EV71 Infection Induces IFNβ Expression in Neural Cells

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  Figure 1. Enterovirus 71 (EV71) induces the expression of IFNβ in neural cells. (A) SF268, IMR32, and SH-SY5Y cells were infected with EV71 at an multiplicity of infection (MOI) of 40, and the expression levels of IFNβ and EV71 vRNA were examined by RT-qPCR at different time points. (B) Human neural stem cells (hNSC)-derived neurons were infected with EV71 at an MOI of 40, and the expression levels of IFNβ and EV71 vRNA were examined by RT-qPCR at different time points. (C) Human NSCs were differentiated into neurons and the expression of MAP2 (i) and Neuron-specific class III beta-tubulin (ii) was assessed by immunofluorescence staining (magnification = 200x). The differentiated neurons were then infected with EV71 at an MOI of 40 for 24 h and double immunofluorescence staining was applied to detect EV71 infection. Arrows point to the cells expressing EV71 3D antigen (red) and MAP2 (green)(iii); a higher magnification of white box is shown (iv). (D) SF268 cells were infected with EV71 at different MOIs for 12 h, total RNA was extracted, and RT-qPCR was applied to detect the expression levels of IFNβ and vRNA. (E) SF268 cells were infected with EV71 2231 and EV71 BrCr at an MOI of 40 for…

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  Figure 2. EV71 infection induces IRF3 phosphorylation. Cells were seeded and infected with EV71 at an MOI of 40 for 12 h. (A) Expression of pIRF3 and viral protein 3D was examined by double immunofluorescence staining. Mock-infected cells were used as negative controls; cells transfected with poly(I:C) were applied as positive controls (magnification = 200×). Arrows indicate the cells were positive stained with anti-EV71 3D and anti-pIRF3 antibodies. (B) Percentages of pIRF3-positive and negative cells were counted in EV71-infected cells. (C) Cells were infected with EV71 at an MOI of 100. Western blot analysis was performed to detect expression of total IRF3 (T-IRF3), phosphorylated IRF3 (P-IRF3), and viral protein 3CD. Expression of β actin was used as a control. Asterisks indicate values that are statistically significant compared to mock-infected cells (*, p < 0.05, **, p < 0.01, ***, p < 0.001).

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  Figure 3. MAVS and TRIF proteins are cleaved in EV71 infected SF268 and human rhabdomyosarcoma (RD) cells. SF268 and RD cells were infected with EV71 at an MOI of 40. (A) Expression levels of MAVS, EV71 VP, and β actin were assessed by western blot. (B) Protein levels of TRIF, EV71 3C, and β actin were examined by immunoblot analysis. (C) Protein lysates were harvested at indicated times from SF268 and RD cells that were mock-infected or infected with EV71. Expression levels of virus protein 3CD, 3D, 3C, and VP were assessed by immunoblot analysis. (D) SF268 and RD cells were infected with EV71 at MOIs of 1 and 40, respectively. After 12 h of infection, expression of MAVS, EV71 VP, and β actin was analyzed by western blotting. (E) SF268 and RD cells were transfected with 3xflag-TRIF-myc and 3C-EGFP plasmids. Immunoblot analysis was performed to detect the expression levels of TRIF and 3C. (F) In vitro cleavage assays were performed to assess the activities of 3C WT and 3CC147S in cleaving TRIF proteins in SF268 and 293T cells. The expression of β actin was used as an internal control.

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  Figure 4. TLR3, TLR8, and MDA-5 are responsible for inducing IFN expression. (A) SF268 cells were infected with EV71 or UV-treated EV71 for 9 h and 12 h. Western blotting was performed to detect expression of EV71 VP and βactin, which was applied as an internal control. IFNβ transcript expression was examined by RT-qPCR. (B) Different amounts of EV71 viral RNA were transfected into SF268 cells for 12 h and expression of IFN was analyzed by RT-qPCR (LF2K= lipofectamin 2000). (C) SF268 cells were transfected with scrambled or siRNA against TLR3, TLR7, TLR8, MDA5, and RIG-I. After 48 h, the cells were infected with EV71 at an MOI of 40 for 12 h. RT-qPCR analysis was then applied to detect the expression levels of IFNβ. The inhibition efficiency of specific siRNAs was examined by western blotting (sc = scramble). Asterisks indicate values that are statistically significant compared to control cells (*, p < 0.05, **, p < 0.01, ***, p < 0.001).

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  Figure 5. Transfection of EV71 viral RNA induces more IFN transcripts in SF268 cells than in RD cells. SF268 and RD cells were infected with EV71 at MOIs of 1 and 40, as indicated, and samples were collected at different time points. (A) Total protein was isolated and subjected to immunoblotting to detect expression of EV71 viral protein 3D and VP; β actin was used as an internal control. RT-qPCR was performed to detect the expression levels of EV71 vRNA and IFNβ. (B) EV71 genomic RNA (0.5 and 0.25 μg) was transfected into SF268 and RD cells, respectively, and the expression levels of EV71 vRNA and IFNβ were examined by RT-qPCR. (C) RD and SF268 cells were transfected with poly(A:U), poly(I:C)_HMW, and motolimod with lipofectamine 2000. Total RNA was collected at 12 h post transfection and RT-qPCR was performed to detect the expression levels of IFNβ. (LF2K = lipofectamine 2000) (D) RT-qPCR was performed to assess the expression levels of TLR3, TLR8, and MDA-5. (E) The expression levels of TLR3, MDA-5, and TLR8 were assessed by western blotting. Asterisks indicate values that are statistically significant compared to mock-infected cells (*, p < 0.05, **, p < 0.01, ***, p < 0.001).

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  Figure 6. EV71 induces expression of interferon-stimulated genes (ISGs) in SF268 cells. (A) SF268 cells were infected with EV71 at an MOI of 40 or transfected with poly(I:C). Total RNA was extracted and the expression levels of MxA, ISG56, and TLR3 mRNA were determined by RT-qPCR. (B) SF268 cells were infected with EV71 at an MOI of 40 in the presence or absence of anti-IFNβ antibodies, after which expression of vRNA was analyzed by RT-qPCR. Asterisks indicate values that are statistically significant compared to mock-infected cells (*, p < 0.05, **, p < 0.01, ***, p < 0.001).