Antiviral Role of IFITM Proteins in Classical Swine Fever Virus Infection

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  Figure 1. Overexpression of IFITMs inhibits classical swine fever virus (CSFV) replication in porcine alveolar macrophages (PAMs). (A) Cell viability of cell lines stably overexpressing IFITMs. (B) Confirmation of CMV-IFITM1, CMV-IFITM2, and CMV-IFITM3 lentivirus transfection by detection of enhanced green fluorescent protein (EGFP) reporter. (a) Mock-transfected PAMs. (b) PAMs transfected with lentiviruses expressing CMV. (c) PAMs transfected with CMV-IFITM1 lentivirus, CMV-IFITM2 lentivirus (d), and CMV-IFITM3 lentivirus (e). Scale bars, 100 μm. (C) Western blot analysis of PAM cell lines stably expressing Flag-tagged IFITM1, IFITM2, or IFITM3 or CMV alone using an anti-Flag antibody. (D) Western blot analysis of IFITMs in PAM cell lines stably expressing Flag-tagged IFITM1, IFITM2, or IFITM3 or CMV alone using an IFITM1-specific antibody and an IFITM2/3 antibody against IFITM2 and IFITM3, respectively. β-actin served as an internal control. (E) CSFV genomic RNA in CMV-IFITM1, CMV-IFITM2, and CMV-IFITM3 cell lines. The CMV, CMV-IFITM1, CMV-IFITM2, and CMV-IFITM3 cell lines were infected with CSFV at a multiplicity of infection (MOI) of 1. CSFV genomic RNA levels were determined by…

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  Figure 2. Knockdown of IFITMs mediated by short hairpin RNA (shRNA) enhances CSFV replication. (A) Cell viability of stable IFITM-knockdown cell line. (B) Confirmation of recombinant IFITM shRNA lentivirus (shIFITM) transfection by detection of EGFP reporter. (a) Mock-transfected PAMs. (b) PAMs transfected with scrambled shRNA lentivirus (shN). (c) PAMs transfected with shIFITM. Scale bars, 100 μm. (C) Western blot analysis of IFITM expression in IFITM-knockdown cells using an IFITM1-specific antibody and an IFITM2/3 antibody against IFITM2 and IFITM3, respectively. β-actin served as an internal control. (D) Western blot analysis of IFITM expression using an anti-Flag antibody in shIFITM cell lines following transfection with CMV-IFITM1, CMV-IFITM2, or CMV-IFITM3 for 48 h. β-actin served as an internal control. (E) CSFV genomic RNA in shIFITM cell lines. The shN and shIFITM cell lines were infected with CSFV at an MOI of 1. CSFV genomic RNA levels were determined by RT-qPCR at 12 and 24 hpi. Data were normalized to β-actin expression. (F) Infectious progeny viral titers in culture medium from shIFITM cells. The viral titers of CSFV in supernatants were quantified and expressed as…

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  Figure 3. Expression of IFITMs is induced by interferon (IFN)-α treatment. RT-qPCR analysis of IFITM1 (A) and IFITM2/3 (B) mRNA levels (normalized to β-actin expression) in PAMs treated with IFN-α (10 ng/mL) at 6, 12, 18, and 24 h post-treatment (hpt). (C) Western blot analysis of IFITM expression in PAMs treated with IFN-α (10 ng/mL) at 6, 12, 18, and 24 hpt. β-actin served as an internal control. (D) CSFV genomic RNA in shIFITM cell lines with IFN-α pre-treatment. The shN and shIFITM cell lines were pre-treated with IFN-α and then infected with CSFV at an MOI of 1. CSFV genomic RNA levels were determined by RT-qPCR at 24 hpi. Data were normalized to β-actin expression. RT-qPCR analysis of IFITM1 (E), IFITM2/3 (F) and Mx1 (G) mRNA levels (normalized to β-actin expression) in shN and shIFITM cells. Data (A,B,D,E–G) represent the mean ± SD of three independent experiments and were measured in technical duplicate. Comparisons between groups were performed by Student’s t test. ** p < 0.01 and *** p < 0.001

