Classical swine fever virus utilizes stearoyl-CoA desaturase 1-mediated lipid metabolism to facilitate viral replication

Fig 1. Screening of anti-CSFV compounds from a lipid metabolism library. (A) Screening process for antiviral compounds. (B) Inhibition effects of the compounds. Each dot represents the inhibition rate of a compound (2 µM) against CSFV (MOI = 1). (C) PK-15 cells infected with CSFV (MOI = 1) for 24 h were collected and analyzed by RT-qPCR to measure CSFV mRNA levels. (D) Antiviral effects of compounds targeting three key rate-limiting enzymes (SCD1, FASN, and ACC) in lipid metabolism. PK-15 cells infected with CSFV (MOI = 1) were treated with inhibitors of rate-limiting enzymes, then fixed and stained with anti-CSFV E2 antibody (green) for fluorescence microscopy. Scale bars = 50 µm. (E through G) Antiviral effects of three SCD1-targeting compounds. PK-15 cells were treated with DMSO or varying concentrations of A939572, MF-438, and SCD1 inhibitor-4 for 24 h after infection with CSFV (MOI = 1). Viral replication was assessed by Western blotting and RT-qPCR. Cell cytotoxicity was quantified using the CCK-8 assay. β-Actin was used as a loading control, and protein expression levels were calculated by determining the ratio of the protein to β-actin using ImageJ 7.0 software. Data are…

Fig 2. SCD1 is a broad-spectrum antiviral target. (A through C) MDBK cells were treated with DMSO or varying concentrations of A939572, MF-438, or SCD1 inhibitor-4 for 24 h after infection with BVDV (MOI = 0.5). Viral replication was assessed by Western blotting and RT-qPCR. The effects of the indicated compounds on cell cytotoxicity were quantified using the CCK-8 assay. (D through I) PK-15 cells were treated with DMSO or varying concentrations of A939572, MF-438, or SCD1 inhibitor-4 for 24 h after infection with JEV or PRV (MOI = 0.1). Viral replication was assessed by RT-qPCR (JEV) or qPCR (PRV) and Western blotting. β-Actin served as a loading control. Protein expression levels were quantified by calculating the ratio of protein to β-actin using ImageJ 7.0 software. Data are presented as the mean ± SD from three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3. The SCD1 inhibitors mainly affect the late stage of CSFV infection. (A through C) PK-15 cells were treated with varying concentrations of A939572, MF-438, or SCD1 inhibitor-4 and infected with CSFV (MOI = 10) for 1 h at 4°C, followed by incubation at 37°C for either 0 hpi (binding) or 1 hpi (entry). Cells were harvested and analyzed by RT-qPCR to measure viral mRNA levels. (D through F) The drug action at specific stages was evaluated using a time-of-addition assay, as described below. (i) 12 h pretreatment: cells were treated with the indicated concentration (1,000 nM) of A939572, MF-438, or SCD1 inhibitor-4 for 12 h before infection with CSFV (MOI = 1); (ii) 1 h entry: cells were treated with a mixture of the indicated SCD1 inhibitors and CSFV (MOI = 1) for 1 h; (iii) post-entry: cells were treated with the indicated SCD1 inhibitors after infection with CSFV; (iv) throughout: cells were treated with the indicated SCD1 inhibitors throughout the entire process. Treated cells were collected at 24 hpi and analyzed by RT-qPCR and Western blotting to assess viral replication. β-Actin served as a loading control. Protein expression levels were quantified by calculating the ratio of…

Fig 4. SCD1 positively regulates CSFV replication. (A) PK-15 cells infected with CSFV (MOI = 1) were collected at the indicated time points and analyzed by Western blotting using rabbit anti-CSFV Npro antibody. (B) The SCD1 content shown in panel A was analyzed through grayscale analysis using ImageJ software. (C) BHK-21 cells infected with JEV (MOI = 0.1) were collected at the indicated time points and analyzed by Western blotting using mouse anti-JEV NS5 antibody. (D) PK-15 cells infected with varying MOIs of CSFV were collected at the indicated time points and analyzed by Western blotting. (E) PK-15 cells transfected with pHA-SCD1 (1, 1.5, and 2 µg) were infected with CSFV (MOI = 1). At 24 hpi, cells were collected and analyzed by Western blotting. (F and G) PK-15 cells transfected with siNC or siSCD1 for 24 h were infected with CSFV (MOI = 1). Cells were collected and analyzed by RT-qPCR and Western blotting. (H through J) PK-15 cells infected with CSFV (MOI = 1) were treated with A939572 (1 µM) and varying concentrations of OA/PA in 2% DMEM for 24 h. Treated cells were collected and analyzed by RT-qPCR and Western blotting. (K through M) PK-15 cells transfected with siSCD1 for 24 h…

