Antiviral Role of Serine Incorporator 5 (SERINC5) Proteins in Classical Swine Fever Virus Infection

Figures

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  FIGURE 1. Serine incorporator 5 (SERINC5) inhibited CSFV growth in PK-15 (A) and 3D4/2 (B) cells. Cells were infected with CSFV (MOI = 0.1) for 24 and 48 h after transfection with p3 × Flag-SERINC5. Statistical analysis of the influence of overexpression of SERINC5 on viral genome replication in PK-15 (C) and 3D4/2 (D) cells. Statistical analysis of the influence of SERINC5 on CSFV Npro protein expression in PK-15 (E) and 3D4/2 (F) cells. The expression of CSFV Npro protein was analyzed by western blotting using anti-CSFV Npro antibody. GAPDH was used as an internal citation control. The relative levels of the targeted proteins were analyzed with ImageJ software, and the ratios were calculated relative to the GAPDH control. Error bars represent the mean ± SD; n = 3; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; NSP > 0.05.

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  FIGURE 2. The RNA interference of SERINC5 enhanced CSFV replication in PK-15 and 3D4/2 cells. siRNA knockdown of SERINC5 in PK-15 (A,C) and 3D4/2 (B,D) cells transfected with siNC or SERINC5 siRNA-1/-2/-3. The expression of SERINC5 was assessed by western blotting (A,B) and qRT-PCR (C,D) at 24 hpi. PK-15 (E,G) and 3D4/2 (F,H) cells were infected with CSFV (MOI = 0.1) for 24 and 48 h after transfection with SERINC5 siRNA. Statistical analysis of the influence of SERINC5 siRNA on viral titers (E,F) and viral genome replication (G,H). The effect of overexpression and siRNA on cell viability of PK-15 (I) and 3D4/2 (J) cells. Cells were transfected with p3 × Flag-CMV, p3 × Flag-SERINC5, siNC and SERINC5 siRNA were evaluated by CCK-8 assay. The relative levels of the targeted proteins were analyzed with ImageJ software, and the ratios were calculated relative to the GAPDH control. Error bars represent the mean ± SD; n = 3; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; NSP > 0.05.

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  FIGURE 3. Classical swine fever virus (CSFV) infection downregulates SERINC5 expression in cells. Statistical analysis of the SERINC5 protein and transcription in PK-15 (A,C) and 3D4/2 (B,D) cells infected with CSFV (MOI = 0.1) for 0, 6, 12, 24, 36, and 48 h by western blotting (A,B) and qRT-PCR (C,D). Statistical analysis of SERINC5 protein and transcription in CSFV infected PK-15 (E,G) and 3D4/2 (F,H) cells at an MOIs of 0, 0.01, and 0.1 for 24 h by western blotting (E,F) and qRT-PCR (G,H). The relative levels of the targeted proteins were analyzed with ImageJ software, and the ratios were calculated relative to the GAPDH control. Error bars represent the mean ± SD; n = 3; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; NSP > 0.05.

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  FIGURE 4. Classical swine fever virus (CSFV) infection downregulates SERINC5 expression in tissues of CSFV-infected pigs. Tissue samples (heart, liver, spleen, lung, kidney, brain, inguinal lymph nodes, mesenteric lymph nodes, thymus, and tonsil) were collected after the pigs were euthanized at 7 dpi. Protein (A) and transcription (B) of SERINC5 in different tissues were analyzed by western blotting and qRT-PCR, respectively. The relative levels of the targeted proteins were analyzed with ImageJ software, and the ratios were calculated relative to the GAPDH control. Error bars represent the mean ± SD; n = 3; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; NSP > 0.05.