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  Figure 4. IFITM expression is downregulated by CSFV. RT-qPCR analysis of IFITM1 (A) and IFITM2/3 (B) mRNA levels (normalized to β-actin expression) in PAMs infected with CSFV at an MOI of 0.1, 1, or 3 for 24 h. Data represent the mean ± SD of three independent experiments and were measured in technical duplicate. Comparisons between groups were performed by Student’s t test. * p < 0.05 and *** p < 0.001 (C) Western blot analysis of IFITM expression in PAMs infected with different doses of CSFV (MOI = 0.1, 1, or 3) for 24 h. β-actin served as an internal control.

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  Figure 5. Distribution of IFITMs in CSFV-infected PAMs. (A) Confirmation of C1-IFITM1, C1-IFITM2, and C1-IFITM3 transfection in PAMs by western blot detection of fusion proteins with an anti-GFP antibody. PAMs were transfected with C1-IFITM1, C1-IFITM2, C1-IFITM3, or C1-EGFP for 48 h and collected for western blot analysis. β-actin served as an internal control. (B) Confocal images of PAMs expressing EGFP-tagged IFITM1, 2, or 3 or EGFP alone. PAMs were transfected with C1-EGFP (a), C1-IFITM1 (b), C1-IFITM2 (c), or C1-IFITM3 (d) for 24 h, followed by mock infection or infection with CSFV (MOI = 1) for another 24 h. Cells were fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bars, 10 μm.

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  Figure 6. IFITMs do not interfere with CSFV binding but restrict CSFV entry. (A) Virus binding assay. PAMs were inoculated with CSFV at an MOI of 1 for 1 h at 4 °C. After washing with ice-cold phosphate-buffered saline (PBS), cell-bound CSFV was quantified by RT-qPCR. Data were normalized to β-actin expression. (B) Entry assay. PAMs were inoculated with CSFV at an MOI of 1 for 1 h at 4 °C. The viral inoculum was removed, and cells were washed with ice-cold PBS and then incubated with culture medium at 37 °C. After 2 h, PAMs were treated with PBS containing proteinase K to strip the CSFV that remained on the cell surface. CSFV genomic RNA levels were determined by RT-qPCR. Data were normalized to β-actin expression. Data represent the mean ± SD of three independent experiments and were measured in technical duplicate. Comparisons between groups were performed by Student’s t test. *** p < 0.001.

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  Figure 7. Colocalization of IFITMs with Rab5, Rab7, and Lamp1. Confocal images of IFITM colocalization with Rab5 (A), Rab7 (B), and Lamp1 (C) in mock-infected and CSFV-infected PAMs. The expression of Rab5, Rab7, and Lamp1 was visualized by immunofluorescence staining with an anti-Rab5, anti-Rab7, or anti-Lamp1 antibody, followed by the Alexa Fluor 594 AffiniPure goat anti-rabbit IgG (H + L) antibody. (A) PAMs were transfected with IFITM-expressed plasmid for 24 h and then mock-infected or infected with CSFV at an MOI of 1. PAMs were fixed at 24 hpi and stained with anti-Rab5 antibody (red). Nuclei (blue) were stained with DAPI. (B) PAMs were transfected with IFITM-expressed plasmid for 24 h, followed by CSFV (MOI = 1) infection for another 24 h. PAMs were stained with anti-Rab7 antibody (red) and DAPI (blue). (C) PAMs transfected with IFITM-expressed plasmid for 24 h were infected with CSFV (MOI = 1) for another 24 h. PAMs were stained with anti-Lamp1 antibody (red). Nuclei (blue) were stained with DAPI. Scale bars, 10 μm. (D) Overlap quantification of IFITMs with Rab5, Rab7, and Lamp1 was performed using Image J software and measured for individual cells. More than 10 cells were analyzed…