Celastrol Suppresses Porcine Deltacoronavirus Replication by Modulating Endoplasmic Reticulum Stress‐Associated Ca 2+ Balance

Figures

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  Figure 1. CE inhibits PDCoV infection in LLC‐PK1 cells in a dose‐dependent manner. (A) 2D and 3D structure of celastrol (CE). (B) LLC‐PK1 cells were treated with CE at the concentrations from 0 to 1 μM for 24 h. The relative cell viability was detected by using CCK‐8 assay. (C–H) The LLC‐PK1 cells were infected by PDCoV at different MOI of 0.1 and 0.5 and followed by addition of CE at the concentration of 0.2, 0.4, and 0.6 μM. (C, D) Samples were collected at 24 hpi, and PDCoV replication indicated by level of PDCoV N was detected by western blotting. The immunoblotting bands were calculated by Image J. (E, F) The transcript level of PDCoV N was measured by qRT‐PCR. (G, H) The virus titers of the cell supernatant were evaluated by TCID50 analysis. Data were shown as means ± SD from three independent experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

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  Figure 2. CE targets multiple stages of PDCoV life cycle. (A, B) The inhibitory effect of CE on PDCoV was further assessed by immunofluorescence staining. PDCoV N protein was stained green and the cellular nuclei were stained blue. Scale bar, 50 μm. (C) The CE was added into LLC‐PK1 cells during the PDCoV infection stages including attachment, entry, and postentry, the transcript of PDCoV N was measured by qRT‐PCR. Data were expressed as means ± SD from three independent experiments. ∗ p < 0.05 and ∗∗ p < 0.01.

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  Figure 3. Network pharmacology analysis of the antiviral activity of CE targeting CoV‐related diarrhea. (A) CE‐, viral diarrhea‐, and CoV‐related genes were collected from indicated databases, and Venn diagram was pictured to reflect intersecting genes. (B) The network of protein–protein interaction was constructed by Cytoscape. (C, D) GO analysis and KEGG enrichment were displayed. (E) The mRNA expressions of CASP3, STAT3, TP53, and IL6 were detected by qPCR. Data were expressed as means ± SD from three independent experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

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  Figure 4. CE significantly reduces PDCoV‐induced calcium accumulation. (A) LLC‐PK1 cells were challenged by PDCoV and treated with CE at the concentration of 0.6 μM. The cells were collected at 24 hpi, and calcium ions were stained green with Fluo‐4 AM. Scale bar, 50 μm. (B) LLC‐PK1 cells were treated with EGTA at the indicated concentrations, the relative cell viability was measured by CCK‐8 analysis. (C) Cells were infected by PDCoV and followed by EGTA treatment for 24 h. The protein level of PDCoV N was analyzed by western blotting and the bands were quantified by Image J. (D) LLC‐PK1 cells were treated with CaCl2 at the indicated concentrations, the relative cell viability was measured by CCK‐8 analysis. (E) The transcript level of PDCoV N was measured by qRT‐PCR. (F) The protein synthesis was analyzed by treating cells with puromycin for 25 min after PDCoV infection and EGTA treatment, and then western blot analysis was applied. (G) The mRNA expression levels of genes encoding IFN‐β, ISG15, and MX1 were determined by qRT‐PCR analysis. Data were expressed as means ± SD from three independent experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

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  Figure 5. CE attenuates PDCoV‐induced ER stress and ER‐associated calcium accumulation. (A–D) LLC‐PK1 cells were infected with PDCoV (MOI = 0.1) and treated with 0.6 μM CE. The cells were processed at 24 hpi. (A) The expression levels of ER stress‐related proteins were detected by western blotting. The bands were analyzed by using Image J. (B) The cells were stained with Fluo‐4 AM and ER‐Tracker simultaneously. The calcium ions were stained green and ERs were stained red. Scale bar, 50 μm. (C) The colocalization of calcium and ER was analyzed by using Image J. (D) The expression levels of SERCA2 and Orai1 were detected by western blotting. The bands were analyzed by using Image J. (E) LLC‐PK1 cells were infected by PDCoV and treated with CaCl2 or EGTA for 24 h. The protein expression of SERCA2 was detected by western blotting and the bands were analyzed by using Image J. Data were expressed as means ± SD from three independent experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

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  Figure 6. Inhibition of ER stress regulates calcium balance to suppress PDCoV replication. (A) The PPI network of ER‐stress‐related proteins and SERCA2 were pictured by STRING database. (B, C) LLC‐PK cells were infected with PDCoV (MOI = 0.1), following by the treatment of 4‐PBA. The cells were processed at 24 hpi. (B) The protein expression of ATF4 and SERCA2 was detected by western blotting. (C) The cells were stained with Fluo‐4 AM and ER‐Tracker simultaneously. The calcium ions were stained green and ERs were stained red. Scale bar, 50 μm. (D–G) LLC‐PK cells were infected with PDCoV (MOI = 0.1), following by the addition of CE and 4‐PBA individually or simultaneously. The cells were harvested at 24 hpi. The mRNA expressions of PDCoV N, IFN‐β, MX1, and ISG15 were analyzed by qRT‐PCR. Data were expressed as means ± SD from three independent experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

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  Figure 7. CE shows potential binding affinity to viral proteins. (A, B) Molecular docking results of CE and S1 subunit or S2 subunit of PDCoV S protein. (C) Molecular docking results of CE and PDCoV RNA‐dependent RNA polymerase (RdRp). (D) Molecular docking results of CE and PDCoV papain‐like protease (PLpro). (E) Molecular docking results of CE and PDCoV N protein. (F) Molecular docking results of CE and PDCoV M protein. (G) Molecular docking results of CE and PDCoV 3‐chymotrypsin‐like protease (3CLpro). (H) Molecular docking results of CE and SARS‐CoV E protein (ID: 2MM4). (I) The vina docking energy for the structures are as shown in the table.