Thapsigargin suppresses porcine hemagglutinating encephalomyelitis virus replication by disrupting host energy metabolism

Figures

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  Fig. 1. Antiviral effects of Tg on PHEV infection in N2a cells. Different concentrations of Tg were added simultaneously with PHEV (MOI = 1) to N2a cells for 1 h of incubation. Then the medium was discarded, and the cells were further cultured for 24 h with fresh medium before being collected. A Schematic diagram of antiviral experiments. B Viral titer and cytotoxic effect of Tg. The black dots and red dots indicate viral titer and cytotoxicity respectively, and the black lines and red lines indicate dose-response curve fits. IC50 and CC50 values were calculated using Prism software. Data represents mean ± SD for n = 3 biological replicates. C Immunofluorescence of PHEV N at the indicated Tg concentrations. At 24 h post infection (hpi), infected cells were subjected to immunostaining for PHEV N antigen (in red) and staining of nuclei (in blue). Scale bars: 100 μm. D PHEV N mRNA levels at different Tg concentrations were detected by qRT-PCR. The results were normalized to GAPDH, and the relative expression levels were calculated using the 2−ΔΔCt method. E The levels of viral proteins (nsp1, nsp8, and N) were analyzed via Western blotting. The ratio of protein band intensities of nsp1,…

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  Fig. 2. The antiviral effect of short-term Tg exposure. N2a cells were pretreated with 0.5 μM Tg or DMSO for 1 h, washed three times with PBS and then infected with PHEV (MOI of 1) at 24, 48 or 60 h post priming. A Experimental workflow. B The cells were collected at 24 hpi, freeze-thawed three times, and centrifuged to harvest supernatant for viral titer detection by TCID50 assay. C At 24 hpi, infected cells were collected and PHEV N mRNA was measured by qRT-PCR. D At 24 hpi, infected cells were collected and PHEV N protein levels were determined by Western blotting. The ratio of protein band intensity of N was normalized by GAPDH. E At 24 hpi, infected cells were subjected to immunostaining for PHEV N antigen (in red) and staining of nuclei (in blue). Scale bars: 100 μm. Significance was assessed using two-tailed, unpaired Student's t tests. ∗∗∗, P < 0.001.

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  Fig. 3. Antiviral effects of Tg on PHEV infection in vivo. Mice were mock infected (M) or intranasally inoculated with 104 TCID50 of PHEV. PHEV-infected mice were orally administered Tg every other day (PT) or the same volume of DMSO (P) for three doses, and the first dose was given 1 day before infection. A Schematic diagram of animal experiments. B–D The mice were monitored daily for body weight, mortality and signs of disease (respiration, general appearance, responsiveness and neurological signs) after infection. B Relative body weight change (n = 10). C Clinical signs (n = 10). D Survival rate (n = 10). E–G Brains in P and PT groups were collected at 5 days postinfection (dpi) to detect viral load. E PHEV N mRNA levels were measured by qRT-PCR. F PHEV N protein levels from three biological replicates were measured by Western blotting. The ratio of protein band intensity was normalized by GAPDH. G Brain samples were subjected to immunostaining for PHEV N antigen (in green) and staining of nuclei (in blue). Scale bars: 2000 μm. The right panels in (G) represent the olfactory bulb (1), cerebral cortex (2), hippocampus (3), piriform cortex (4), brainstem (5), and cerebellum (6). Scale…

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  Fig. 4. Tg cannot inactivate PHEV directly, but suppress genome replication and transcription. A–F PHEV was co-incubated with different concentrations of Tg at 37 °C for 1 h and then centrifuged in an ultrafiltration tube, and N2a cells were infected with the Tg-treated PHEV for 24 h. A Schematic diagram of the infection experiments. B The cells were collected at 24 hpi, freeze-thawed three times, and centrifuged to harvest supernatant for viral titer detection by TCID50 titration assay in triplicate. C Immunofluorescence assay of the PHEV N protein. At 24 hpi, infected cells were subjected to immunostaining for PHEV N antigen (in red) and staining of nuclei (in blue). Scale bars: 50 μm. D Percentage of N-positive cells from three different fields of view in immunofluorescence assays were analyzed by ImageJ software. E PHEV N mRNA was measured by qRT-PCR. The results were normalized to GAPDH, and the relative expression levels were calculated using the 2−ΔΔCt method. F Western blotting of PHEV N protein levels. The ratio of protein band intensities of nsp1, nsp8, and N were normalized by GAPDH. P values were calculated using one-way analysis of variance (ANOVA). ns, not significant. G, H…

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  Fig. 5. Transcriptomic characteristics of mock-infected (M), PHEV-infected (P), Tg-treated (T), and Tg-treated and PHEV-infected (PT) N2a cells. A–E N2a cells were mock-infected (M), PHEV-infected (P), Tg-treated (T), and Tg-treated and PHEV-infected (PT) for 24 h and collected for transcriptomic sequencing (3 biological replicates in each group). A Principal component analysis (PCA). B Clustering analysis. C Top 30 enriched KEGG pathways between the P and PT groups. D–E Gene set enrichment analysis (GSEA) of the potentially altered pathways between the P and PT groups.

