The Triticum Mosaic Virus Internal Ribosome Entry Site Relies on a Picornavirus-Like YX-AUG Motif To Designate the Preferred Translation Initiation Site and To Likely Target the 18S rRNA

Abstract

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.

Keywords: 5′ UTR; IRES; YX-AUG; cap independent; plant virus; potyvirus; translation.

FIG 1

The 5′ and 3′ borders of the TriMV 5′ UTR are necessary for the TriMV IRES activity. (A) Schematic diagram of the bicistronic dual-luciferase reporter RNA and the positions of the insertions of the RNA element tested. Translation of the renilla luciferase gene is cap mediated, but translation of the downstream firefly luciferase gene can be directed only by internal initiation driven by the RNA sequence inserted into the intergenic region. The internal initiation ability is quantified as the ratio of firefly luciferase to renilla luciferase activities. The ratio of firefly to renilla luciferase activities in wheat germ extract of the bicistronic mRNAs was determined by a m7 GpppG cap at the 5′ end and the TriMV 5′ UTR (construct 1-739) or the nonfunctional TriMV reverse complementary sequence (construct 739-1) or the deletion mutants in the intergenic region. (B) Schematic diagram of monocistronic firefly luciferase reporter RNA containing a stable hairpin insertion (ΔG = –34 kcal) immediately at the 5′ end of the mRNA (12). The relative luciferase activities in wheat germ extract of the TriMV 5′ UTR deletion mutants with the strong hairpin is shown and is relativized to that of the TriMV wild-type sequence. (C) Relative luciferase activity in oat protoplasts of the reporter mRNAs containing the full-length TriMV leader (construct 1-739 SL) or the deletion mutant missing the last 30 nt (construct 1-709 SL) as 5′ UTR with the strong hairpin, normalized to values for a m7GpppG-capped polyadenylated renilla mRNA used as an internal control, which we coelectroporated at a 1:10 ratio. As a control, we included the full-length TriMV leader sequence as 5′ UTR in the absence of the strong stem-loop (construct 1-739 noSL) and the nonfunctional TriMV reverse complementary sequence (construct 739-1 SL).

FIG 2

The functional AUG is part of a functional equivalent of the picornavirus YX-AUG motif. (A) The relative luciferase activity in wheat germ extract of the TriMV 5′ UTR with the deletion of the last 30 nucleotides (1-709) or with the last 30 nt replaced with unrelated human β-globin sequence (30-nt b-globin) is relativized to that of the TriMV wild-type sequence in the presence of the strong hairpin. (B) The TriMV 5′ UTR sequence (GenBank accession number FJ669487) bears an Y₁₆X₁₁-AUG like motif at positions nt 710 to 739, upstream of the correct AUG. The Y is a 16-nt polypyrimidine tract. X is the spacer sequence with 11 random nucleotides. These motifs are commonly found in picornavirus IRESes, exemplified with the motifs in PV type I IRES Y₉X₁₈-^aAUG-X₁₅₄-^bAUG, and in the EMCV type II IRES Y₉X₁₀-^aAUG-X₅-^bAUG (10). The motifs contain a cryptic ^aAUG triplet, which is out of frame of the downstream correct ^bAUG. The truncated motifs are missing all the sequences downstream of the cryptic AUG. The AUG of the luciferase corresponds to the boxed AUG codon. (C) The relative luciferase activity in wheat germ extract (on the left) and in oat protoplasts (on the right) of the chimeric TriMV 5′ UTRs with the last 30 nt being replaced with the mammalian YX-AUG motif counterparts is relativized to that of the TriMV wild-type sequence in the SL-mRNA reporter. For the oat protoplast assays, the reporter mRNAs were coelectroporated with a m7GpppG capped polyadenylated renilla mRNA used as an internal control at a 1:10 ratio. (D) The relative luciferase activity in wheat germ extract of the TriMV 5′ UTR sequence with an extended (X17, X25, and X122) or reduced (X2) spacer length is relativized to that of the TriMV wild-type sequence, in the presence of the strong hairpin. (E) The relative luciferase activity in wheat germ extract of the TriMV 5′ UTR sequence with an upstream AUG inserted 2 bases downstream of the CU-tract at position nt 727 and placed out of frame or in frame with the downstream luciferase AUG.

FIG 3

The TriMV YX-AUG motif likely functions as target sites for the 18S rRNA binding (A) Sequence showing the two adjacent putative binding sites (box A and box B) of the TriMV IRES at positions nt 711 to 720 and nt 721 to 728 to a highly conserved region of the 18S rRNA at positions nt 1123 to 1140. (B) The mutated bases within the TriMV box A 18S rRNA target site that either partially or fully decreased (AΔ3, AΔ4, AΔ6, and AΔ10), increased (A-15/BΔ5) base pairing interaction or strengthened (A10-GC with the wobble base pairs within box A replaced with GC pairs) are shaded. The wild-type sequence is annotated as “wt A-10/B-8”. The relative luciferase activity in wheat germ extract (on the left) and in oat protoplasts (on the right) of the different TriMV mutants is relativized to that of the TriMV wild-type sequence. For the oat protoplast assays, the reporter mRNAs were coelectroporated with a m7GpppG capped polyadenylated renilla mRNA used as an internal control at a 1:10 ratio. (C) The mutated bases within the TriMV box A and box B 18S rRNA target sites that reduced the overall base pairing interaction and the CU-richness of the region are shaded. The wild-type sequence is annotated as “wt A-10/B-8”. The relative luciferase activity of the TriMV 5′ UTR with different mutations across the box A and box B 18S rRNA binding sites in wheat germ extract is relativized to that of the wild-type sequence in the presence of the strong hairpin. (D). trans-Inhibition assay of the TriMV 5′ UTR SL-mRNA with increasing molecular excess of antisense single-stranded DNA oligonucleotides targeting the box A (anti-BoxA) or both box A and box B (anti-BoxB) 18S rRNA binding sites in wheat germ extract. As a control DNA oligonucleotides targeting unrelated human β-globin sequence was added. A 0- to 100-fold molar excess of the antisense oligonucleotides was added to the reaction.

