Fig 2: N4BP3 is a positive regulator of the RIG-I-MAVS antiviral signal pathway. (A) N4BP3 interacts with RIG-I and MAVS. 293T were transfected with the indicated plasmid (3 µg each). 24 h after transfection, cells were left uninfected or infected with SeV before harvested and lysed to perform Co-immunoprecipitation assays with anti-HA, following immunoblotting assay with anti-Flag. Expression levels of the proteins were analyzed by immunoblot analysis of the lysates with anti-HA and anti-Flag antibodies. (B) N4BP3 promotes the production of IFN-ß. Transfect different doses (0, 0.01, 0.1, 0.5, 1.0, and 1.5 µg) of N4BP3 expression plasmid into 24-well plates. After 12 h of transfection, the cells were left uninfected or infected with SeV for 12 h, and the concentration of the cytokines in the culture supernatant was measured by ELISA. The data table shown were presented as mean ± SD, n = 3, *P < 0.05, **P < 0.01. (C) N4BP3 promotes the activation of the IFN-ß promoter and ISRE induced by SeV. The IFN-ß promoter or ISRE luciferase plasmid (0.05 µg) was transfected into 293T cells (~1 × 105) cultured in a 24-well plate, and different doses (0, 0.01, 0.1, 0.5, 1.0, and 1.5 µg each) of N4BP3 expression plasmids were transfected simultaneously. After 12 h of transfection, cells were left uninfected or infected with SeV for 12 h, and the dual-luciferase reporter gene experiment was performed. The data table shown were presented as mean ± SD, n = 3, *P < 0.05, **P < 0.01. (D) Overexpression of N4BP31 enhances MAVS-mediated activation of IFN-ß promoter and ISRE. Before the reporter gene detection, MAVS (0.5 µg) and different doses of N4BP3 (0, 0.01, 0.1, 1.0, and 1.5 µg each) and reporter gene plasmids (0.05 µg each) were transfected into 293T cells for 20 h. The data table shown were presented as mean ± SD, n = 3, *P < 0.05, **P < 0.01. (E) Under SeV induction, N4BP3 enhances the expression of IFNB1, ISG56, and CXCL10 at the mRNA level. 293T cells were transfected with different doses (0, 0.5, and 1.0 µg each) of the indicated plasmid. After 12 h of transfection, the cells were left uninfected or infected with SeV for 12 h and then subjected to qRT-PCR detection, while the actin was used for normalization. The data table shown were presented as mean ± SD, n = 3, *P < 0.05, **P < 0.01. (F) N4BP3 promotes SeV-induced IRF3 dimer formation. 293T cells were transfected with N4BP3 plasmids (0.5, 1.0, and 2.0 µg each). 12 h after transfection, the cells were left uninfected or infected with SeV for 12 h. IRF3 dimers or monomers were analyzed by Native PAGE for western blot analysis. (G) N4BP3 promotes MAVS-mediated IRF3 dimer formation and phosphorylation of P65. 293T cells were transfected with different doses (0, 0.5, 1.0, 1.5, and 2.0 µg each) of N4BP3 and MAVS (1.5 µg each) plasmids for 20 h. Western blot analysis is like panel (F).

Fig 3: dsRNA accumulation and IFNB1 expression in response to HNRNPC repression and silencing of UPF1 and STAU1 Immunofluorescence analysis of the dsRNA in MCF7 cells after knock-down of HNRNPC only or double knock-down of HNRNPC & UPF1 or HNRNPC & STAU1, with 4',6-diamidino-2-phenylindole (DAPI) staining (blue) and anti-dsRNA antibody J2 (green).mRNA expressions of UPF1, STAU1, STAU2, HNRNPC, and IFNB1 upon knock-down of UPF1, STAU1, and STAU2 or double knock-down of HNRNPC & UPF1, HNRNPC & STAU1, or HNRNPC & STAU2 in MCF7 cells. Data represent mean ± SD.

