NLRC5 Regulates Enterovirus 71 Infection Through an IFN-β-Dependent Pathway

Figures

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  Figure 1. Expression of NLRC5 activated by EV71 infection. (A,B) HE staining of brain tissue in the infected and control groups. Scale bars = 100 μm. (C) GSEA revealed that the NLR signal pathway was significantly upregulated. (D) Expression of NLRC5 in RNA-seq. (E) Expression of NLRC5 in different tissues. (F–I) Western blot and RT-qPCR analysis of relative NLRC5 expression in RD and Hela cells at different hours with 1 MOI EV71. *: p < 0.05, **: p ≤ 0.01, ***, p ≤ 0.001, NS: p ≥ 0.05.

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  Figure 2. The EV71-mediated NLRC5 response is dependent on viral replication. (A) NLRC5 activation mediated by EV71-infected RD cells showed a viral dose-dependent effect. (B) Graph showing 1MOI UV-inactivated virus or HeatKill-inactivated virus with wild-type-virus-infected RD cells; the NLRC5 mRNA expression was detected 6 h later. *: p < 0.05, NS: p ≥ 0.05.

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  Figure 3. NLRC5 inhibits the replication of EV71 (A–C). Transfected Hela cells with 1 μg, 2 μg, and 3 μg of Flag-NLRC5 plasmid, followed by EV71 infection 24 h later. At 12 h post-infection (hpi), cell samples and cultured supernatants were collected to asses EV71 VP1 capsid protein expression and viral titre analysis using a TCID50 assay, respectively (D). Hela cells were transfected with a Flag-NLRC5 or the control plasmid and then infected with EV71. Protein samples were then harvested at the indicated time points post-infection for immunoblot analysis of VP1 expression (E). The pRHF-EV71 5′UTR plasmid and Flag-NLRC5 plasmid (100 ng, 200 ng, 500 ng, 1000 ng) were con-transfected into 293T cells. At 48 h post-transfection, cell lysates were collected to determine Rluc and Fluc luciferase activity using a dual-luciferase reporter assay (F–H). Hela cells were transfected with varying concentrations (10, 20 or 50 nm) of siNLRC5 and subsequently infected with EV71. At 12 h post-infection, protein samples were harvested and analyzed for VP1 expression (G). Viral titres in the culture supernatants were quantified using a TCID50 assay (H). *: p < 0.05, **: p ≤ 0.01, ***, p ≤ 0.001, ****: p ≤…

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  Figure 4. EV71 activates NLRC5 through the IFN-β pathway. (A) Cells were treated with graded concentrations of IFN-β for 8 h, followed by RNA extraction. (B,C) IFN-β expression profiles in RD and Hela cells following EV71 infection. (D) Time-dependent expression of RIG-I after EV71 infection. (E,F) Relative mRNA expression levels of IRF3 and IRF7 in EV71-infected RD and Hela cells. (G,H) Protein expression levels of IRF3 in EV71-infected RD and Hela cells. (I) Expression of interferon pathway-related proteins in EV71-infected Vero cells. (J) Expression of STAT1, NLRC5, and MHC-I in Vero cells in response to IFN-β stimulation. *: p < 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001, NS: p ≥ 0.05.

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  Figure 5. EV71 upregulates MHC-I expression through the IFN-β-activated NLRC5 pathway. (A) RNA sequence profiling of MHC-I-related genes in brain tissues from EV71-infected versus control neonatal mice. (B) Flow cytometric analysis of MHC-I expression in Hela cells infected with EV71 (MOI = 1) for 12 h. (C,D) Expression of NLRC5 and MHC-I in RD cells (C) infected for 6 h and Hela cells (D) infected for 12 h. (E) Subcellular localization of NLRC5 in Hela cells infected with EV71 (MOI = 0.1) for 12 h, compared with uninfected controls. Scale bars = 10 μm. (F) MHC-I expression levels under EV71 infection conditions after IFN-β treatment (5 ng/mL, 24 h). (G) MHC-I expression in Hela cells transfected with NLRC5 followed by EV71 infection. *: p < 0.05, **: p ≤ 0.01, ****: p ≤ 0.0001, NS: p ≥ 0.05.

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  Figure 6. The negative feedback effect of NLRC5 on IFN-β expression. (A) Inhibitory effect of NLRC5 on RIG-I and IRF3. RD cells were transfected with 1 μg, 2 μg, and 3 μg of Flag-NLRC5 plasmid, and protein samples were collected 24 h later to assess the expression of RIG-I and IRF3. (B,C) NLRC5 suppresses IFN-β expression. RD cells were transfected with increasing doses of Flag-NLRC5, stimulated with poly(I:C) for 12 h, and subjected to RT-qPCR analysis of IFN-β mRNA (B). HEK293T cells were co-transfected with an IFN-β–luc reporter plasmid and 100 ng, 200 ng, and 500 ng of Flag-NLRC5 and stimulated with poly(I:C) after 24 h. Luciferase activity was measured at 48 h using a dual-luciferase assay system (C). *: p < 0.05, **: p ≤ 0.01.