Protein Interactions Network of Porcine Circovirus Type 2 Capsid With Host Proteins

Figures

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  FIGURE 1. Characterization of PCV2 Cap-interacting cellular proteins. (A) Mock infected PK-15 cells or infected with PCV2 at MOI of 1.0 were harvested 48 hpi and coimmunoprecipitation assay was performed by purified anti-Cap IgG. PCV2 Cap-interacting host proteins were eluted with protein A/G sepharose and analyzed on SDS-PAGE followed by silver staining. β-actin was used as a internal loading control. For (A), lane 1, protein molecular weight ladder; lane 2, Mock-infected; lane 3, PCV2-infected. (B) Venn diagram of the identified protein candidates interacting with PCV2 Cap from mock infected, PCV2 infected and reported previously, respectively. Blue, orange, and gray colors indicate proteins from mock infected, PCV2 infected and reported previously, respectively. Common proteins within the data sets have been indicated in the colored intersections. Proteins have been represented as the respective NCBI gene names (Supplementary Table S1).

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  FIGURE 2. Construction and analysis of the protein-protein interactions (PPIs) network using STRING database. Each edge color indicates a different method of PPIs prediction as indicated below the figure. The map of PCV2 Cap-interacting host proteins further interacting with the other proteins of our data were constructed and plotted by using the network analyzer tool of “Cytoscape version 3.7.1.” The corresponding symbols indicating different protein classes have been mentioned on the figure. Proteins have been represented as the respective NCBI gene names (Supplementary Table S2).

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  FIGURE 3. Gene ontology analysis of identified PCV2 Cap-host interactome. (A,B) Representative overrepresented GO terms of protein clusters and the GO distribution of all number of proteins in the PCV2-infected cells versus mock-infected were classified into three categories using software Cytoscape 3.7.1 with plugin GOclue and were shown for significantly enriched terms based on biological process (BP), molecular function (MF), cellular component (CC), p-value < 0.05. The Roman numerals represented the detailed GO terms as shown in Supplementary Tables S3, S5.

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  FIGURE 4. KEGG pathway enrichment analysis. (A,B) Graph shows the enriched pathways targeted by PCV2 Cap-interacting proteins, analyzed by KEGG functional annotation pathway database (Supplementary Tables S4, S5) using software Cytoscape 3.7.1 with plugin GOclue (Supplementary Figures S1A,B). Schematic representation of the MAPK signaling pathway (imported from KEGG: map04010) and the spliceosome pathway targeted by PCV2 Cap-interacting proteins (imported from KEGG: map03040). Proteins interacting with PCV2 Cap within the entire pathway are depicted red color.

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  FIGURE 5. Validation of Cap-host protein interactions. (A,B) HEK-293T cells were co-transfected with plasmids expressing FLAG-hnRNPC, FLAG-NPM1, FLAG-DDX21, FLAG-IPO5, FLAG-EIF4A2, FLAG-SNAI2, FLAG-MX2, or FLAG-IC1 and plasmids expressing GFP-Cap (A), Myc-Cap (B), respectively. Among them, GFP-Cap (A), Myc-Cap (B), FLAG-hnRNPC (A,B) co-transfected with empty vector were served as negative controls and GFP-Cap (A), Myc-Cap (B) co-transfected with FLAG-IC1 were served as positive controls, respectively. The cell lysates were immunoprecipitated with FLAG beads, anti-GFP, or anti-Myc mAbs, separated by SDS-PAGE, western-blotted and detected with corresponding antibodies, respectively. β-actin was served as internal loading control. (C) Purified His-sumo-Cap was separately added to the GST, GST-hnRNPC, GST-NPM1, GST-DDX21 proteins and His-IC1 was also separately added to the GST, GST-dCap proteins and then subjected to GST pull-down assays and immunoblotted with corresponding antibodies, respectively. (D) PCV2 Cap-host interactions network. Interaction map of PCV2 Cap with interacting host proteins was constructed by software Cytoscape 3.7.1. Proteins were classified on the basis of their…