Fig. 6: The M358L mutation in nsP3 inhibited the activation of PKR and STAT1, further inhibiting IFN-mediated antiviral responses.

a HCT-116 cells were infected with M1-GFP and M1-NS3M (MOI = 10), and the infection rate was determined by flow cytometry. b The expression of GFP in infected cells in (a) was detected. MFI, mean fluorescence intensity. c A modified plaque formation assay was performed and plaques were counted with a fluorescence microscope 48 hours after infection. d Schematic of the process for identifying interactions with nsP3 WT and nsP3 M358L. e This Venn diagram shows the host proteins that interact with nsP3 WT and nsP3 M358L. f, g Bar graph of enriched terms across the 59 nsP3 WT interactors and 22 nsP3 M358L interactors, colored by p values. The top 10 enriched pathways are shown. See also Table S1 and S2. h Coimmunoprecipitation was conducted with an anti-Flag antibody or isotype control IgG prior to immunoblot analysis with anti-nsP3 and anti-PKR antibodies. Representative images of n  =  3. i, j HCT-116 cells were treated with control, M1-GFP or M1-NS3M for 4 and 24 hours (MOI = 10), and the levels of proteins in the cell lysates was examined by Western blotting (left). Quantification of p-PKR and p-STAT1 (right). k Quantification of PKR and STAT1 expression in (i, j). l The transcript levels of ISGs and viral RNA were quantified by qRT‒PCR after 24 h infection with control, M1-GFP or M1-NS3M (MOI = 1). (m-o) qRT-PCR (m) and western blotting (n) were used to evaluate the shRNAs knockdown efficiency of PKR. PKR knockdown cells were infected with M1-GFP and M1-NS3M (MOI = 1) and the infection rate was determined by flow cytometry (o). p, q Proteins were detected by Western blotting in CCD-18Co cells after infection with M1-GFP or M1-NS3M (MOI = 1). Quantification of p-PKR and p-STAT1. Statistical significance was calculated using Two-way ANOVA with Sidak’s multiple comparisons test (a, b, i, j, o, q) or One-way ANOVA with Tukey’s multiple comparisons test (k–l), and adjusted P values are indicated. Data are shown as mean ± SD from three biological replicates. Source data are provided as a Source Data file.