Middle East respiratory syndrome coronavirus ORF4b protein inhibits type I interferon production through both cytoplasmic and nuclear targets

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel and highly pathogenic human coronavirus and has quickly spread to other countries in the Middle East, Europe, North Africa and Asia since 2012. Previous studies have shown that MERS-CoV ORF4b antagonizes the early antiviral alpha/beta interferon (IFN-α/β) response, which may significantly contribute to MERS-CoV pathogenesis; however, the underlying mechanism is poorly understood. Here, we found that ORF4b in the cytoplasm could specifically bind to TANK binding kinase 1 (TBK1) and IκB kinase epsilon (IKKε), suppress the molecular interaction between mitochondrial antiviral signaling protein (MAVS) and IKKε, and inhibit IFN regulatory factor 3 (IRF3) phosphorylation and subsequent IFN-β production. Further analysis showed that ORF4b could also inhibit IRF3 and IRF7-induced production of IFN-β, whereas deletion of the nuclear localization signal of ORF4b abrogated its ability to inhibit IRF3 and IRF7-induced production of IFN-β, but not IFN-β production induced by RIG-I, MDA5, MAVS, IKKε, and TBK-1, suggesting that ORF4b could inhibit the induction of IFN-β in both the cytoplasm and nucleus. Collectively, these results indicate that MERS-CoV ORF4b inhibits the induction of type I IFN through a direct interaction with IKKε/TBK1 in the cytoplasm, and also in the nucleus with unknown mechanism. Viruses have evolved multiple strategies to evade or thwart a host's antiviral responses. A novel human coronavirus (HCoV), Middle East respiratory syndrome coronavirus (MERS-CoV), is distinguished from other coronaviruses by its high pathogenicity and mortality. However, virulence determinants that distinguish MERS-CoV from other HCoVs have yet to be identified. MERS-CoV ORF4b antagonizes the early antiviral response, which may contribute to MERS-CoV pathogenesis. Here, we report the identification of the interferon (IFN) antagonism mechanism of MERS-CoV ORF4b. MERS-CoV ORF4b inhibits the production of type I IFN through a direct interaction with IKKε/TBK1 in the cytoplasm, and also in the nucleus with unknown mechanism. These findings provide a rationale for the novel pathogenesis of MERS-CoV as well as a basis for developing a candidate therapeutic against this virus.

Figure 1. ORF4b predominantly localizes to the nucleolus and inhibits the induction of the IFN-β in a dose-dependent manner.

(A) Nuclear localization of ORF4b protein. HeLa cells were transfected with an expression plasmid for HA-tagged ORF4b protein, and then stained for ORF4b with anti-HA antibody (green) at 24 h post-transfection. Nuclei were stained with DAPI (blue). Cells were analyzed by confocal microscopy using a 100× objective, and representative images are shown. Scale bar, 20 μm. (B) ORF4b could not induce apoptosis. HeLa cells were transfected with an expression plasmid for HA-tagged ORF4b protein, and cell lysates were analyzed by Western blotting with anti-Caspase-3 and anti-PARP antibodies. FL indicates full-length and CF indicates cleaved fragment. (C) expression of ORF4b protein in cultured cells. The 293T cells were transfected with increasing amounts of an expression plasmid for HA-tagged ORF4b protein, and cell lysates were analyzed by Western blotting. (D) ORF4b inhibits the induction of IFN-β in a dose-dependent manner. 293T cells were co-transfected with pGL3-IFNβ-luc, the internal control pRL-SV40, and increasing amounts of plasmid expressing ORF4b. At 24 h post-transfection, cells were infected with Sendai virus, then cells were harvested at 24 h post-infection and analyzed for Firefly and Renilla luciferase. Data are representative of three independent experiments with triplicate samples.

Figure 2. ORF4b can inhibit IFN-β expression by targeting MDA5, TBK1, and IKKε.

(A) Association of ORF4b protein with MDA5, TBK1, and IKKε. The 293T cells were co-transfected with expression plasmids for HA-tagged ORF4b protein and the indicated transducer proteins including GFP-tagged MAVS and IRF3, FLAG-tagged IKKε, TBK1, MDA5, and RIG-I. Input cell lysates and immunoprecipitates were analyzed by Western blotting (WB) with anti-GFP (α-GFP), anti-FLAG (α-FLAG), and anti-HA (α-HA) antibodies. (B) Co-localization of ORF4b protein with MDA5, TBK1, and IKKε. HeLa cells were co-transfected with expression plasmids for HA-tagged ORF4b protein and the indicated expression plasmids for Flag-tagged MDA5, TBK1, and IKKε. Cells were then stained for ORF4b and MDA5/TBK1/IKKε with anti-HA and anti-FLAG antibodies, respectively. The green (ORF4b) and red (MDA5/TBK1/IKKε) fluorescent signals were merged. Nuclei were stained with DAPI (blue). Cells were analyzed by confocal microscopy using a 100× objective, and representative images are shown. Scale bar, 20 μm. (C) association of ORF4b protein with endogenous MDA5, TBK1, and IKKε. 293T cells were transfected with ORF4b expressing plasmid and immunoprecipitated by anti-HA monoclonal antibody, and the co-immunoprecipitation was assessed by Western blotting using anti-MDA5, IKKε, and TBK1 monoclonal antibody. (D) ORF4b could also inhibit interferon-inducing activity of IRF-3 and IRF7. Experiments were carried out as in Fig. 1D except that 293T cells were not stimulated with SeV but co-transfected with plasmids expressing MDA5, RIG-I, MAVS, TBK1, IKKε, IRF3 and IRF7. Data are representative of three independent experiments with triplicate samples.