FIG 4

The TriMV YX-AUG motif can be substituted with putative 18S rRNA binding sites from unrelated viruses. The sequence of the viral motifs used to swap the last 30 nt of the TriMV 5′ UTR is shown. Sequences are from Blackcurrant reversion virus (BRV) RNA 2 (accession no. NC_003502.1; nt 115 to 161), Hibiscus chlorotic ringspot virus (HCRV; accession no. X86448; nt 2494 to 2512), and Barley yellow dwarf virus (accession no. NC_004750.1; nt 4837 to 4853). (A) The relative luciferase activity in wheat germ extract of the chimeric TriMV 5′ UTRs with the last 30 nt being replaced with unrelated viral sequence with reported putative 18S binding sites is relativized to that of the TriMV wild-type sequence in the SL-mRNA reporter. (B) The relative luciferase activity in wheat germ extract of the chimeric TriMV 5′ UTRs with the last 30 nt replaced with the 19-nt HCRV sequence (+19nt HCRV) or a modified 19-nt HCRV sequence (+19nt mutated HCRV) with mutations that disrupted the putative 6-nt complementary sequence to the 18S rRNA is relativized to that of the TriMV wild-type sequence in the SL-mRNA reporter. (C) trans-Inhibition assay of the chimeric TriMV+19nt HCRV SL-mRNA with increasing molecular excess of antisense single-stranded DNA oligonucleotides targeting the 19-nt HCRV sequence (anti-HCRV) in wheat germ extract. As controls, DNA oligonucleotides targeting unrelated human β-globin sequence and the native TriMV box A + box B were added. A 0- to 100-fold molar excess of the antisense oligonucleotides was added to the reaction.

FIG 5

Sequence alignment of different potyvirus 5′ UTRs bearing multiple AUGs. Highlighted in gray shades are the CU-rich tract identified to close proximity of the last AUG codon. Putative motifs found closest to the first AUG codon are indicated by asterisks. Sequence symbols: *, putative motif found in the first AUG; …, stretch of non-polypyrimidine-rich sequence.

FIG 6

A 35-nt sequence from the BVMoV leader sequence can functionally substitute for the TriMV YX-AUG function in translation. (A) Boxed are the two uncoupled putative box A′ and box B′ binding sites within the BVMoV 5′ UTR sequence at position nt 240 to 247 and nt 258 to 265 (accession number KY491536.1) upstream of the fourth AUG. The relative luciferase activity of the TriMV 5′ UTR with the last 30 nt swapped with a 35-nt sequence of the BVMoV in wheat germ extract (on the left) and in oat protoplasts (on the right) is relativized to that of the TriMV wild-type sequence in the presence of the strong hairpin. For the oat protoplast assays, the reporter mRNAs were coelectroporated with a m7GpppG-capped polyadenylated renilla mRNA used as an internal control at a 1:10 ratio (B). trans-Inhibition assay of the chimeric TriMV 5′ UTR-BVMoV SL-mRNA construct with increasing molecular excess of antisense single-stranded DNA oligonucleotides targeting the box A′ alone (anti-BoxA′), box B’ (anti-BoxB′) alone, both box A′ and box B′ (anti-BoxB′), and box A and box B of the native TriMV (anti-BoxA+BoxB-TriMV) 18S rRNA binding sites in wheat germ extract. As a control, DNA oligonucleotides targeting unrelated human β-globin sequence were added. A 0- to 100-fold molar excess of the antisense oligonucleotides was added to the reaction.

FIG 7

The BVMoV 5′ UTR-driven translation may not be dependent of the YX-AUG like motif. (A) Sequence of the 276-nt BVMoV 5′ UTR (accession number KY491536.1). In gray are the multiple AUGs. Boxed are the two putative 18S rRNA binding sites. The BVMoV YX-AUG like motif covers region nt 242 to 275. (A). The relative luciferase activities in wheat germ extract of the BVMoV 5′ UTR reporter mRNA with (BVYM 5′ UTR SL) or without (BVMoV 5′ UTR) the strong hairpin are relativized to that of an m7GpppG-capped polyadenylated mRNA control with vector sequence. (B) trans-Inhibition assay of the BVMoV 5′ UTR mRNA construct with increasing molecular excess of antisense single-stranded DNA oligonucleotides targeting the box A′ alone (anti-BoxA′), both box A′ and box B′ (anti-BoxB′) 18S rRNA binding sites in wheat germ extract. As a control, DNA oligonucleotides targeting unrelated human β-globin sequence were added. A 0- to 100-fold molar excess of the antisense oligonucleotides was added to the reaction. (C) The relative luciferase activities in wheat germ extract of the BVMoV 5′ UTR reporter mRNAs with the last 36 nt swapped with the 30-nt TriMV YX-AUG motif with or without the strong hairpin are relativized to that of the native BVMoV 5′ UTR reporter mRNA.