Toll-Like Receptor 3 Is Involved in Detection of Enterovirus A71 Infection and Targeted by Viral 2A Protease

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  Figure 1. TLR3 detects EV-A71 infection to trigger type I IFN-mediated antiviral immunity. (A) HEK293 cells were transfected with an IFN-β luciferase reporter plasmid, plus either a control vector (pcDNA6.0) or a Flag-tagged RIG-I construct. After 24 h, the transfected cells were either left untreated, treated with poly(I:C) (0.2 µg/mL) transfection, or infected with an influenza A virus (IAV/WSN/33, 50 HA/well) or EV-A71 (4643 strain, MOI, 0.1 and 1). After another 40 h, the treated cells were harvested to analyze the IFN-β promoter activity. The EV-A71 used for most experiments was the 4643 strain, unless otherwise indicated. (B) Similar to Panel A, His-tagged MDA-5 was used to assess the IFN-β promoter activity in transfected HEK293 cells upon EV-A71 (MOI, 0.1) infection. (C–E) HEK293 cells were transfected with either a control vector or a Flag-tagged TLR3 plasmid with an IFN-β (C,D) or an ISRE (E) luciferase reporter plasmid. After 24 h, the transfected cells were either left untreated, treated with poly(I:C) (50 µg/mL), or infected with EV-A71 (MOI, 0.1). After another 24 h (C) or 40 h (D,E), the treated cells were harvested for luciferase assays. (F) HEK293 cells were transfected…

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  Figure 2. EV-A71-derived, double-stranded RNA is a ligand for TLR3 detection. (A–D) HEK293 cells were transfected with either a control vector or Flag-tagged TLR3, plus an IFN-β reporter plasmid (A,C) or an ISRE reporter plasmid (B,D). After 24 h, the transfected cells were either left untreated or infected with EV-A71 (MOI, 0.1) or heat-inactivated EV-A71 (iEV-A71) (MOI, 0.1). After another 40 h, the treated cells were harvested to analyze the IFN-β or ISRE promoter activity (A,B). Control RNA (ctrl, 2 µg/mL) and EV-A71-infected RNA (two doses, 1 and 2 µg/mL) harvested from HEK293 cells and EV-A71-infected HEK293 cells, respectively, were used to stimulate the transfected cells by DOTAP lipofection. After another 20 h, the treated cells were harvested to analyze the IFN-β or ISRE promoter activity (C,D). (E) HEK293 cells were transfected with Flag-tagged TLR3. Control RNA (1 µg/mL) and EV-A71-infected RNA (1µg/mL) were used to stimulate the transfected cells by DOTAP lipofection. After another 20 h, IFN-β in the supernatants was measured using ELISA. (F) Like Panel A, HEK293 cells were transfected as indicated. EV-A71-infected RNA left untreated or treated with RNase III or Shrimp…

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  Figure 3. TLR3 downstream signaling is essential for triggering a type I IFN response to EV-A71 infection. (A,B) HEK293 cells were treated with the indicated siRNAs targeting Trif, TAPE, and MAVS for 48 h, and the cells were subsequently transfected with Flag-tagged TLR3 and an IFN-β (A) or ISRE (B) reporter plasmid. These cells were infected with EV-A71 (MOI, 0.1) for 40 h, and were then harvested for luciferase assays. (C) Like panel A, EV-A71-infected RNA was used to stimulate cells by DOTAP lipofection, and then the IFN-β promoter activity was measured using luciferase assays. (E,F) HEK293 cells were treated as in Panel B, with no transfection of the ISRE reporter plasmid. Supernatants from the treated cells were measured using ELISA for the production of IFN-β (D), RANTES (E), and IP-10 (F). (G–I) Western blot analyses of endogenous TAPE (G), TRIF (H), and MAVS (I) in HEK293 cells treated with indicated siRNAs or control siRNA. ND means “not detected.” * p < 0.05, Student’s t-test. Data are representative of at least two or three independent experiments.