Figure 3. 4b(Δ2-38) localizes to the cytoplasm and cannot inhibit IRF3 induced production of IFN-β.

(A) 4b(Δ2-38) localizes to the cytoplasm. Scale bar, 10 μm. (B) expression of ORF4b and ORF4b(Δ2-38) proteins in cultured cells. (C) 4b(Δ2-38) inhibits interferon-inducing activity of RIG-I/MDA5, MAVS, and TBK1/IKKε, but not IRF-3 and IRF7. Data are representative of three independent experiments with triplicate samples. *P < 0.05; **P < 0.01 versus empty (Students’ t-test). (D) 4b(Δ2-38) interacts with MDA5, IKKε and TBK1. (E) co-localization of 4b(Δ2-38) protein with MDA5, TBK1, and IKKε. Scale bar, 20 μm. Experiments were carried out as in Figs 1A,C and 2A,B,D respectively, except that 4b(Δ2-38) was accessed.

Figure 4. ORF4b has a stronger interaction with the amino terminus of MDA5, and does not disrupt formation of the complex between MDA-5 and MAVS.

(A) ORF4b protein mainly interacts with the amino terminus MDA5. The 293T cells were co-transfected with expression plasmids for HA-tagged ORF4b protein, and the indicated expression plasmids for MDA5C containing the amino-terminal domain (aa 1-287) or MDA5H containing the carboxyl-terminal domain (aa 287-1025). Input cell lysates (left panel) and immunoprecipitates (right panel) were analyzed by Western blotting (WB) with anti-FLAG (α-FLAG) and anti-HA (α-HA) antibodies. (B) ORF4b does not disrupt the formation of complex between MDA5 and MAVS. The 293T cells were co-transfected with expression plasmids for FLAG-tagged MDA5 and GFP-tagged MAVS and plasmid expressing HA-tagged ORF4b or empty vector. Input cell lysates (left panel) and immunoprecipitates (right panel) were analyzed by Western blotting (WB) with anti-MAVS (α-MAVS), anti-FLAG (α-FLAG), and anti-HA (α-HA) antibodies.

Figure 5. Inhibition of IRF3 phosphorylation and nuclear translocation in cells expressing ORF4b.

(A) ORF4b inhibits the phosphorylation of IRF3. The 293T cells were co-transfected with different combinations of expression plasmids for HA-tagged ORF4b protein and FLAG-tagged TBK1/IKKε. Cell lysates were subjected to SDS-PAGE for subsequent analysis with anti-FLAG (α-FLAG), anti-HA(α-HA), anti-phosphoIRF3 (α-p-IRF3), anti-IRF3 (α-IRF3), and anti-β-Actin (α-Actin) antibodies. (B) ORF4b inhibits nuclear translocation of IRF3. Hela cells were co-transfected with different combinations of expression plasmids for HA-tagged ORF4b protein and FLAG-tagged TBK1/IKKε. Cells were then stained for ORF4b and TBK1/IKKε with anti-HA and anti-FLAG antibodies, respectively. The green (IRF3), red (TBK1/IKKε), and blue (ORF4b) fluorescent signals were merged. Cells were analyzed by confocal microscopy using a 100× objective, and representative images are shown. Scale bar, 20 μm. (C,D) 4b(Δ2-38) inhibits the phosphorylation and nuclear translocation of IRF3. Experiments were carried out as in Fig. 5AB, except that 4b(Δ2-38) was accessed.

Figure 6. ORF4b does not affect the interaction between IKKε/TBK1 and their IRF substrates.

The 293T cells were co-transfected with expression plasmids for FLAG-tagged IKKε/TBK1, plasmids expressing HA-tagged IRF3 (A) or HA-tagged IRF7 (B), together with a plasmid expressing HA-tagged ORF4b or empty vector. Input cell lysates (left panel) and immunoprecipitates (right panel) were analyzed by Western blotting (WB) with anti-FLAG (α-FLAG) and anti-HA (α-HA) antibodies.

Figure 7. ORF4b suppresses the formation of complex between IKKε and MAVS.

The 293T cells were co-transfected with expression plasmid for FLAG-tagged IKKε, plasmid expressing GFP-tagged, together with a plasmid expressing HA-tagged ORF4b or empty vector. Input cell lysates (left panel) and immunoprecipitates (right panel) were analyzed by Western blotting (WB) with anti-FLAG (α-FLAG) and anti-MAVS (α-MAVS) antibodies.