Fig 5. SCD1 is recruited to the viral replication complex by CSFV p7. (A) PK-15 cells infected with CSFV (MOI = 1) for 24 h were fixed and stained with mouse anti-dsRNA monoclonal antibody (J2), ER-Tracker Red dye, and rabbit anti-SCD1 antibody for confocal microscopy. Scale bars = 10 µm. (B) PK-15 cells infected with CSFV (MOI = 1) for 24 h were fixed and stained with mouse anti-dsRNA monoclonal antibody (J2), Golgi-Tracker Red dye, and rabbit anti-SCD1 antibody for confocal microscopy. Scale bars = 10 µm. (C) Co-localization analysis of panels (A, B) was performed using Pearson’s correlation coefficient. (D) PK-15 cells infected with CSFV for 24 h were collected, and ER/VRC proteins were extracted using a commercial kit. Western blotting was performed to determine the relative levels of SCD1 protein. (E) PK-15 cells were co-transfected with pHA-SCD1 and different CSFV plasmids for 24 h. Cells were then fixed and stained with appropriate antibodies for confocal microscopy. Scale bars = 10 µm. Co-localization analysis was performed using ImageJ. (F, G) 293T cells were co-transfected with pHA-SCD1 and pGFP-SCD1 for 24 h, harvested for Co-IP using anti-GFP/HA antibodies, and the resulting…

Fig 6. CSFV infection induces the endoplasmic reticulum stress pathway IRE1α/XBP1 to regulate SCD1. (A and B) PK-15 cells infected with CSFV (MOI = 1) or BHK-21 cells infected with JEV (MOI = 0.1) were collected at the indicated time points and analyzed by Western blotting using rabbit anti-CSFV Npro, mouse anti-JEV NS5, or rabbit anti-IRE1α antibodies. (C) PK-15 cells infected with varying MOIs of CSFV were collected at the indicated time points and analyzed by Western blotting. (D) Schematic overview of the IRE1α/XBP1/SCD1 signaling pathway. (E and F) PK-15 cells transfected with siNC, siIRE1α, or siXBP1 for 24 h were infected with CSFV (MOI = 1). Cells were then collected and analyzed by RT-qPCR and Western blotting. (G and H) PK-15 cells transfected with p3×Flag-IRE1α and p3×Flag-XBP1, respectively, and infected with CSFV (MOI = 1) for 24 h. Cells were then collected and analyzed by RT-qPCR and Western blotting. (I through K) PK-15 cells infected with CSFV (MOI = 1) were treated with IRE1α/XBP1 inhibitors for 24 h. Cells were then collected and analyzed by RT-qPCR and Western blotting. PK-15 cells infected with CSFV (MOI = 1) were treated with IRE1α/XBP1 inhibitors for 24 h. Cells…

Fig 7. The IRE1α/XBP1/SCD1 axis affects the synthesis of triglyceride and lipid droplets during CSFV infection. (A) Triglyceride detection. PK-15 cells transfected with siNC, siSCD1, siIRE1α, or siXBP1 for 24 h were infected with CSFV (MOI = 1). At 24 hpi, cells were digested with 0.25% trypsin, centrifuged at 1,000 g for 5 min, and the supernatant was discarded. Triglyceride content was measured using a commercial detection kit. (B) PK-15 cells infected with CSFV (MOI = 1) were treated with the indicated inhibitors for 24 h. Triglyceride content was measured using a commercial detection kit. (C through E) PK-15 cells transfected with siNC, siSCD1, siIRE1α, or siXBP1 for 24 h were infected with CSFV (MOI = 1). At 24 hpi, cells were fixed and stained with rabbit anti-SCD1, anti-IRE1α, anti-XBP1 antibodies, mouse anti-dsRNA monoclonal antibody, or BODIPY 493/503 for confocal microscopy. Scale bars = 10 µm. (F) Integrated density of LDs from panels (C through E) was analyzed using ImageJ 7.0 and GraphPad Prism 9 software. (G through J) PK-15 cells infected with CSFV (MOI = 1) were treated with the indicated inhibitors for 24 h. Cells were then fixed and stained with rabbit anti-SCD1…

Fig 8. Schematic model depicting the IRE1α/XBP1 pathways and p7 regulating SCD1 during CSFV infection. The replication of CSFV is regulated by the endoplasmic reticulum stress pathway IRE1α/XBP1, which controls the synthesis of SCD1; the IRE1α/XBP1/SCD1 pathway regulates the synthesis of TGs and LDs, thereby affecting CSFV replication. Furthermore, the non-structural protein p7 of CSFV interacts with SCD1 in the endoplasmic reticulum and hijacks SCD1 to the CSFV VRC to promote viral replication.