Prolyl endopeptidase is a multifunctional host factor required for FMDV infection

Figures

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  Fig. 1. PREP-KO significantly inhibits FMDV infection. A The sequencing results near the PREP gene, with the sgRNA sequences highlighted in red, the protospacer adjacent motif (PAM) sequence sites highlighted in green, the black boxes indicating the inserted base, and the red characters “-” indicating the deleted bases. B Western blot analysis of FMDV VP1 protein expression levels in PREP-KO and sgNC IBRS-2 cells after infection with both high titer (MOI = 1) and low titer (MOI = 0.01) of FMDV at two distinct time points, 4.5 h and 16.5 h. C Viral quantification was performed using plaque assay. PREP-KO and sgNC IBRS-2 cells were seeded into 12-well culture plates and infected with FMDV at two different MOIs (MOI = 0.01 and MOI = 1) for 16.5 h and 4 h. Plates were stained with 1% crystal violet to view plaques. D Immunofluorescence analysis of the FMDV VP1 protein expression levels in PREP-KO and sgNC IBRS-2 cells after infection with FMDV (MOI = 1) at 4 hpi. Scale bars = 125 μm. Left: Immunofluorescence analysis. Right: The normalized mean fluorescence intensity analysis. E Virus titer analysis of PREP-KO and sgNC IBRS-2 cells after infection with FMDV (MOI = 1) at 6 hpi. F Western blot…

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  Fig. 2. The type I IFN signaling pathway is significantly activated in PREP-KO IBRS-2 cells. A Volcano plots of DEGs (P-value ≤ 0.05 and an absolute fold-change of ≥ 1) between PREP-KO and sgNC IBRS-2 cells. B GO function enrichment analysis of DEGs between PREP-KO and sgNC IBRS-2 cells. C KEGG pathway analysis of DEGs between PREP-KO and sgNC IBRS-2 cells. D The heatmap displays the top 50 genes that are significantly upregulated in PREP-KO IBRS-2 cells. E RT-qPCR analysis of the relative mRNA levels of IFN-β and ISGs in PREP-KO and sgNC IBRS-2 cells. F RT-qPCR analysis of the relative mRNA levels of OASL and MX1 in PREP-KO and sgNC IBRS-2 cells treated with IgG or varying volumes of porcine IFN-β antibody. sgNC, sgRNA negative control; KO, knock out. Data are represented as means ± SD. n = 3 (E and F). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. P values were determined by two-tailed Student′s t-tests

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  Fig. 3. PREP-KO IBRS-2 cells upregulate RIG-I mRNA expression by decreasing PUM1 stability. A Western blot analysis of the expression levels of key molecules involved in the type I IFN signaling pathway in PREP-KO and sgNC IBRS-2 cells. B Western blot analysis of RIG-I protein expression levels in GFP-PREP overexpression and WT IBRS-2 cells. C RT-qPCR analysis of relative RIG-I, MDA5 and MAVS mRNA expression levels in PREP-KO and sgNC IBRS-2 cells. D RT-qPCR analysis of relative PUM1 mRNA expression levels in PREP-KO and sgNC IBRS-2 cells. E Western blot analysis of PUM1 protein expression levels in PREP-KO and sgNC IBRS-2 cells. F RT-qPCR analysis of the relative mRNA expression levels of IFN-β, RIG-I, MDA5 and OASL in IBRS-2 cells transfected with either control siRNA (siNC) or PUM1-targeting siRNA (siPUM1). G Co-immunoprecipitation analysis of lysates from HEK293T cells co-transfected with PREP-Myc and PUM1-Flag. H Western blot analysis of PUM1 expression levels in PREP-KO and sgNC IBRS-2 cells treated with CHX (100 µg/mL) for the indicated times. I Western blot analysis of PUM1 expression levels in PREP-KO and sgNC IBRS-2 cells maintained in the presence of DMSO, MG132 (4 or 20 µM),…