Fig 4: Knockdown of N4BP3 inhibits RIG-I-MAVS antiviral signaling pathway. (A) The effect of N4BP3 shRNA plasmid on the expression of endogenous N4BP3. Co-transfected into 293T cells with control or N4BP3 shRNA plasmid and HA-Actb. Western blot analysis was performed with corresponding antibodies. (B) Knockdown of N4BP3 inhibits SeV-induced activation of IFN-ß promoter and ISRE. 293T cells were transfected with the indicated N4BP3 shRNA (1.5 µg) and reporter gene plasmids (0.05 µg). After 12 h of transfection, the cells were left uninfected or infected with SeV for 12 h before luciferase assays were performed. The data table shown were presented as mean ± SD, n = 3, *P < 0.05, **P < 0.01. (C) Knockdown of N4BP3 inhibits SeV-induced IRF3 dimer formation and phosphorylation of P65. 293T cells were transfected with control siRNA plasmid or designated N4BP3 siRNA plasmid (4.0 µg each). 12 h after transfection, the cells were left uninfected or infected with SeV for 12 h. IRF3 dimers or monomers were analyzed by Native PAGE for western blot analysis. (D) Under SeV induction, knocking down N4BP3 blocked the expression of IFNB1, ISG56, and CXCL10 at the mRNA level. Transfected 293T cells with the indicated shRNA plasmid. After 12 h of transfection, the cells were left uninfected or infected with SeV for 12 h and then subjected to qRT-PCR detection. The data table shown were presented as mean ± SD, n = 3, *P < 0.05, **P < 0.01.

Fig 5: Host Transcriptional Response to Respiratory Infection in Human Lung Epithelium-Derived Cell Lines(A) Virus replication levels in infected cells. RNA-seq was performed on poly(A)-enriched total RNA, and the percentage of virus-aligned reads (over total reads) is indicated for each sample. Error bars represent standard deviation from three independent biological replicates (except for IAV infection, where data are representative of independent biological duplicates). The cell types used for each infection is indicated (+) at the bottom of the figure. All infections were performed at a high MOI (MOI, 2–5), except for *, which indicates an MOI of 0.2.(B) Read coverage along the SARS-CoV-2 genome for mCherry- or ACE2-expressing A549 cells. The graph indicates the number of viral reads per position of the virus genome in A549 cells transduced with adenovirus (AdV)-based vectors expressing mCherry (MOI, 0.2; light blue) or ACE2 (MOI 0.2, salmon (*); MOI 2, dark red). A scaled model of the SARS-CoV-2 genome and its genes is depicted below (generated in BioRender).(C) Western blot analysis of mCherry- or ACE2-expressing A549 cells infected with SARS-CoV-2. Whole-cell lysates were analyzed by SDS-PAGE and blotted for ACE2, SARS-CoV-2 nucleocapsid (N), and actin.(D) qRT-PCR analysis of mCherry- or ACE2-expressing A549 cells infected with SARS-CoV-2 (MOI, 0.2). The graph depicts the relative amount of SARS-CoV-2 Envelope (E), non-structural protein 14 (nsp14), and human IFNB transcripts normalized to human a-tubulin. Error bars represent the standard deviation of the mean log2(fold change) of three independent biological replicates.(E) Principal-component analysis (PCA) for the global transcriptional response to respiratory viruses. Sparse PCA depicts global transcriptome profiles of the samples in (A). Cell types used for infection are represented by different shapes (circle, A549; square, A549-ACE; diamond, Calu-3; triangle, MRC5).(F) Heatmap depicting the expression levels of differentially expressed genes (DEGs) of the samples in (A) belonging to the indicated GO biological processes (GO: 0034097, GO: 0045087, GO: 0009615, GO: 0006954). The graph depicts the log2(fold change) of DEGs of infected compared with mock-treated cells. The included genes have a log2(fold change) of more than 2 and a p-adjusted value of less than 0.05. Data from SARS-CoV-1 and MERS-CoV infections correspond to GEO: GSE56192.