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  Fig. 6. Metabolic characterization of mock-infected (M), PHEV-infected (P), Tg-treated (T), and Tg-treated and PHEV-infected (PT) N2a cells. N2a cells were mock-infected or PHEV-infected for 24 h without or with the addition of Tg (1 μM), and collected for metabolic analysis using the Seahorse Cell Glycolysis/Mito Stress Test Kit. A Extracellular acidification rate (ECAR). B O2 consumption rate (OCR). C glycolysis. D Glycolytic capacity. E Glycolytic reserve. F Basal respiration. G Maximal respiration. H Spare respiration capacity. P values were calculated using one-way analysis of variance (ANOVA). ns, not significant; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001. Results are representative of data from three independent experiments, and data are expressed as mean ± SD.

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  Fig. 7. PHEV infection depends on glycolysis and OXPHOS. A‒C The inhibition of energy metabolism attenuates PHEV replication. N2a cells were pretreated with different inhibitors for 1 h, then cells were inoculated with PHEV and cultured for another 24 h in fresh medium. A Schematic of the mechanism of action of the metabolism modulator. B Viral load determination by qRT-PCR of infected N2a cells treated with glycolysis inhibitors (n = 3). C Viral load determination by qRT-PCR of infected N2a cells treated with OXPHOS inhibitors (n = 3). D Immunofluorescence of 2-NBDG. Glucose uptake was determined with 2-NBDG (a fluorophore-tagged glucose tracer). E Fluorescence intensity of 2-NBDG. F, G Glucose rescue experiment. N2a cells were pre-treated with Tg (0.01 μM) for 1 h and infected with PHEV (MOI of 1) for 24 h in fresh media by exogenous addition of Glucose (40 mM). F Immunofluorescence of PHEV N. G Viral N protein analysis by Western blotting. 2-DG, 2-deoxy-d-glucose; F6P, fructose 6-phosphate; F1,6-BP, fructose-1,6-bisphosphate; F2,6-BP, fructose-2,6-bisphosphate; PFKFB3,6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; LDH, lactate dehydrogenase. P values were calculated using…

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  Fig. 8. Proposed model for the anti-PHEV mechanism of Tg. PHEV infection is dependent on energy metabolism of host cells. Tg inhibits the SERAC ATPase pump and induces ER Ca2+ depletion, which further leads to a reduction in glycolysis and OXPHOS, thus inhibiting viral replication.

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  Supplementary Figure S1. Effect of Tg on PHEV infection in PK-15 cells. A–C PK-15 cells were infected with PHEV at an MOI of 1 and incubated with different concentrations of Tg. After adsorption at 37 °C for 1 h, the medium was replaced with fresh medium, and the cells were incubated for another 24 h. A Immunofluorescence of PHEV N at the indicated concentrations of Tg. At 24 hpi, infected cells were subjected to immunostaining for PHEV N antigen (in red) and staining of nuclei (in blue). Scale bars: 100 μm. B The levels of viral proteins (nsp1, nsp8, and N) were analyzed via Western blotting. C Viral infection percentage and cytotoxic effect of Tg at the indicated concentrations. The black dots and red dots indicate percentage of viral infection and cytotoxicity respectively, and the black lines and red lines indicate dose-response curve fits. IC50 and CC50 values were calculated using Prism software. Data represents mean ± SD for n = 3 biological replicates.

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  Supplementary Figure S2. Tg exerts broad-spectrum antiviral activity. A–E Cells were pretreated with 1 μM Tg or DMSO for 1 h and infected with different viruses. At 24 hpi, the cells were harvested, and viral N mRNA and titer was assayed. A PHEV-JT06, B PHEV-CC/2008, C TGEV, D PEDV, and E PDCoV. Significance was assessed using two-tailed, unpaired Student's t tests. ∗∗∗, P < 0.001.

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  Supplementary Figure S3. Tg does not affect the adsorption, internalization or release steps of the PHEV infection cycle. A and B N2a cells were prechilled at 4 °C for 1 h, and PHEV was added at an MOI of 5 with Tg or vehicle (DMSO) at 4 °C for another hour. After the cells were washed with precooled PBS three times, fresh medium was added, and the cells were incubated at 37 °C for 2 h. The relative level of bound viral particles was quantified via qRT-PCR. A Schematic diagram of the adsorption assay. B PHEV N mRNA was measured by qRT-PCR in the adsorption assay. C and D N2a cells were prechilled at 4 °C for 1 h and then incubated with PHEV (MOI of 5) at 4 °C for 1 h. Then, the cells were washed three times with precooled PBS to remove the unbound viruses. Afterward, Tg was added to the cells at a final concentration of 1 μM, and the cells were incubated at 37 °C for 2 h before qRT-PCR analysis. C Schematic diagram of the internalization assay. D PHEV N mRNA was measured by qRT-PCR in the internalization assay. E and F N2a cells were infected with PHEV (MOI of 1), and the medium was replaced with fresh medium containing Tg (1 μM) at 10 hpi. After 15, 30, 45 and 60 min, the amount of released viral…

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  Supplementary Figure S4. Cell viability of metabolic inhibitors. N2a cells were treated with DMSO or different chemicals for 1 h, and cell viability was assessed by a CCK-8 assay. P values were calculated using one-way analysis of variance (ANOVA). ns, not significant; ∗, P < 0.05; ∗∗, P < 0.01.