Porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between IRF3 and TBK1

Abstract

Porcine epidemic diarrhea virus (PEDV), a porcine enteropathogenic coronavirus, causes lethal watery diarrhea in piglets and results in large economic losses in many Asian and European countries. A large-scale outbreak of porcine epidemic diarrhea occurred in China in 2010, and the virus emerged in the United States in 2013 and spread rapidly, posing significant economic and public health concerns. Previous studies have shown that PEDV infection inhibits the synthesis of type I interferon (IFN), and viral papain-like protease 2 has been identified as an IFN antagonist. In this study, we found that the PEDV-encoded nucleocapsid (N) protein also inhibits Sendai virus-induced IFN-β production, IFN-stimulated gene expression, and activation of the transcription factors IFN regulatory factor 3 (IRF3) and NF-κB. We also found that N protein significantly impedes the activation of the IFN-β promoter stimulated by TBK1 or its upstream molecules (RIG-I, MDA5, IPS-1, and TRAF3) but does not counteract its activation by IRF3. A detailed analysis revealed that the PEDV N protein targets TBK1 by direct interaction and that this binding sequesters the association between TBK1 and IRF3, which in turn inhibits both IRF3 activation and type I IFN production. Together, our findings demonstrate a new mechanism evolved by PEDV to circumvent the host's antiviral immunity.

Importance: PEDV has received increasing attention since the emergence of a PEDV variant in China and the United States. Here, we identify nucleocapsid (N) protein as a novel PEDV-encoded interferon (IFN) antagonist and demonstrate that N protein antagonizes IFN production by sequestering the interaction between IRF3 and TBK1, a critical step in type I IFN signaling. This adds another layer of complexity to the immune evasion strategies evolved by this economically important viral pathogen. An understanding of its immune evasion mechanism may direct us to novel therapeutic targets and more effective vaccines against PEDV infection.

FIG 1

N protein significantly inhibits SEV-induced expression of IFN-β and ISGs. HEK-293T cells grown in 24-well plates were transfected with 1 μg of a plasmid encoding PEDV N protein (pCAGGS-HA-N) or an empty vector, and 24 h later, the cells were infected with SEV (10 hemagglutinating activity units/well). Twelve hours after infection, the cells and supernatants were collected separately. (A and C to E) Total RNA was extracted from cells, and the expression levels of the IFN-β (A), ISG56 (C), ISG54 (D), ISG20 (E), and GAPDH genes were evaluated by quantitative real-time RT-PCR. The results are expressed as increases in mRNA levels relative to those in cells transfected without SEV infection and were normalized to the expression level of the GAPDH housekeeping gene. (B) The harvested supernatants were used to detect IFN-β by an ELISA. Anti-HA antibody was used to confirm the expression of PEDV N protein, and anti-β-actin antibody was used to detect β-actin, which served as a protein loading control. The results are representative of data from three independent experiments. WB, Western blot.

FIG 2

N protein independently inhibits promoter activation of IFN-β, IRF3, and NF-κB. HEK-293T cells were cotransfected with IFN-β–Luc (A), IRF3-Luc (B), or NF-κB–Luc (C) together with the pRL-TK plasmid and increasing quantities (0, 0.2, 0.4, or 0.8 μg) of plasmid pCAGGS-HA-N. Twenty-four hours after the initial transfection, the cells were infected with SEV. Luciferase assays were performed 12 h after infection. The results represent the means and standard deviations of data from three independent experiments. The relative firefly luciferase activity was normalized to the Renilla reniformis luciferase activity, and the untreated empty vector control value was set to 1. The expression of PEDV N protein was confirmed by immunoblotting with an anti-HA antibody.

