Duck Enteritis Virus VP16 Antagonizes IFN‐ β ‐Mediated Antiviral Innate Immunity

Figures

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  Figure 1. The immune evasion of DEV in vitro. (a) DEFs were cultured in 6-well plates and infected with WT DEV (1 MOI) when they grew up to 90%. At 4 hours, 12 hours, and 24 hours, the infected cells were collected and real-time qPCR was performed to determine the transcriptional levels of duck IFN-β in the DEF. (b) DEFs were seeded in 24-well plates and transfected with 500 ng of the duIFN-β-Luc (duck IFN-β promoter reporter plasmid), together with 50 ng of pRL-TK (Renilla luciferase plasmid, Promega, America) and pCAGGS empty vector or plasmids encoding the indicated protein (duck IRF7, duck STING, or duck cGAS, 500 ng/well). Cells were mock infected or infected with 0.5 MOI WT DEV 12 hours posttransfection, and firefly luciferase activities were measured at 48 h postinfection; the data were analyzed by GraphPad Prism software, and results were presented using two-way ANOVA (n = 3) and considered significant (∗∗∗∗P < 0.0001).

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  Figure 2. DEV VP16 is having a demonstrable effect on IFN-β activation and IFN-β-related ISGs. (a) Duck IFN-β mRNA relative levels in DEF transfected 4 μg or 10 μg pCAGGS-UL48-HA, stimulated with 25 μg/mL poly (I:C) for over 36 hours at 12 hours posttransfection, were presented by real-time qPCR. The expression of VP16 was analyzed by western blotting using anti-HA and anti-β-actin (as a control) (CST, America). In this group, an increased amount of VP16-HA expression plasmid was used. (b) DEFs seeded in 24-well plates were transfected with pCAGGS or pCAGGS-UL48-HA (500 ng/well), together with duIFN-β-Luc (500 ng/well) and pRL-TK (50 ng/well) for 12 hours; then, 25 μg/mL poly (I:C) was transfected into cells for another 24 hours. Firefly luciferase activities were measured and the data were analyzed by GraphPad Prism software, and results were presented using two-way ANOVA (n = 3). (c, d) Quantitative real-time PCR analysis of duck OASL and duck Mx mRNA levels in DEF cells which express VP16-HA or not. Duck ISG mRNA relative expression was upregulated by (c) poly (I:C) or (d) poly (dA:dT). Experiments were shown using two-way ANOVA (n = 3). Significant, ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P <…

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  Figure 3. DEV VP16 could inhibit IFN-β promoter activity at IRF level but not IRF1. DEFs were cultured in 24-well plates and then cotransfected with duIFN-β-Luc (500 ng), pRL-TK (50 ng), and appointed plasmids encoding (a) STING and cGAS, (b) TBK1, (c) IRF7, and (d) IRF1. Luciferase assays were performed as described for Figure 1 to measure the activation of the IFN-β promoter.

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  Figure 4. A sketch of DNA sensing pathway mainly induced by herpesvirus and how the VP16 of DEV exerts function in immune evasion strategy.

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  Figure 5. DEV VP16 associates with duck IRF7 but not with duck IRF1. HEK293T cells were cotransfected with pCAGGS-UL48-HA and pCAGGS-IRF7-Flag or pCAGGS-IRF1-Flag. (a) At 48 hours after transfection, cells were resolved to coimmunoprecipitation and immunoblot analysis with the indicated antibodies. (b) Colocalization of UL48 (green) and IRF7 (red) in HEK293T cells, the nuclei were stained by DAPI (1 : 1000) (blue). (c) Localization of UL48 (green) and IRF1 (red) in HEK293T cells. Nuclei stained by DAPI were shown in blue.

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  Figure 6. N-terminal of VP16 plays the most effective role in the inhibition of IFN-β promoter activity. (a) Construction of different kinds of truncated VP16 protein. (b, c) Transfection of DEFs that were cultured in 24-well plates with duIFN-β-Luc (500 ng), pRL-TK (50 ng), and VP16 or plasmids encoding truncated VP16, the same with that described in Figure 2(a).