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  Fig. 4. PREP-KO inhibits FMDV replication. A Confocal microscopy for detection of FMDV binding stage in PREP-KO and sgNC IBRS-2 cells. The cells were infected with FMDV (MOI = 10) on ice for 1 h, cells were then washed twice with PBS. The samples were subjected to immunofluorescence assay for detection of FMDV VP1 protein (green). Scale bars = 20 μm. Left: Confocal microscopy analysis. Right: The normalized mean fluorescence intensity analysis. B Confocal microscopy for detection of FMDV internalization stage in PREP-KO and sgNC IBRS-2 cells. The cells were infected with FMDV (MOI = 10) on ice for 1 h and transferred to 37˚C for 30 min. Cells were then washed twice with PBS and treated with 500 ng/mL proteinase K on ice for 1 h. The samples were subjected to immunofluorescence assay for detection of FMDV VP1 protein (green) and early endosomes (red). Scale bars = 20 μm. Left: Confocal microscopy analysis. Right: The colocalization analysis. C Absolute RT-qPCR analysis of FMDV binding stage in PREP-KO and sgNC IBRS-2 cells, along with mock-infected 293 T cells. D Absolute RT-qPCR analysis of FMDV internalization stage in PREP-KO and sgNC IBRS-2 cells. E Virus titer analysis of PREP-KO and…

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  Fig. 5. The FMDV replication is inhibited in PREP inhibitor baicalein treated cells. A Western blot analysis of FMDV VP1 protein expression levels in baicalein- and DMSO-treated IBRS-2 cells after infection with FMDV (MOI = 0.01) at different time points post infection. The IBRS-2 cells were pretreated with 100 µM of the PREP inhibitor baicalein for 3 h prior to FMDV infection, and samples were collected at 0, 6, 8, 10, and 12 hpi for analysis. B, C, D RT-qPCR analysis of FMDV RNA levels in baicalein- and DMSO-treated cells after infection with FMDV (MOI = 0.01) at different time points post infection. The cells were pretreated with 100 µM of the PREP inhibitor baicalein for 3 h prior to FMDV infection, and samples were collected at different time points post infection for analysis. IBRS-2 cells (B); PK-15 cells (C); iPAM cells (D). E, G Western blot analysis of FMDV VP1 protein expression levels in cells treated with baicalein at different time points after infection with FMDV (MOI = 0.01). Samples were collected at 24 hpi for analysis. IBRS-2 cells (E); PK-15 cells (G). F, H Virus titer analysis of FMDV titers in cells treated with baicalein at different time points after infection…

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  Fig. 6. Inhibition or knockout of PREP increases the survival rate of mice following FMDV challenge. A Preventive strategy for baicalein treatment and FMDV challenge in mice. B Survival curves for 30 mg/kg or 50 mg/kg baicalein- and DMSO-treated mice infected with FMDV (n = 12 mice per group). C RT-qPCR analysis of the FMDV viral load in different tissues from 50 mg/kg baicalein- and DMSO-treated mice after FMDV infection. D H&E staining analysis of different tissues from 50 mg/kg baicalein- and DMSO-treated mice after FMDV infection. Scale bars = 100 μm. The red arrows in DMSO group indicate inflammatory cellular infiltration in hearts and kidneys, shallow staining of liver cytoplasm, exfoliated cells, prominent fibrosis in livers and alveolar wall thickening in lungs; the red arrows in baicalein group represent normal areas of tissues. E Therapeutic strategy for FMDV challenge and baicalein treatment in mice. F Survival curves for 50 mg/kg baicalein- and DMSO-treated mice infected with FMDV (n = 12 mice per group). G Survival curves for Prep−/− and WT mice infected with FMDV (n = 8 mice per group). H Absolute RT-qPCR analysis of FMDV RNA levels in MEF cells from Prep−/− and WT mice…

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  Fig. 7. PREP is a multifunctional host factor required for FMDV infection. In IBRS-2 cells, PREP interacts with PUM1, while PREP-KO reduces the stability of PUM1. The destabilization of PUM1 enhances porcine RIG-I promoter activity, upregulating RIG-I and MDA5, thereby activating the type I IFN signaling pathway. In other FMDV-susceptible cells, PREP-KO inhibits the FMDV life cycle at the replication stage