Fig 2: Signaling pathways involved in HEV-induced IFN response.Immunoblots of RIG-I, MDA5, MAVS and β-actin in HepG2 cells expressing gene-specific shRNA, or GFP (Ctrl). (B-C) IFN-β promoter activity in HepG2 cells with different gene knockdown following Sendai virus (SeV) infection (B) or poly IC transfection (C). Cells were transfected with IFN-β-Luc and TK-RLuc (for normalization of transfection efficiency) 20 h prior to SeV infection or poly IC transfection. Cells were lysed and luciferase activity was determined 20 h after SeV infection or 12 h after poly IC transfection. Data are presented as fold changes relative to non-treated cells. Shown are representative results from two independent experiments each performed in triplicate. (D-F) Effect of RIG-I, MDA5 or MAVS knockdown on HEV replication and host IFN responses. Control and shRNA-expressing HepG2 cells were inoculated with HEV (1000 GE/cell). IFN-λ mRNA expression (D), HEV RNA abundance (E), and HEV-positive foci (F) were determined at 5 days after infection. The results show the mean ± SEM of 2 independent experiments performed in duplicate each. * P<0.05; ** P<0.01. (G) Immunoblots of IRF-3, IRF-7 and β-actin in HepG2 cells transduced with lentiviruses expressing GFP (Ctrl) or gene-specific shRNA. (H-J) Effect of IRF-3 or IRF-7 knockdown on HEV replication and IFN responses. Control and shRNA-expressing cells were inoculated with HEV (1,000 GE/cell). IFN-λ mRNA expression (H), HEV RNA abundance (I), and HEV-positive foci (J) in different cells were determined after 5 days. The results show the mean ± SEM of 2 independent experiments each performed in duplicate wells. * P<0.05; ** P<0.01. Scale bar (upper panel in F), 100 μm.

Fig 3: MDA5/LGP2 Are the Dominant RNA Sensors Responsible for Innate Immune Induction in Calu-3 Cells Infected with SARS-CoV-2(A) siRNA-mediated knockdown efficiency in Calu-3 cells was evaluated using qRT-PCR with specific primers.(B) The siRNA-transfected Calu-3 cells were infected with SARS-CoV-2 with MOI = 0.125. At 48 h post-infection, the total RNA was extracted using the NucleoSpin 96 RNA extraction kit. The IFN-β mRNA was quantified by quantitative RT-PCR. Data are expressed as fold change relative to non-treated cells.(C) The percentage of infection was calculated as the ratio between the number of infected cells stained for SARS-CoV-2 NP and the total amount of cells stained with DAPI. Data are from three independent experiments with three technical replicates.(D) Immunoblots of parental, MDA5, RIG-I, MAVS, and β-actin in the CRISPR-knockout (KO) Calu-3 cells.(E) The CRISPR-knockout cells were infected with SARS-CoV-2 with MOI = 0.125. At 48 h post-infection, cells were fixed, immunostained with rabbit-anti-SARS-CoV-2 NP antibody, and imaged using Celigo. The percentage of infection was calculated as the ratio between the number of infected cells stained for SARS-CoV-2 NP and the total amount of cells stained with DAPI. Data are from two independent experiments with three technical replicates.(F) IFN-β mRNA induction in the CIRPSR-KO cells infected with SARS-CoV-2.(G) Representative immunofluorescence images are shown.(H) The CRISPR-KO cells were transfected with either PolyI:C (5 μg/mL) or total RNA extracted from infected cells (10 μg/mL). 8 h post-transfection, the cells were lysed to measure IFN-β mRNA production using qRT-PCR.