Systematic Proteomic Characterization of EV‐A71‐Infected Mice Identifies Dynamic Molecular Changes and Therapeutic Targets

Figures

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  FIGURE 1. Basic and functional analyses of the transcriptome and proteome. (A and B) Volcano plot. The upregulated molecules are shown in red, the downregulated molecules are shown in blue, and the molecules with no significant difference are shown in gray. (A) Volcano plot showing differential gene expression. (B) Volcano plot showing differential protein abundance. (C) Venn diagram of the transcriptome and proteome. The left circle represents the differentially expressed transcripts, and the right circle represents the DEPs. The left picture shows the results at 2 dpi, and the right picture shows those at 5 dpi. (D and E) Functional enrichment analyses for shared and unique molecules in the transcriptome and proteome at 2 dpi (D) and 5 dpi (E). The top 10 KEGG pathways enriched in each group are integrated and presented in the bubble plots.

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  FIGURE 2. Characterization of temporal changes in proteome after EV‐A71 infection. (A) Clusters depicting protein abundance at 0, 2, and 5 dpi. The DEPs at each time point were integrated into a union set. On the basis of the protein FC at each time point, fuzzy C‐means were used to separate the DEPs into six clusters. (B) Heatmap of KEGG functional enrichment analysis results for each cluster in (D). Each cluster represents the top 5 pathways, and the color of the square represents the degree of enrichment (p value). (C–E) The differential expression of enriched proteins in the complement and coagulation cascades (C), phagosome (D), and DNA replication (E) pathway. (F) PPI network of proteins in the enriched KEGG pathways in the cluster shown in (A). The red‐to‐blue color gradient represents the log2 FC of the corresponding proteins at each time point. EV‐A71, enterovirus A71.

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  FIGURE 3. EV‐A71 virus infection triggers NETs. (A) Immunofluorescence analysis in skeletal muscle. Skeletal muscle sections from EV‐A71‐infected and NC mice at Day 5 were stained with an anti‐MPO antibody (green), an anti‐histone H3 antibody (red), and DAPI (blue). The image was captured using 10× (bar = 250 µm) and 40× (bar = 75 µm). Seven‐day‐old neonatal BALB/c mice were intraperitoneally injected with 100 µL of EV‐A71 per mouse. Mice in the LY6G‐Knockout group were administered 100 µL of anti‐LY6G (10 mg/kg) on alternate days, whereas mice in the isotype group were given rat IgG2a isotype control (10 mg/kg) on alternate days. Both groups were observed daily (n = 8) and dissected and sampled for blood and muscle at 5 dpi (n = 6). (B) The body weights changes, clinical scores, and survival rates were recorded from 1 to 14 dpi. (C) The viral loads in blood and muscle. (D) Pathological grading in LY6G‐knockout and isotype groups. (E) Skeletal muscle sections from LY6G‐knockout and isotype groups were stained with H&E. Black arrows represent muscle fiber necrosis; yellow arrows represent connective tissue hyperplasia; red arrows represent inflammatory cell infiltration. The image was…

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  FIGURE 4. Phosphoproteomic profiling revealing dysregulated pathways and kinases after EV‐A71 infection. (A) Volcano plot showing the activation and inhibition of phosphorylation sites. (B) Differentially expressed phosphosites at each time point were integrated into a union set. On the basis of the phosphosite FC at each time point, fuzzy C‐means was used to separate the differentially expressed phosphosites into six clusters. (C) Heatmap of KEGG functional enrichment analysis results for each cluster in (B). Each cluster represents the top 5 pathways, and the color of the square represents the degree of enrichment (p value). (D) PPI network of phosphosites and proteins in the enriched top 5 KEGG pathways in the cluster shown in B. The red‐to‐blue color gradient represents the log2 FC of the corresponding proteins at each time point. Heatmap of predicted kinases that may have had significantly altered activity after EV‐A71 infection; intersection (E) and union (F) of two time points. Kinase activity is represented by NES values. EV‐A71, enterovirus A71.

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  FIGURE 5. Mapping regulated kinases to kinase inhibitors identifies EV‐A71 therapies. (A) Drugs (left) mapped to kinases (right) whose activity was regulated by EV‐A71 infection. Lines connecting them indicate known kinase targets for each drug. (B) Western blotting images showing kinase activation in control (left) and EV‐A71‐infected (right) mice. (C) RD cells were pretreated with drugs at the indicated doses and then infected with EV‐A71 for 72 h. The viral load in the cell supernatants was determined. The inhibitory effect of drug treatment on EV‐A71 replication. The graphs show the results for regorafenib, zanubrutinib, sorafenib, vandetanib, pralsetinib, ponatinib, lenvatinib, selpercatinib, nintedanib, dasatinib, baricitinib, tofacitinib, acalabrutinib, avitinib, nilotinib, ellagic acid, ATP, and lycorine (n = 3). (D) Viral load in the cell supernatants of the vandetanib (4 µM), nintedanib (4 µM), dasatinib (4 µM), avitinib (8 µM), nilotinib (8 µM), and lycorine (8 µM) treatment groups and the control group. All data are represented as the mean ± SEM, and one‐way ANOVA was used for statistical analysis. EV‐A71, enterovirus A71. *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 6. Kinase inhibitor drug inhibited viral replication in mice. Ten‐day‐old BALB/c mice were infected with 100 LD50 or 0.1 LD50 EV‐A71 virus by intraperitoneal injection. Vandetanib (10 mg/kg/day), nintedanib (5 mg/kg/day), dasatinib (10 mg/kg/day) and avitinib (5 mg/kg/day) were administered by gavage, and nilotinib (5 mg/kg/day) and lycorine (0.6 mg/kg/day) were administered intraperitoneally for 2 h prior to infection and then daily. PBS was used as treatment in the NC group. Both groups were observed daily (100 LD50, n = 8) and dissected and sampled for blood and muscle at 5 dpi (0.1 LD50, n = 6). The body weights changes (A), clinical scores (B), and survival rates (C) were recorded from 1 to 12 dpi. Blood (D) and muscle (E) samples were collected to detect the viral loads at 5 dpi. (F and G) Skeletal muscle sections from vandetanib‐, nintedanib‐, dasatinib‐, avitinib‐, nilotinib‐, lycorine‐, and PBS‐treated mice were stained with H&E. Black arrows represent muscle fiber necrosis; Blue arrow nucleolysis, rare calcification; yellow arrows represent connective tissue hyperplasia; red arrows represent inflammatory cell infiltration. The image was captured using 10× (bar = 200 µm)…