Porcine Deltacoronavirus Nsp13 Suppresses the Assembly of the MAVS‐TBK1‐IRF3 Complex and IRF9 Nuclear Translocation

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  Figure 1. Nsp13 inhibits IFN‐I signaling and promotes viral replication. (A, B) HEK‐293T cells were cotransfected with pIFN‐β‐Luc, pRL‐TK, and increasing amounts of Nsp13 expression plasmid (200, 400, and 600 ng). At 24 h posttransfection, cells were stimulated with 500 U/mL SeV (A) or 100 ng/mL poly (I:C) (B). Luciferase activity was measured 24 h later. Firefly luciferase activity was normalized to Renilla luciferase activity. (C, D) Dual‐luciferase reporter assay using PDCoV Nsp13 and SARS‐CoV‐2 Nsp13 at different concentrations (300, 600, and 900 ng). (E–G) Relative mRNA expression of ISG56, CXCL10, and IFIT1 in SeV‐stimulated HEK‐293T cells with or without Nsp13 overexpression was analyzed by qRT‐PCR. GAPDH served as an internal control. (H, I) Western blot analysis of TBK1 and IRF3 phosphorylation. Whole‐cell lysates were collected at 24 h and probed for p‐TBK1 (S172), TBK1, p‐IRF3 (S396), and total IRF3. (J) HEK‐293T cells were transfected with PDCoV Nsp13 (or empty vector) together with IRF3 and then stimulated with SeV. Indirect immunofluorescence and confocal microscopy showed predominantly nuclear IRF3 in control cells, whereas IRF3 remained largely cytoplasmic in…

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  Figure 2. Nsp13 suppresses IFN‐β promoter activation induced by RLR signaling components in a dose‐dependent manner. (A–F) HEK‐293T cells were cotransfected with pIFN‐β‐Luc, pRL‐TK, and plasmids encoding RIG‐IN (A), MDA5 (B), MAVS (C), TBK1 (D), IKKε (E), or IRF3 (F), along with increasing amounts (0, 100, 200, 300 ng) of Nsp13 expression plasmid. Luciferase assays were performed 24 h posttransfection. Data are shown as mean ± SD from three independent experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 (one‐way ANOVA).

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  Figure 3. TBK1‐Nsp13 interaction is mediated by the CTD and the 1B domain. (A, B) Co‐immunoprecipitation (Co‐IP) of HA‐Nsp13 with Flag‐tagged signaling proteins (RIG‐I, MDA5, MAVS, TBK1, IRF3, STING) expressed in HEK‐293T cells. Flag‐tagged proteins were immunoprecipitated with anti‐Flag affinity gel and probed with anti‐HA. (C) His‐pulldown assay using purified His‐Nsp13 protein incubated with lysates from Flag‐TBK1 or GFP‐vector‐expressing HEK‐293T cells. (D) Confocal microscopy of LLC‐PK1 cells transfected with HA‐Nsp13 and Flag‐TBK1 for 24 h. Cells were immunostained with anti‐HA (red), anti‐Flag (green), and DAPI (blue). Colocalization was observed in the cytoplasm. Scale bar = 10 μm. (E, F) HEK‐293T cells were cotransfected with HA‐Nsp13 and full‐length or truncated TBK1 constructs (D1–D4). Co‐IP was performed using anti‐Flag. The strongest binding was observed with TBK1‐D4 containing the CTD. (G, H) HEK‐293T cells were transfected with Flag‐TBK1 and full‐length or truncated Nsp13 constructs (Nsp13‐D1, Nsp13‐D2). At 24 h posttransfection, Co‐IP was performed using anti‐Flag antibody, and coprecipitated Nsp13 was detected by western blotting. Both truncates containing the 1B domain…

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  Figure 4. Nsp13 competitively disrupts TBK1‐IRF3 and TBK1‐MAVS complex formation. (A, B) HEK‐293T cells were cotransfected with Flag‐TBK1, GFP‐IRF3 (A) or GFP‐MAVS (B), and increasing amounts of HA‐Nsp13 (1.0, 2.0 µg). Co‐IP was performed using anti‐Flag affinity gel. Immunoblots revealed that increasing levels of Nsp13 impaired the TBK1‐IRF3 and TBK1‐MAVS interactions in a dose‐dependent manner. Data are representative of three independent experiments. (C, D) Densitometric quantification of the western blot results shown in (A–B). Data are presented as mean ± SD from three independent experiments. ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001.

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  Figure 5. Differential effects of PDCoV and SARS‐CoV‐2 Nsp13 on TBK1 stability and ubiquitination. (A, B) HEK‐293T cells were cotransfected with GFP‐TBK1 and increasing amounts of HA‐PDCoV‐Nsp13 or HA‐SARS‐CoV‐2‐Nsp13. Whole‐cell lysates (WCL) were subjected to immunoblotting (IB) with the indicated antibodies. (C, D) Densitometric quantification of the western blot results shown in (A, B). Data are presented as mean ± SD from three independent experiments. (E, F) HEK‐293T cells were cotransfected with Flag‐TBK1, Myc‐tagged ubiquitin (Myc‐Ub), and increasing amounts of HA‐PDCoV‐Nsp13 or HA‐SARS‐CoV‐2‐Nsp13. Flag‐TBK1 was affinity‐precipitated using anti‐Flag agarose beads (co‐IP) and analyzed by immunoblotting (IB) with the indicated antibodies. GAPDH served as a loading control. (G, H) Densitometric quantification of the western blot results shown in (E, F). Data are presented as mean ± SD from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

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