Cellular RNA Helicase DDX1 Is Involved in Transmissible Gastroenteritis Virus nsp14-Induced Interferon-Beta Production

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  Figure 1. Transmissible gastroenteritis virus (TGEV) dose-dependently triggers activation of the IFN-β signal pathway and suppresses the replication of VSV. PK-15 cells were cotransfected with pRL-TK and IFN-β-Luc (A), IRF3-Luc (C), or NF-κB-Luc (D), followed by infection with increasing doses of TGEV [multiplicity of infection (MOI) = 0.02, 0.1, or 0.5] at 12 h post-transfection. The cells lysates were collected for dual luciferase assays at 24 hpi. (B) PK-15 cells were infected with TGEV as described in panel A and harvested at 24 hpi. Cell RNAs were extracted for RT-qPCR to examine the mRNA expression levels of IFN-β. The mRNA expression levels were normalized to porcine GAPDH transcripts. Values are the mean ± SD of three independent tests. *P < 0.05, **P < 0.01, or ***P < 0.001 compared with the mock infection group. (E) PK-15 cells were infected or mock infected with TGEV (MOI = 0.02, 0.1, 0.5). The supernatants were collected for UV-irradiation and then transferred onto fresh PK-15 cells, followed by infection with vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP) at 24 h later. Cells were observed with fluorescence microscope at 16 hpi. The supernatants from…

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  Figure 2. Effect of transmissible gastroenteritis virus (TGEV) proteins on IFN-β, IRF3, and NF-κB promoter. HEK-293T cells were cotransfected with pRL-TK and IFN-β-Luc (A), IRF3-Luc (C), or NF-κB-Luc (E) together with each expression plasmid of TGEV proteins. Also, PK-15 cells were cotransfected with pRL-TK and IFN-β-Luc (B), IRF3-Luc (D), or NF-κB-Luc (F) together with each expression plasmid of TGEV proteins. The cell lysates were harvested for dual luciferase assays at 30 h post-transfection. Values are the mean ± SD of three independent tests. ***P < 0.001 or **P < 0.01 compared with empty vector group.

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  Figure 3. Transmissible gastroenteritis virus nsp14 triggers IFN-β and NF-κB activation. HEK-293T cells were cotransfected with pRL-TK plasmid and IFN-β-Luc (A), IRF3-Luc (B), or NF-κB-Luc (C) luciferase reporter plasmids together with increasing doses (0.2, 0.4, 0.8 µg) of pCAGGS-HA-nsp14 or empty vector. Also, PK-15 cells were cotransfected with pRL-TK plasmid and IFN-β-Luc (D), IRF3-Luc (E), or NF-κB-Luc (F) luciferase reporter plasmids together with increasing doses (0.2, 0.4, 0.8 µg) of pCAGGS-HA-nsp14 or empty vector. The cells lysates were harvested for dual luciferase assays at 30 h post-transfection. Values are the mean ± SD of three independent tests. **P < 0.01 or ***P < 0.001 compared with empty vector group. (G) HEK-293T cells were transfected with increasing quantities (1, 2, 4 µg) of pCAGGS-HA-nsp14 or empty vector for 30 h, and then subjected to immunoblotting with antibodies specific for endogenous IRF3, phosphorylated IRF3 (p-IRF3), p65, or p-p65. Anti-HA mouse antibody was used to confirm the expression of nsp14. β-actin expression was used as a loading control. The ratio of phosphorylated/total p65 and phosphorylated/total IRF3 was analyzed using ImageJ Software. (H)…

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  Figure 4. Interaction between transmissible gastroenteritis virus nsp14 and DDX1. (A,B) HEK-293T cells were cotransfected with pCAGGS-HA-nsp14 and/or pCMV-Tag2B-DDX1 and/or empty vector(s). Cell lysates were treated with RNase and co-immunoprecipitated with anti-HA (A) or anti-Flag (B) mAbs followed by immunoblot of nsp14 and DDX1 using anti-HA and -Flag mAbs to assess the interaction between nsp14 and DDX1 protein. (C) Colocalization of DDX1 with nsp14. PK-15 cells were transfected with pCAGGS-HA-nsp14 or empty vector. Then, the cells were fixed at 30 h post-transfection and immunostained with anti-HA mouse antibody and anti-DDX1 rabbit antibody to detect the colocalization of DDX1 with nsp14.

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  Figure 5. Silencing DDX1 decreases nsp14-stimulated IFN-β and NF-κB activation. (A,B) Assessment of the silencing efficiency of hsiDDX1. HEK-293T cells were transfected with 50 nM/well of hsiDDX1 or siNC for 36 h each. The expression levels of human DDX1 were determined by RT-qPCR (A) and western blot assay (B). (C,D) HEK-293T cells were cotransfected with psiDDX1 or siNC, together with pCAGGS-HA-nsp14 or empty vector, pRL-TK, IFN-β-Luc (C), or NF-κB-Luc (D). Dual luciferase assays were performed at 30 h post-transfection. (E) HEK-293T cells were cotransfected with psiDDX1 or siNC, together with pCAGGS-HA-nsp14 or empty vector and harvested at 30 h post-transfection. Cell RNAs were extracted for RT-qPCR to examine the mRNA expression levels of IFN-β. The mRNA expression levels were normalized to human GAPDH transcripts. All data represent the mean ± SD of three independent experiments. *P < 0.05 or ***P < 0.001 compared with the siNC group.

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  Figure 6. Involvement of DDX1 in the antiviral response triggered by transmissible gastroenteritis virus (TGEV). (A,B) Assessment of the silencing efficiency of psiDDX1. PK-15 cells were transfected with 50 nM/well of psiDDX1 or siNC for 36 h each. The expression levels of porcine DDX1 were determined by RT-qPCR (A) and western blot assay (B). (C,D) PK-15 cells were cotransfected with psiDDX1 or siNC, along with pRL-TK, IFN-β-Luc (C), or NF-κB-Luc (D). At 12 h post-transfection, cells were mock infected or infected with TGEV [multiplicity of infection (MOI) = 0.5]. Dual luciferase assays were performed at 24 hpi. (E) PK-15 cells were transfected with psiDDX1 or siNC, and 12 h later, cells were mock infected or infected with TGEV (MOI = 0.5) for 24 h. Then, the cells were collected for western blot assay with specific antibodies against p65, p-p65, DDX1, or TGEV N protein, using β-actin expression as a loading control. The ratio of phosphorylated/total p65 was analyzed using ImageJ Software. (F–J) PK-15 cells were treated as described for (E) and collected at 24 hpi. Cell RNAs were extracted for RT-qPCR to examine the mRNA expression levels of IFIT1 (F), IFIT2 (G), IFIT3 (H), IL-6 (I), and…