hnRNPC facilitates coronavirus replication by directly binding the frameshift-stimulatory element of viral genomic RNA

Figures

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  Fig 1. 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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  Fig 2. SARS-CoV-2 FSE RNA binds to ASN7 and ASN83 of hnRNPC. (A) Domain mapping of Flag-tagged full-length (FL) and truncated hnRNPC. (B) RNA binding activity of FL and truncated hnRNPC with SARS-CoV-2 FSE RNA in Huh7 cells. (C) Binding mode between SARS-CoV-2 FSE RNA and hnRNPC predicted by molecular docking. (D) Schematic representation of wild-type and mutated hnRNPC proteins. (E) RNA binding activity of WT and mutated hnRNPC with SARS-CoV-2 FSE RNA in Huh7 cells. 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.

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  Fig 3. hnRNPC binds to U-rich sequences of SARS-CoV-2 FSE RNA. (A) Schematic view of FL and deleted fragments of SARS-CoV-2 FSE RNA. (B) Two-dimensional representations of the SARS-CoV-2 FSE RNA pseudoknot. (C) Representative immunoblot examining hnRNPC protein in input and immunoprecipitation (IP) samples for RNA immunoprecipitation (RIP) assay (left panel). FL and deleted SARS-CoV-2 FSE RNA quantified by qRT-PCR after enrichment from RIP (right panel). (D) RNA pull-down assay showing the binding activity of hnRNPC with FL or deleted SARS-CoV-2 FSE RNA. (E) SPR assays monitoring hnRNPC binding to RNA. U-rich single-stranded RNA served as the positive RNA control. For the RNA sequences used in the SPR assays, see also S4 Table. 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.

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  Fig 4. 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).

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  Fig 5. 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.

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  Fig 6. 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.

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  Fig 7. hnRNPC acts as a host cofactor for HCoV-OC43 infection. (A-B) RNA levels quantified by qRT-PCR (A) and protein levels detected by immunoblotting (B) in H460 cells after 48 and 72 hours of HCoV-OC43 infection (MOI = 0.01). (C) Protein levels measured by immunoblotting in H460 cells transfected with siRNA. (D-E) mRNA levels quantified by qRT-PCR (D) and protein levels detected by immunoblotting (E) in siRNA-treated H460 cells after HCoV-OC43 infection (MOI = 0.1) for 24, 48 and 72 hours. 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 (A-B); unpaired, two-tailed Student’s t-test (D-E).

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  Fig 8. Elbasvir specifically inhibits –1 PRF efficiency of coronaviruses by blocking the binding of hnRNPC to FSE RNA. (A) Schematic representation of the high-throughput compound screening procedure. (B) Binding sites and LibDockScores of compound with hnRNPC. (C) Binding affinity between hnRNPC and Elbasvir analyzed by SPR. (D) Binding mode between Elbasvir and hnRNPC predicted by molecular docking. (E) Antiviral efficacy of Elbasvir against HCoV-OC43 and HCoV-229E detected by CCK-8 assay at 72 hours of drug treatment. MOI = 1. Molnupiravir was served as a positive drug control. CC50, half-maximal cytotoxic concentration. EC50, half-maximal effective concentration. SI, selection index (CC50 / EC50). (F-H) Elbasvir reduced the efficacy of –1 PRF reporters for SARS-CoV-2 (F), HCoV-OC43 (G), and HCoV-229E (H) in Huh7 cells detected by immunoblotting. Merafloxacin (MER) and Carrimycin (CAM) were served as positive drug controls. (I-K) FSE RNA levels of SARS-CoV-2 (I), HCoV-OC43 (J), and HCoV-229E (K) quantified by qRT-PCR after enrichment from RIP in Huh7 cells treated with Elbasvir. Data were shown as mean ± s.d. with individual data points. Statistical analyses were performed using…

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  Fig 9. 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).