Fig 1: hnRNPC specifically promotes coronavirus frameshifting.(A) Representative images of mCherry-FSECoV-2-EGFP reporters in Huh7 cells transfected with protein overexpression plasmid. Scale bar, 100 μm. (B) Effects of protein overexpression on mCherry-FSECoV-2-EGFP reporters in Huh7 cells detected by fluorescence. (C-E) Protein levels measured by immunoblotting in Huh7 cells transfected with mCherry-FSECoV-2-EGFP (C), mCherry-FSEOC43-EGFP (D), and mCherry-FSE229E-EGFP reporters (E), respectively. (F) Frameshifting reporter RNAs were translated in RRL in the presence of increasing concentrations of hnRNPC ranging from 0 to 4 µM and measured by immunoblotting. Data were shown as mean ± s.d. with individual data points. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s post hoc comparison test.
Fig 2: Schematic diagram of hnRNPC facilitating SARS-CoV-2 replication and serving as a therapeutic target.hnRNPC directly binds to the SARS-CoV-2 FSE RNA to promote SARS-CoV-2 –1 PRF and enhance viral replication (Left). Elbasvir inhibits SARS-CoV-2 replication by directly binding to hnRNPC and blocking the interaction between hnRNPC and the viral FSE RNA (Right).
Fig 3: Effect of coronavirus infection on hnRNPC stability.(A-B) Protein levels measured by immunoblotting in HCoV-OC43-infected H460 cells (A) and HCoV-229E-infected Huh7 cells (B) at 48 hours post infection. (C) Schematic of the hnRNPC mRNA stability assay. (D-E) mRNA stability in HCoV-OC43-infected H460 cells (D) and HCoV-229E-infected Huh7 cells (E). MOI = 1. (F) Schematic of the protein stability assay. (G-H) Protein levels measured by immunoblotting in HCoV-OC43-infected H460 cells (G) and HCoV-229E-infected Huh7 cells (H) treated with cycloheximide (CHX). MOI = 1. Data were shown as mean ± s.d. with individual data points. Statistical analyses were performed using unpaired, two-tailed Student’s t-test (A-B), or paired, two-tailed Student’s t-test (D, E, G, H).
Fig 4: HCoV-OC43 infection facilitates hnRNPC entry into the cytoplasm.(A-B) Protein levels measured by immunoblotting in whole cell, nuclear and cytoplasmic fractions of H460 cells infected with HCoV-OC43 (MOI = 0.01) at 48 hpi (A) and 72 hpi (B). GAPDH and Lamin B1 served as cytoplasmic and nuclear endogenous controls, respectively. (C) Co-localization image assessed with specific probes against HCoV-OC43 FSE RNA (green) and the specific antibody against hnRNPC (red). Scale bar, 10 μm. Nuclei were stained with DAPI (blue). Fluorescence intensity was calculated using ImageJ software. Data were shown as mean ± s.d. with individual data points. Statistical analyses were performed using an unpaired, two-tailed Student’s t-test.
Fig 5: Discovery of protein interactors of the SARS-CoV-2 FSE RNA.(A) Schematic of in vitro interactome capture from protein interactors of biotinylated IVT SARS-CoV-2 FSE RNA. (B) Host proteins identified from HCoV-OC43-infected H460 cells and HCoV-229E-infected Huh7 cells. (C) RNA pull-down assays. hnRNPC proteins were detected by immunoblotting in Huh7 cell lysates. (D-F) Graphic illustration of RNA immunoprecipitation (RIP) experiments (D) using anti-hnRNPC (E) or anti-hnRNPC2 (F) antibody to capture IVT FSE RNA. Representative immunoblot showing hnRNPC protein in input and immunoprecipitation (IP) samples for RIP assay (left panel). SARS-CoV-2 FSE RNA was quantified by qRT-PCR after enrichment from RIP (right panel). (G) SPR analysis of affinity binding between hnRNPC and RNA. Data were shown as mean ± s.d. with individual data points. Statistical analyses were performed using an unpaired, two-tailed Student’s t-test.
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