Several viruses encode an internal ribosome entry site (IRES) at the 5' end of their RNA, which, unlike most cellular mRNAs, initiates translation in the absence of a 5' m7GpppG cap. Here, we report a uniquely regulated translation enhancer found in the 739-nucelotide (nt) sequence of the Triticum mosaic virus (TriMV) leader sequence that distinguishes the preferred initiation site from a plethora of IRES-encoded AUG triplets. Through deletion mutations of the TriMV 5' untranslated region (UTR), we show that the TriMV 5' UTR encodes a cis-acting picornaviral Y₁₆-X₁₁-AUG-like motif with a 16-nt polypyrimidine CU-tract (Y₁₆), at a precise, 11-nt distance (X₁₁) from the preferred 13th AUG. Phylogenetic analyses indicate that this motif is conserved among potyviral leader sequences with multiple AUGs. Consistent with a broadly conserved mechanism, the motif could be functionally replaced with known picornavirus YX-AUG motifs and is predicted to function as target sites for the 18S rRNA by direct base pairing. Accordingly, mutations that disrupted overall complementarity to the 18S rRNA markedly reduced TriMV IRES activity, as did the delivery of antisense oligonucleotides designed to block YX-AUG accessibility. To our knowledge, this is the first report of a plant viral IRES YX-AUG motif, and our findings suggest that a conserved mechanism regulates translation for multiple economically important plant and animal positive single-stranded RNA viruses.IMPORTANCE Uncapped viral RNAs often rely on their 5' leader sequences to initiate translation, and the Triticum mosaic virus (TriMV) devotes an astonishing 7% of its genome to directing ribosomes to the correct AUG. Here we uncover a novel mechanism by which a TriMV cis-regulatory element controls cap-independent translation. The upstream region of the functional AUG contains a 16-nt polypyrimidine tract located 11 nt from the initiation site. Based on functional redundancy with similar motifs derived from human picornaviruses, the motif is likely to operate by directing ribosome targeting through base pairing with 18S rRNA. Our results provide the first report of a broad-spectrum mechanism regulating translation initiation for both plant- and animal-hosted picornaviruses.

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