Viperin Inhibits Enterovirus A71 Replication by Interacting with Viral 2C Protein

Figures

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  Figure 1. EVA71 induces viperin expression in vitro. (A,B) RD, (C,D) SK-N-SH, and (E,F) CCF-STTG1 cells were infected with EVA71 at a multiplicity of infection (MOI) = 1 for the indicated time points. The relative expression of viperin was detected using quantitative polymerase chain reaction (q-PCR) using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the control. After the cells were infected with EVA71 at an MOI = 1, levels of viperin mRNA and viral RNA of EVA71 were detected using real-time q-PCR at the indicated time points. Data are representative of at least three independent experiments performed in triplicate (mean ± SD of fold change). * p < 0.05 and ** p < 0.01. (G) Western blot analysis of viperin protein, VP1 protein, and β-actin of EVA71-infected CCF-STTG1 cells at the indicated time points.

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  Figure 2. EVA71 induced viperin expression in vivo. Two-week-old BALB/c mice were infected with high-virulence EVA71 (GZ-CII strain), and the relative expression levels of (A) viperin and (B) EVA71 viral RNA in brain tissues were measured using q-PCR with GAPDH as a control. Data are representative of at least three independent experiments performed in triplicate (mean ± SD of fold change). ** p < 0.01. (C) Double-labeling immunohistochemistry analysis of EVA71 VP1 and viperin in the brain tissue was performed. Green fluorescence represents viperin protein, red fluorescence represents EVA71 VP1 protein, and blue fluorescence represents nuclei. The scale bar in the images is 20 μm. The enlarged images corresponding to the rectangle areas are shown next to the original image.

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  Figure 3. Viperin suppresses EVA71 replication. 293T cells were transfected with FLAG or FLAG-viperin for 24 h and then infected with EVA71 at an MOI = 1 for the indicated times. Subsequently, expression levels of VP1 were detected by (A) western blotting and (B) q-PCR with GAPDH as a control. (C) Synthesized siRNAs—Si-viprin-1, Si-viprin-2, and Si-viprin-3—or negative control (NC) were used to transfected 293T cells. At 24 h post-transfection, cells were treated with type I interferon for 6 h and lysed to determine siRNA efficiency by detecting viperin protein through western blotting. (D) 293T cells were then transfected with viperin siRNA or negative control for 24 h followed by infection with EVA71 at an MOI = 1 for the indicated times. Expression levels of VP1 were measured using western blotting. (E) q-PCR analysis with GAPDH as control to detect the viral RNA of EVA71 of viperin siRNA transfected 293T cells at indicated time points. Data are representative of at least three independent experiments performed in triplicate (mean ± SD of fold change). * p < 0.05 and ** p < 0.01.

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  Figure 4. Viperin colocalization with EVA71 2C in the endoplasmic reticulum (ER). 293T cells were transfected with (A) GFP-viperin, (B) HA-2C, or (C) co-transfected with HA-2C and GFP-viperin for 24 h. The cells were fixed and stained with ER-Tracker Blue-White for ER labeling and then visualized under a fluorescent microscope. In the above experiments, viperin (green) and HA-2C (red) are shown, as well as ER (blue) counterstained with ER-Tracker. The enlarged images corresponding to the rectangle areas are shown next to (A and B) or below (C). Scale bar is 10 μm.

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  Figure 5. EVA71 2C interacted with viperin. FLAG-viperin was (A) co-transfected with HA-2C into 293T cells or (B) transfected alone into 293T cells that were then infected with EVA71 at an MOI = 1. After 24 h, the cells were lysed and analyzed by co-immunoprecipitation (co-IP) and immunoblotting assays. (C) HA-2C, HA-3D, and the empty expressing vector pCAGGS-HA were co-transfected with FLAG-viperin into 293T cells for 24 h, and the cells were lysed and analyzed by co-immunoprecipitation (co-IP) and immunoblotting assays with indicated antibodies.

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  Figure 6. The N-terminal domain is responsible for viperin binding to 2C. (A) Schematic diagram of a series of viperin deletion mutants. (B) FLAG-viperin, ΔSAM3+4, ΔSAM2+3, ΔSAM1+2, ΔSAM, ΔC, or ΔN were co-transfected with HA-2C into 293T cells for 24 h. The cells were then lysed, followed by co-IP and immunoblotting assays. (C) 293T cells were co-transfected HA-2C with ΔC, ΔSAM, viperin, ΔN, ΔN-42, ΔN-50, ΔN-65, or ΔN-50 for 24 h, and then the cells were lysed and used for co-IP and immunoblotting.

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  Figure 7. The N-terminal domain was responsible for viperin inhibition of EVA71 replication. 293T cells were transfected with FLAG-viperin, ΔN, ΔSAM, ΔC, FLAG-N-viperin, or FLAG-C-viperin for 24 h, and then infected with EVA71 at an MOI = 1 for 12 h. The cells were lysed and EVA71 (A) RNA levels were measured using q-PCR, while (B) VP1 protein levels in the infected cells were analyzed using western blotting. Data are representative of at least three independent experiments performed in triplicate (mean ± SD of fold change). ** indicates p < 0.01, *** indicates p < 0.001 and “NS” indicates there was no significant difference between the two groups.