) is a dicistrovirus. Its positive-sense single-stranded RNA genome contains two internal ribosomal entry sites (IRESs). The 5′ untranslated region (5′UTR) IRES(5′UTR) mediates translation of non-structural proteins encoded by ORF1 whereas the well-known intergenic region (IGR) IRES(IGR) is required for translation of structural proteins from open reading frame 2 in the late phase of infection. Concerted action of both IRES is essential for host translation shut-off and viral translation. IRES(IGR) has been extensively studied, in contrast the IRES(5′UTR) remains largely unexplored. Here, we define the minimal IRES element required for efficient translation initiation in drosophila S2 cell-free extracts. We show that IRES(5′UTR) promotes direct recruitment of the ribosome on the cognate viral AUG start codon without any scanning step, using a Hepatitis-C virus-related translation initiation mechanism. Mass spectrometry analysis revealed that IRES(5′UTR) recruits eukaryotic initiation factor 3, confirming that it belongs to type III class of IRES elements. Using Selective 2′-hydroxyl acylation analyzed by primer extension and DMS probing, we established a secondary structure model of 5′UTR and of the minimal IRES(5′UTR). The IRES(5′UTR) contains a pseudoknot structure that is essential for proper folding and ribosome recruitment. Overall, our results pave the way for studies addressing the synergy and interplay between the two IRES from CrPV.