Swine Interferon-Inducible Transmembrane Proteins Potently Inhibit African Swine Fever Virus Replication

Figures

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  Figure 1. The expression level of SwIFITMs. (A) Expression levels of SwIFITMs in different porcine tissues were normalized by β-actin mRNA. (B) Expression levels of SwIFITMs in PAMs were normalized by β-actin mRNA and SwIFITM3 served as a control. (C) Expression levels of SwIFITMs in PAMs after treatment with IFN-α for 24 h were compared with untreated PAM mRNA, Mx1 served as a control. (D) Expression levels of SwIFITMs in PAMs after being infected with ASFV for 24 h were compared with untreated PAM mRNA. (E) Expression levels of SwIFITMs in PAMs after being infected with ASFV (0.1 MOI) each 6 h from 0 to 36 hpi. All samples were analyzed in triplicate, and three independent experiments were performed. * p<0.05; ** p<0.01; *** p<0.001 vs negative control group. Error bars indicate standard deviation.

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  Figure 2. Subcellular localization of SwIFITMs in PK-15 cells and Vero cells. The cell nucleus, expression of SwIFITM1a, -1b, -2, -3, and -5 proteins, and LAMP1-positive late endosomes (red) in (A) PK-15 cells and (B) Vero cells are indicated by blue, green, and red, respectively.

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  Figure 3. Knockdown of SwIFITMs enhances ASFV replication. (A) SwIFITM mRNA expression levels were evaluated in PAMs 24 h after transfection of 50 nmol of siRNA1a, 1b, 2, or negative control. (B) SwIFITM mRNA expression levels were evaluated in PAMs 24 h after transfection of 50 nmol of siRNA3 or negative control. (C) SwIFITM mRNA expression levels were evaluated in PAMs 24 h after transfection of 50 nmol of siRNA5 or negative control. siRNAs were transfected into PAMs separately for 24 h and then infected with 0.1 MOI of ASFV; the virus replication level of ASFV was determined by (D) RT-qPCR and (E) western blotting with β-actin used as a control at 36 hpi; (F) WB quantification by densitometry for each viral protein and β-actin, and the density quantitative ratios of each viral protein and β-actin were calculated; (G) the viral titration of progeny virus was tested in HAD assay. Samples were analyzed in triplicate, and three independent experiments were performed. ** p < 0.01; *** p<0.001; **** p<0.0001 vs negative control group. Error bars indicate standard deviation.

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  Figure 4. Overexpression of SwIFITMs Inhibits ASFV replication. pEF-SwIFITMs with FLAG tags were transfected into WT Vero cells separately for 24 h and then infected with 0.1 MOI of ASFV; (A) the cells viability was detected by cell proliferation assay; (B, C) the virus replication levels of ASFV were determined by western blotting at 36 hpi, and band densitometry for each protein was determined, and the density quantitative ratios of each viral or SwIFITM protein and β-actin were calculated, ratio of SwIFITM protein and β-actin served as a control; (D) the expression levels of p72 protein were detected by RT-qPCR with β-actin used as a control at 36 hpi; (E) the viral titration of progeny virus was tested in HAD assay. Samples were analyzed in triplicate, and three independent experiments were performed. ** p<0.01; *** p<0.001; vs empty vector control group. Error bars indicate standard deviation.

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  Figure 5. Generation and validation of csIFITM knockout Vero cell lines. (A) Three different sgRNA targeting sites and the result of sanger sequencing with WT and csIFITMs− Vero cells. (B) Expression of endogenous csIFITMs was detected by western blotting with β-actin used as a control. (C) Proliferation of csIFITMs−, control, and untreated Vero cells were detected by the CCK-8 Cell Counting method. The csIFITMs−, control, and untreated Vero cells were infected with 0.1 MOI of ASFV; (D, E) the virus replication levels of ASFV were determined by western blotting at 36 hpi, and band densitometry for each protein was determined, and the density quantitative ratios of each viral or SwIFITM protein and β-actin were calculated, ratio of SwIFITM protein and β-actin served as a control; (F) the expression levels of p72 protein were detected by RT-qPCR with β-actin used as a control at 36 hpi; (G) the viral titration of progeny virus was tested in HAD assay. Samples were analyzed in triplicate, and three independent experiments were performed. ** p < 0.01; *** p<0.001 vs empty vector control group. Error bars indicate standard deviation.

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  Figure 6. Antiviral effect of SwIFITMs on ASFV. pEF-SwIFITMs with FLAG stag were transfected into csIFITMs− Vero cells separately for 24 h and then infected with 0.1 MOI of ASFV; (A) the cells viability was detected by cell proliferation assay; (B, C) the virus replication levels of ASFV were determined by western blotting at 36 hpi, and band densitometry for each protein was determined, and the density quantitative ratios of each viral or SwIFITM protein and β-actin were calculated, ratio of SwIFITM protein and β-actin served as a control; (D) the expression levels of p72 protein were detected by RT-qPCR with β-actin used as a control at 36 hpi; (E) the viral titration of progeny virus was tested in HAD assay. Samples were analyzed in triplicate, and three independent experiments were performed. * p < 0.05; ** p < 0.01; *** p<0.001 vs empty vector control group. Error bars indicate standard deviation.