FIG 3

N protein blocks the phosphorylation and nuclear translocation of endogenous IRF3. (A) HEK-293T cells were mock transfected or transfected with an expression plasmid encoding HA-tagged N protein 24 h before being infected with SEV or not for 8 h. Cell lysates were collected for immunoblot analysis with antibodies directed against phosphorylated IRF3 (Ser396), IRF3, HA, or β-actin. (B) HEK-293T cells were transfected with plasmid pCAGGS-HA-N or an empty vector. At 24 h after transfection, the cells were infected with SEV for 8 h. After the fixation and permeation of the cells, an immunofluorescence analysis was performed to detect endogenous IRF3 (red) and N protein (green) with rabbit anti-IRF3 and mouse anti-HA antibodies, respectively. DAPI staining (blue) indicates the locations of the cell nuclei. Fluorescent images were acquired with a confocal laser scanning microscope (Fluoview ver. 3.1; Olympus, Japan). Cells transfected with an empty vector or mock infected with SEV were used as the negative controls.

FIG 4

N protein disrupts the TBK1/IKKε-mediated IFN signaling pathway. HEK-293T cells were cotransfected with IFN-β–Luc, the pRL-TK plasmid, and pCAGGS-HA-N together with constructs expressing RIG-I/RIG-IN, MDA-5, IPS-1, TRAF3, IKKε, TBK1, or IRF3. Luciferase assays were performed 28 h after transfection. The results represent the means and standard deviations of data from three independent experiments. The relative firefly luciferase activity was normalized to the Renilla reniformis luciferase activity, and the untreated empty vector control value was set to 1. Western blotting with anti-HA antibody shows expression of PEDV N protein, and Western blotting for β-actin served as a protein loading control.

FIG 5

PEDV N protein interacts with TBK1. (A) HEK-293T cells were transfected with expression constructs encoding HA-tagged PEDV N protein and Flag-tagged RIG-I, MDA-5, IPS-1, TRAF3, IKKε, TBK1, and IRF3. The cells were lysed 28 h after transfection and subjected to immunoprecipitation with anti-HA antibody. The whole-cell lysates (WCL) and immunoprecipitation (IP) complexes were analyzed by immunoblotting (IB) using anti-Flag, anti-HA, or anti-β-actin antibodies. Because three different Flag-tagged control vectors were used to construct the expression plasmids, they were detected in three different gels. (B) Expression plasmids encoding Flag-tagged TBK1 and HA-tagged N protein were cotransfected, and immunoblotting analyses were performed as described above for panel A except for immunoprecipitation with anti-Flag antibody. (C) HEK-293T cells were transfected with expression plasmids encoding HA-tagged N protein and Flag-tagged TBK1. The cells were then fixed for an immunofluorescence assay to detect N protein (red) and TBK1 (green) with anti-HA and anti-Flag antibodies, respectively, 28 h after transfection. DAPI staining (blue) indicates the locations of the cell nuclei. Fluorescent images were acquired with a confocal laser scanning microscope (Fluoview ver. 3.1; Olympus, Japan).

FIG 6

N protein inhibits TBK1-mediated IRF3 phosphorylation and sequesters the interaction between IRF3 and TBK1. (A) HEK-293T cells were cotransfected with an empty vector or expression plasmids encoding HA-tagged N protein and Flag-tagged TBK1. Twenty-four hours after transfection, the cells were mock infected or infected with SEV for 8 h, followed by immunoblotting assays with antibodies directed against phosphorylated IRF3 (Ser396), IRF3, HA, Flag, or β-actin. (B) HEK-293T cells were transfected with increasing amounts (0, 1.5, 3, or 6 μg) of a plasmid expressing HA-tagged N protein. Twenty-four hours after transfection, the cells were mock infected or infected with SEV for 8 h. The cell lysates were immunoprecipitated with rabbit anti-TBK1 or rabbit normal IgG antibody (lane 2). They were then analyzed by immunoblotting (IB) with antibodies directed against TBK1, IRF3, or HA. The whole-cell lysates (WCL) were used to analyze the expression of TBK1, IRF3, and N protein by immunoblotting using anti-TBK1, anti-IRF3, and anti-HA antibodies, respectively.