Concerted action of two 3′ cap-independent translation enhancers increases the competitive strength of translated viral genomes

Figures

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  Figure 1.. Contributions of 3′CITEs for translation of PEMV2 gRNA in vitro. (A) Genome organization of PEMV2. Dashed line denotes the kissing-loop interaction between the kl-TSS and 5′ proximal hairpin 5H2. The approximate locations of the three 3′CITEs are shown. (B) Secondary structures of the 3′CITEs and the 5′ terminal 89 nt. Dashed lines denote tertiary interactions including the long-distance interaction (left) and pseudoknots (middle and right) between residues shown in red. Mutations introduced into the apical loops involved in the kissing-loop interaction are shown. (C) In vitro translation of wild-type (WT) and mutant gRNAs in wheat germ extracts (WGE). ΔK, ΔP, and ΔT are deletions of the kl-TSS, PTE and TSS, respectively. (D) In vitro translation of WT and mutant gRNAs that are missing the long-distant interaction. Assays in (C) and (D) were repeated once, with similar results.

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  Figure 2.. PTE can inhibit translation by sequestering eIF4G. (A) Pm2 mutation that disrupts eIF4E binding (17) is shown. Residues in red participate in the internal pseudoknot. (B) In vitro translation of WT and mutant gRNAs in WGE. (C) In vitro translation of ΔKΔPΔT, ΔKΔT and ΔKΔT/Pm2 gRNAs. (D) Trans-inhibition of WT gRNA translation in WGE. Fragments (10-fold molar ratio) containing either the WT PTE or PTE Pm2 were added to reaction mixtures containing WT gRNA. None, no added fragment. (E) Trans-inhibition of mutant gRNA translation in WGE. (F) Purified Arabidopsis eIF4E, eIF4G or eIF4F (200 nM) were added to WGE translation mixes containing ΔKΔPΔT or ΔKΔT gRNAs. None, no added translation factors. Data are from three independent experiments. Unpaired t-test was used to analyze the statistical significance for B and C, and one-way ANOVA was used to analyze the significance of D–F. One or two asterisks indicate statistical difference with P < 0.05 or 0.01, respectively.

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  Figure 3.. Competition for translation between PEMV2 and capped or uncapped reporter transcripts in WGE. PEMV2 (0.5 pmol) was translated either alone or with (0.5 pmol) of capped GloLuc (left) or uncapped TZ10ΩLuc (right). Data are from three independent experiments. Unpaired t-test was used for the statistical analysis. One or two asterisks indicate statistical difference with P < 0.05 or 0.01, respectively.

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  Figure 4.. Transferring the long-distance interaction from the kl-TSS to the PTE. (A) Diagram of the alterations made to 5H2, kl-TSS and PTE. PTE with the kl-TSS loop sequence is denoted as PTE*. WT interacting sequences are denoted as red circles and presence of a mutation in the sequence is denoted by a blue outline. The PTE mutation (Pm2) that disrupts eIF4E binding is denoted by a gray circle with a blue outline. (B) Diagram of WT PEMV2 with interaction between the kl-TSS and 5H2 shown. (C) Diagrams of gRNA mutants generated. (D) In vitro translation of mutant gRNAs depicted in (C). Relative levels of p33 are quantified. (E) Relative virus accumulation in protoplasts. Total RNA was extracted at 24 h post-inoculation and analyzed by northern blotting. Data are from three independent experiments. For (D) and (E), unpaired t-test was used to analyze statistical significance. One or two asterisks indicate statistical difference with P < 0.05 or 0.01, respectively.

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  Figure 5.. Effect of exchanging positions of the kl-TSS and PTE. (A) Diagrams of WT and mutant gRNAs with exchanged positions of the kl-TSS and PTE. WT interacting sequences are denoted as red circles and mutant sequences have a blue outline. (B) In vitro translation of gRNAs in WGE. (C) Relative virus accumulation in protoplasts (see legend to Figure 4). (D) Secondary structures in the 3′UTR of WT and PxK gRNAs. Nucleotide flexibilities were determined using SHAPE structural probing and quantified using semiautomated footprinting analysis software. Nucleotides with reactivity of ≥0.6 are in red (medium to high flexibility), those with values ranging from 0.3 to 0.6 are green (low to medium flexibility) and those with values <0.3 are black (inflexible). (E) SHAPE phophorimages showing flexibility alterations in the terminal loop of the 5′ hairpin in the kl-TSS (boxed). G, U, C, A nucleotide ladder lanes; D, DMSO-treated control; N, NIMA-treated.

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  Figure 6.. Toeprinting analysis of ribosomes associating with the p33 initiation codon. ΔK, ΔP and ΔT are deletions of the kl-TSS, PTE and TSS, respectively. AUG to ACG has a mutation in the p33 initiation codon. Bracket denotes the p33 AUG toeprint products and relative levels from three independent experiments are given at the bottom. Position of the A in the p33 initiation AUG codon is numbered 1, and numbering from this position is shown at left. G, U, C, A nucleotide ladder lanes. Asterisk denotes an additional toeprint that likely also corresponds with ribosomes occupying the p33 AUG.

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  Figure 7.. Kl-TSS and PTE increase the number of gRNAs available for translation, but have no effect on full-translation time and polysome distribution. (A) Luciferase construct 5′89+3U that contains the PEMV2 5′ terminal 89 nt and 3′UTR flanking firefly luciferase ORF. Secondary structure of kl-TSS and mutant Km2, which contains two mutations in the loop involved in the kissing-loop interaction. (B) Kinetic curves of real-time translation of firefly luciferase in WGE over a 60 min translation period. Uncapped RNAs tested were 5′89+3U, 5′89+3UKm2 and TZ10ΩLuc. (C) Second derivatives of the translation curves from the first 15 min. Solid lines represent the original kinetic curves of luciferase activity. Dashed lines represent the second derivatives of the original kinetic curves. The numbers shown above the dashed lines indicate the average full-translation time corresponding to the Gaussian peaks. (D) Polysome distribution on uncapped, fluorescein-labeled 5′89+3U, 5′89+3UKm2 and TZ10ΩLuc luciferase reporter constructs. RNAs (50 nM) were incubated in WGE for 25 min and then subjected to sedimentation analysis. Ultraviolet absorbance profiles (upper curves) reflect mainly the distribution of…