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  Figure 4. TLR3 senses EV-A71 infection in primary macrophages and dendritic cells. (A–C) Mouse bone marrow-derived macrophages (BMMs) transfected with control siRNA or two different TLR3 siRNAs were infected with EV-A71 (MP4 strain, MOI, 3) for 6 h, and then mRNA expression levels of Ifnb (A), Il6 (B), and Tlr3 (C) genes were measured using qRT–PCR. ** p < 0.005 and *** p < 0.0005 (Student’s t-test). Data are representative of at least three independent experiments. Expression is normalized to ml32 and set at 1 in unstimulated cells with control siRNA. (D,E) Human monocyte-derived dendritic cells (MoDCs) transfected with control siRNA or TLR3 siRNA were infected with EV-A71 (MOI, 3) for 6 h, and then mRNA expression levels of Ifnb (D) and Tlr3 (E) genes were measured using qRT-PCR. * p < 0.05 (Student’s t-test). NS stands for “not significant.” Data are representative of at least three independent experiments. Expression is normalized to actin and set at 1 in unstimulated cells with control siRNA.

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  Figure 5. The TLR3 protein level is decreased upon EV-A71 infection. (A) HEK293 cells transfected with Flag-tagged TLR3 were left untreated or infected with EV-A71 (MOI, 1 and 3). After 24 h, the treated cells were harvested for the Western blot analysis of TLR3 protein levels. (B) Similar to panel A, EV-A71 (4643 strain, MOI, 3) and EV-A71 (MP4 strain, MOI, 1) were used to infect TLR3-transfected HEK293 cells for the indicated times. These cells were then harvested for the Western blot analysis of TLR3. (C) Like Panel B, the treated cells were harvested at the indicated times to analyze the mRNA levels of Tlr3, EV-A71, and β-Actin by RT-PCR. (D,E) SK-N-SH cells were left untreated or infected with EV-A71 (4643 strain, MOI, 0.2) for 4 h, 8h, and 16 h, and the cells were harvested for the Western blot analysis of the endogenous TLR3 protein level. (F) SK-N-SH cells were left untreated or infected with EV-A71 (4643 strain, MOI, 0.1), and the cells were harvested at the indicated times (6 h and 16 h) to analyze the mRNA levels of Tlr3 and β-Actin by qRT-PCR. (G) SK-N-SH cells were left untreated or infected with EV-A71 (4643 strain, MOI, 0.1 and 1) for 6 h, and the cells were harvested to…

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  Figure 6. EV-A71 protease 2A targets TLR3 to inhibit type I IFN induction. (A) HEK293 cells were transfected with Flag-tagged TLR3 alone or together with increasing amounts of either Flag-tagged EV-A71 2A or Flag-tagged EV-A71 3C. After 20 h, the transfected cells were harvested for the Western blot analysis using the indicated antibodies. (B) HEK293 cells were transfected with Flag-tagged TLR3 alone or together with Flag-tagged EV-A71 2A. After 4 h, the transfected cells were treated with a pan-caspase inhibitor Z-VAD-FMK (100 μM) or a proteasome inhibitor MG132 (20 μM). After another 16 h, the treated cells were harvested for the Western blot analysis using the indicated antibodies. (C) HEK293 cells were transfected with Flag-tagged TLR3 alone or together with V5-tagged EV-A71 2A or V5-tagged EV-A71 2A C110A. After 48 h, the transfected cells were harvested for the Western blot analysis using the indicated antibodies. (D) 293T cells were transfected with FLAG-TLR3. Cell lysates were subjected to the immunoprecipitation and incubated with purified EV-A71 2A protease at 37 °C for 4 h; treated cell lysates were then subjected to Western blotting. (E,F) HEK293 cells were transfected with an…

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  Figure 7. A model for the detection of EV-A71 infection by TLR3. Upon infection into host cells, EV-A71 generates dsRNA during viral replication. Endosomal TLR3 in host cells detects EV-A71 dsRNA, possibly via autophagy, to recruit the adaptor TRIF and TAPE, which further trigger signaling pathways to IFN-β activation and subsequent expression of IFN-stimulated genes (ISGs). To promote EV-A71 survival in host cells, EV-A71 uses its 2A protease to target TLR3 for degradation to counteract type I IFN-mediated antiviral responses.