Cleavage of AUF1 by Coxsackievirus B Affects DDX5 Regulatory on Viral Replication through iTRAQ Proteomics Analysis

Figures

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  Figure 1. GO term enrichment analyses of DEPs in the AUF1-overexpressing group vs. the control group. GO term analyses of proteomics that were cataloged as (a) biological process (BP); (b) molecular function (MF); and (c) cellular component (CC). The top 20 significant GO term enrichment results are shown (P < 0.05). Terms enriched in both upregulated and downregulated protein sets are marked with a gradient color (red to green); terms also enriched in the upregulated protein set are marked in red, while green indicates the downregulated protein set. All of the BP, MF, and CC terms were ranked in terms of enrichment of the differentially expressed proteins, and the top 20 are presented here.

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  Figure 2. KEGG enrichment analyses of DEPs associated with the AUF1 overexpression group vs. the control group. KEGG analyses of proteomics that were cataloged as (a) top pathways enriched with all the DEPs. Upregulated pathways are marked in red, while downregulated pathways are marked in green. Those enriched in both up- and downregulated pathways are marked with a gradient color (red to green); (b) top upregulated pathways enriched by KEGG are listed; (c) Top downregulated pathways enriched by KEGG are listed. Each dot represents the pathways, the color represents the P value, and the size of the dot represents the number of DEPs enrolled. The proportion of genes is represented by the x-axis.

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  Figure 3. Protein–protein interaction network (STRING) obtained from analyzing the products of DEGs. (a, b, and c) The nodes were analyzed every two factors. (d) Thirty-eight DEPs were mapped in the PPI networks. Using Cytoscape 3.6.1, DEPs were mapped in PPI networks. The thresholds of the three parameters were set as follows: degree =50, betweenness =10000, closeness =3.25E-4, and 95% confidence level (STRING score) =0.4. The thickness of the lines indicates the number of interactions that are detailed in STRING (http://string-db.org).

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  Figure 4. The clusters in the network (k = 3). We numbered the clusters from 1 to 29 in descending order according to the MCODE score analyzed with the Cytoscape plugin. (a) Clusters that had more hub nodes were selected. (b) Clusters that had a higher proportion of all genes were selected. (c) Clusters were selected that had a larger value for the sum of parameter “degrees.” (d) Cluster 1, which contained the most hubs, was selected, and hubs are highlighted in yellow background. The red dashed box shows the hub nodes connected with AUF1 directly and mainly played roles in RNA regulation. Red lines indicate the interactions between the hubs.

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  Figure 5. GO term and KEGG enrichment in cluster 1 and the DDX-centric subnetwork. (a) DDX-centric subnetwork module. (b) Top of GO BP enrichment in cluster 1. (c) Top of GO MF enrichment in cluster 1. (d) Top of GO CC enrichment in cluster 1. (e). KEGG enrichment in cluster 1. Each dot represents the pathways, the color represents the P value, and the size of the dot represents the number of DEPs enrolled. The proportion of genes is represented by the x-axis.

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  Figure 6. Correlations and validation of AUF1 regulation of DDX5 w/w.o viral infection. (a) Heatmap of correlations between proteins in cluster 1 and analysis of bidirectional hierarchical clustering. (b) The RT–qPCR assay was used to examine the DDX5 mRNA expression level with or without AUF1 overexpression (t test, P < 0.01). GAPDH was used as an internal control. (c) DDX5 protein expression levels were detected with or without silencing AUF1 with siRNA by Western blot analysis. β-Tubulin was used as a loading control. (d) DDX5 protein levels were detected under the condition that AUF1 was cleaved during CVB infection at different MOIs and infection hours. VP1 was used for viral detection, and β-tubulin was used as a loading control. (e) CVB VP1 was examined in HeLa cells with or without EGFP-DDX5 overexpression by Western blot analysis. (f) CVB genome RNA expression levels in HeLa cells with or without EGFP-DDX5 overexpression were detected by RT-qPCR.

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  Figure 7. Schematic diagram of the interaction between DDX5, AUF1, and CVB. AUF1 is cleaved under CVB infection; thus, the inhibitory effect of AUF1 on DDX5 was suppressed. DDX5 further increases and improves CVB replication.