A ribosome-bound pseudoknot in the HCV coding region stimulates viral growth by tuning viral translation

Figures

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  Figure 1.. Pseudoknot1 (pk1) forms a compact structure and is critical for full-length HCV replication (A) Schematic of the full-length HCV luciferase construct. Arrows indicate the genomic positions of structure-disrupting locked nucleic acids (LNA). (B) Secondary structure map of HCV NS3 region labeled in vivo SHAPE reactivity (data from Wan et al.,24 JVI). The pk1 LNA is indicated by a green line, and the control LNA, targeting a downstream single-stranded region, is shown by a pink line. (C) Replication time courses for wild-type (WT) HCV treated with control LNA or pk1-targeting LNA, measured by Gluc activity. Data are represented as mean ± SD of three biological replicates. (D and E) Left: sequence of the WT or mutated pseudoknotted stem. Right: structural representation of pk1 or the unzip mutant labeled with in vitro SHAPE reactivity, obtained after folding in different concentrations of Mg2+. The nucleotide position of the pseudoknotted left arm is 28–31 (position 4,046–4,049 in Jc1); the nucleotide position of the pseudoknotted right arm is 76–79 (position 4,099–4,102 in Jc1). (F and G) Bar plot of the in vitro SHAPE reactivities obtained from pk1 and the pk1 unzip mutant under…

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  Figure 2.. In vitro pull-down experiments suggest that pk1 interacts with the ribosome in a structure-specific manner (A) Workflow of the in vitro MS2 pull-down approach used to discover proteins that bind pk1 RNA. (B) Mass spectrometry quantification comparing pk1 pull-down versus MS2 control pull-down, pk1-unzip mutant versus MS2 control pull-down, and pk1 pull-down versus pk1-unzip pull-down. The top 15 significantly enriched proteins (padjust < 0.05) are labeled. Mass spectrometry pull-down experiments were performed in triplicate. (C) Western blot analysis of RPL9 and RPL4 from pk1, pk1-unzip, and MS2 control pull-down elution samples. Experiments were performed in triplicate, with one representative replicate shown. (D) Quantification of the western blots from (C). Data are represented as mean ± SD of three biological replicates. Statistical significance was determined using an unpaired Student’s t test with equal variance (**p < 0.01 and ***p < 0.001). (E) Negative stain images of pull-down eluates from pk1, pk1-unzip, MS2 control, and resin-only control samples. Ribosome-like particles are indicated with white arrowheads in the pk1-unzip and MS2 control samples. In the pk1…

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  Figure 3.. pk1 plays a critical role in translation inhibition and viral growth (A) Schematic of the WT pseudoknot and structure-disrupting mutants. (B) Luciferase activity of IRES-driven constructs measured over time; construct designs shown on the left. (C) Normalized luciferase activity of various IRES-driven constructs at different time points, relative to the random control; construct designs shown on the left. (D) Workflow for full-length HCV growth assay. (E) Measurement of WT and mutant viral growth by luciferase activity at 24, 48, and 72 h post-transfection. Data are represented as mean ± SD of three biological replicates. Statistical significance was determined using an unpaired Student’s t test with equal variance (**p < 0.01, ***p < 0.001, and ****p < 0.0001).

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  Figure 4.. Pk1 inhibits translation activity independent of its location by activating protein cofactors (A–C) Top: Schematic of the dual luciferase constructs used to test pk1’s regulation of upstream and downstream protein expression. Bottom: Gaussia and Firefly luciferase activities for pk1 and random control constructs, measured at 2 h post-transfection and normalized to the random control. (D) qPCR quantification of Actin mRNA or luciferase construct RNA amount relative to 18S rRNA for each sample, normalized to the random control RNA. (E) Left: siRNA knockdown workflow. Right: western blot analysis of ZNF598, GIGYF2, and GAPDH confirming siRNA knockdown efficiency. (F) Gaussia and Firefly luciferase activities after siRNA treatments for pk1 and random control constructs, measured at 2 h post-transfection and normalized to the random control. The inhibitory effect in the siCtrl condition for the pk1-middle construct was weaker than in previous experiments (Figure 3), possibly reflecting variability in RNA input associated with the siRNA transfection setup and limited time point sampling. (G) Graphic showing the proposed pk1 inhibiting translation initiation mechanism. Data are…

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  Figure 5.. Pk1 rebalances HCV RNA from active translation to RNA synthesis (A) Workflow of polysome fractionation and RNA quantification for HCV-transfected cells. (B) Top: chromatogram showing the separation of monosome and polysome fractions. Middle: distribution of Actin mRNA across all fractions in WT and PUR-treated control. Bottom: distribution of HCV RNA across all fractions in WT virus and PUR-treated control. (C) Top: chromatogram showing the separation of monosome and polysome fractions. Middle: distribution of Actin mRNA across all fractions in WT and unzip mutant virus. Bottom: distribution of HCV RNA across all fractions in WT virus and unzip mutant virus. (D) Graphic showing the proposed pk1 acting mechanism.

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  Figure 6.. Pk1-like structures exist broadly in diverse virus families, and they exhibit a specific type of tertiary conformation (A) Workflow for identifying pk1-like elements across RNA virus genomes. (B) Top: pie chart summarizing the family distribution of input viruses. Middle: pie chart showing the family distribution of viruses identified after step 2. Bottom: pie chart displaying the family distribution and location of pk1-like elements after step 3. (C) Examples of high-confidence pk1-like elements located near viral protein boundaries. (D) Left: secondary structure representation of a pk1-like motif in Simian pegivirus and the design of the unzip mutant. Right: translation luciferase activity illustrating the function of pk1-like element in translation elongation, measured at 2 h post-transfection. Construct design is shown on the right. Data are represented as mean ± SD of three biological replicates. Statistical significance was determined using an unpaired Student’s t test with equal variance (**p < 0.01, ***p < 0.001, and ****p < 0.0001). (E) AlphaFold3 prediction of tertiary structures for pk1, pk1-like elements, and a canonical frameshifting pseudoknot. The corresponding…