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  FIGURE 5. Serine incorporator 5 (SERINC5) interacted with the signaling adaptor MDA5. (A) Co-IP assay revealed the interactions of SERINC5 with MDA5 in HEK-293T cells. Cells were transfected with p3 × Flag-SERINC5 and HA-labeled proteins of the type I IFN signaling pathway (HA-RIG-I, HA-MDA5, HA-MAVS, HA-TBK1, HA-IRF3, and HA-IRF7). The cell lysates were harvested and immunoprecipitated with anti-Flag mAb, and analyzed by western blotting with anti-HA and anti-Flag mAb. (B) Y2H analysis of the interactions between SERINC5 and MDA5. The fusion plasmids of AD-SERINC5/BD-MDA5 or AD-MDA5/BD-SERINC5 were transfected into yeast strain Y2H Gold. The vectors of BD-Lamin/AD-T and BD-p53/AD-T were introduced into yeast strain Y2H Gold as negative and positive controls, respectively. (C) Confocal microscopy analysis of interactions of SERINC5 with MDA5 in PK-15 cells. PK-15 cells were transfected with p3 × Flag-SERINC5 and HA-MDA5 for 24 h. The cells were incubated with anti-Flag mAb and anti-HA mAb and detected by appropriate secondary antibodies followed by observation on a confocal fluorescence microscope. In the merged images, the yellow represents protein colocalization. Scale bar; 10 μm.

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  FIGURE 6. Serine incorporator 5 (SERINC5) positively regulates the type I IFN signaling pathway. (A) PK-15 cells were transfected with p3 × Flag-SERINC5 followed by infection with CSFV (MOI = 0.1) for 24 and 48 h. The expression of IFNα, IFNβ, TNFα, IL-18, and IL-6 mRNA was determined by qRT-PCR. (B) HEK-293T cells were transfected with p3 × Flag-SERINC5 together with the IFNβ, ISRE or NF-κB luciferase reporter and pTK-Rluc for 24 h. The cells were then infected with SeV for another 24 h. Luciferase activities were detected via a dual-luciferase reporter assay. pTK-Rluc was used as an internal control. (C) HEK-293T cells were transfected with p3 × Flag-SERINC5 along with the IFNβ or ISRE luciferase reporter and pTK-Rluc. Luciferase activities were detected after incubation with poly (I:C) (10 mg/mL) or 5′-ppp-RNA (2 mg/mL) for 24 h. pTK-Rluc was used as an internal control. The protein expression of p3 × Flag-SERINC5 in HEK-293T cells was assayed by western blotting. GAPDH was used as a loading control. Error bars represent the mean ± SD; n = 3; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; NSP > 0.05.

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  FIGURE 7. Melanoma differentiation-associated protein 5 (MDA5) activation of the type I IFN signaling pathway is enhanced by SERINC5. HEK-293T cells were co-transfected with p3 × Fag-SERINC5 (0, 50, 100, and 200 ng), HA- RIG-I, MDA5, MAVS, IRF3, and IRF7. Luciferase activities of IFNβ (A) and ISRE (B) were detected by a dual-luciferase reporter assay after transfection with the IFNβ or ISRE luciferase reporter and pTK-Rluc for 24 h. pTK-Rluc was used as an internal control. PK-15 cells were co-transfected with p3 × Flag-SERINC5 (0, 50, 100, and 200 ng) together with HA-MDA5 (200 ng), HA-RIG-I (200 ng), or HA-CMV (200 ng) for 24 h. The gene expression of IFNα (C), IFNβ (D), Mx1 (E), and OAS1 (F) were analyzed by qRT-PCR. Error bars represent the mean ± SD; n = 3; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; NSP > 0.05.

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  FIGURE 8. Inhibition of MDA5 expression reduced the antiviral activity of SERINC5. The siRNA silencing efficiency of MDA5 (A,C) and RIG-I (B,D) in PK-15 cells were assessed by western blotting (A,B) and qRT-PCR (C,D). The influence of SERINC5 anti-CSFV activity after transfection with siRNAs targeted MDA5 and RIG-I siRNA. PK-15 cells were infected with CSFV (MOI = 0.1) for 24 h after co-transfection with MDA5 siRNA, siNC with p3 × Flag-SERINC5 or p3 × Flag-CMV (E,G), RIG-I siRNA, siNC with p3 × Flag-SERINC5 or p3 × Flag-CMV (F,H). The viral titers (E,F) were reported as TCID50, and viral genome copy numbers (G,H) were analyzed by qRT-PCR. The relative levels of the targeted proteins were analyzed with ImageJ software, and the ratios were calculated relative to the GAPDH control. Error bars represent the mean ± SD; n = 3; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; NSP > 0.05.