Marek’s disease virus protein kinase US3 inhibits DNA-sensing antiviral innate immunity via abrogating activation of NF-κB

Figures

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  Fig 1. MDV US3 inhibits IFN-β and IL-6 production induced by ISD, cGAS-STING, and DNA virus. (A) IFN-β-Luc reporter was co-transfected with ISD and a US3 expression plasmid or empty vector into DF-1 cells; 24 h later, cells were harvested and subjected to a dual-luciferase reporter assay. (B) DF-1 cells were transfected with US3 expression plasmid or empty vector for 24 h and then transfected with ISD; IFN-β mRNA levels were measured by qPCR 8 h later, and IFN-β protein levels were measured by ELISA 24 h later. (C, D) DF-1 cells were transfected with US3 expression plasmid or empty vector for 24 h and then transfected with ISD; IL-6 mRNA levels were measured by qPCR 12 h later, and IL-6 protein levels were measured by ELISA 24 h later. (E) The cGAS and STING expression plasmids were co-transfected with US3 plasmid or an empty vector, and the IFN-β-luc reporter into DF-1 cells, and IFN-β promoter luciferase activity was measured 24 h later. (F) The cGAS and STING expression plasmids were co-transfected with US3 expression plasmid or empty vector into DF-1 cells, and IFN-β mRNA and protein levels were measured by qPCR and ELISA 24 h post-transfection. (G) DF-1 cells were transfected with…

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  Fig 2. MDV US3 inhibits activation of NF-κB. (A) The cGAS and STING expression plasmids were co-transfected with the NF-κB-Luc reporter, as well as US3 expression plasmid or empty vector, into DF-1 cells; 24 h later, cells were subjected to the dual-luciferase reporter assay. (B–E) NF-κB-Luc reporter was transfected with plasmids expressing IKKα (B), IKKβ (C), p65 (D), or p50 (E) together with US3 plasmid or an empty vector. Dual-luciferase reporter assays were performed 24 h post-transfection. **P < 0.01.

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  Fig 3. MDV US3 kinase activity is essential for inhibiting DNA sensing signaling. (A, B) The cGAS and STING expression plasmids were co-transfected with IFN-β-Luc (A) or NF-κB-Luc (B) reporters, as well as plasmid encoding US3 or a US3 kinase-dead mutant US3K220A or empty vector, into DF-1 cells; 24 h later, cells were subjected to the dual-luciferase reporter assay. (C, D) NF-κB-Luc reporter was transfected with plasmids expressing p65 (C) or p50 (D) together with US3, US3K220A expression plasmid or empty vector. Dual-luciferase reporter assays were performed 24 h post-transfection. ns, no significant difference, **P < 0.01.

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  Fig 4. MDV US3 interacts with p65 and p50. (A, B) HEK293T cells were transfected with the indicated plasmids for 36 h before coimmunoprecipitation and immunoblot analyses with the indicated antibodies. (C, D) DF-1 cells were transfected with US3-Flag expression plasmid or an empty vector, and 36 h posttransfection, a coimmunoprecipitation assay was performed with an anti-Flag antibody. (E, F) HEK293T cells were co-transfected with US3-Flag and p65RHD-HA (E) or p50RHD-HA (F) plasmids. At 36 h post-transfection, cells were harvested, and coimmunoprecipitation assays were performed using anti-Flag antibodies.

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  Fig 5. MDV US3 phosphorylates p65 and p50 and reduces their nuclear translocation. (A, B) The p65-HA (A) or p50-HA (B) expression plasmid was co-transfected with an empty vector, US3- or US3K220A-expressing plasmid to DF-1 cells. Forty-eight hours later, cells were lysed and left untreated or treated with Lambda protein phosphatase (Lambda PP) for further Western blot analyses with indicated antibodies. (C) DF-1 cells were transfected and treated with ISD, and the cell lysates were separated into cytoplasmic and nuclear extracts. The p65 and p50 protein levels in the cytoplasm and nucleus were analyzed by Western blot. (D) CEFs were infected with wild-type MDV (MDV-WT) or the US3-deficient MDV (MDV-dUS3) for 24 h. The cell lysates were separated into cytoplasmic and nuclear extracts and analyzed with immunoblot assays with the indicated antibodies. The data represent results from one of the triplicate experiments. *P < 0.05, **P < 0.01.

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  Fig 6. US3 deficiency facilitated IFN-β induction and attenuated MDV replication in chickens. (A) CEFs were infected with wild-type MDV (MDV-WT) or the US3-deficient MDV (MDV-dUS3) for 12 h before the analysis of IFN-β, ZAP, and IFITM3 mRNA levels. (B) The growth properties of MDV-WT and MDV-dUS3 in CEF cell cultures. (C) The indicated cells were infected with wild-type MDV or MDV-dUS3, respectively, and the MDV viral titers were tested with qPCR after 6 days of infection. (D–F) Chickens were infected with MDV-WT and MDV-dUS3, and IFN-β (D), IL-6 (E), ZAP, and IFITM3 (F) production in chickens were measured by qPCR at the indicated time points during MDV infection. (G) Chickens were infected with MDV-WT and MDV-dUS3, and virus genome copy numbers in the spleen were monitored at the indicated time points during MDV infection. ns, no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001.