Interaction Network of Porcine Circovirus Type 3 and 4 Capsids with Host Proteins

Figures

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  Figure 1. Characterization of PCV3 or PCV4 Cap-interacting cellular proteins. (A) Empty vector, PCV3, or PCV4 Cap-transfected PK-15 cells were harvested at 48 hpt, and a co-immunoprecipitation assay was performed using anti-FLAG beads. PCV3 or PCV4 Cap-interacting host proteins were eluted and analyzed via SDS-PAGE followed by Coomassie blue staining. Lane 1, protein molecular weight ladder; lane 2, empty vector-transfected; lane 3, PCV3 Cap-transfected; lane 4, PCV4 Cap-transfected. (B) Venn diagram of the identified protein candidates interacting with PCV3 or PCV4 Cap from the empty vector-transfected, PCV3 Cap-transfected, and PCV4 Cap-transfected cells, respectively. Blue, orange, and gray colors indicate proteins from the empty vector-transfected, PCV3 Cap-transfected, and PCV4 Cap-transfected cells, respectively. Common proteins within the data sets are indicated in the colored intersections. Proteins were represented as their respective NCBI gene names (Supplementary Table S1).

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  Figure 2. Construction and analysis of the protein-protein interaction network using the STRING database. Each edge color indicates a different method of protein–protein interaction prediction, as indicated below the figure. A map of the interaction of 278 identical bound host proteins shared amongst PCV3 and PCV4 Caps with the other proteins in our data was constructed and plotted using the network analyzer tool of the Cytoscape software, version 3.7.1. The corresponding symbols indicating the different protein classes are shown. Proteins are represented by their respective NCBI gene names (Supplementary Table S2).

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  Figure 3. Gene ontology analysis of the identified PCV3 and PCV4 Cap-host interactome. (A,B) Representative overrepresented GO terms of protein clusters and the GO distribution of all proteins in PCV3 and PCV4 Cap-transfected cells versus empty vector-transfected cells were classified into three categories using the GOclue plugin in Cytoscape software, version 3.7.1. Shown are the significantly enriched terms based on biological process (BP), molecular function (MF), and cellular component (CC) with p-values < 0.05. The Roman numerals represent the detailed GO terms, as shown in Supplementary Table S4.

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  Figure 4. KEGG pathway enrichment analysis. (A,B) Graphs showing the enriched pathways targeted by the PCV3 and PCV4 Cap-interacting proteins, as analyzed via KEGG functional annotation (Supplementary Table S5) using the GOclue plugin in Cytoscape software, version 3.7.1.

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  Figure 5. Validation of Cap-host protein interactions. (A,B) HEK293T cells were co-transfected with plasmids expressing FLAG-NPM1, FLAG-NCL, FLAG-DDX21, FLAG-hnRNPA2/B1, FLAG-YTHDF1, FLAG-YBX1, FLAG-RNF2, or FLAG-STAT6 and plasmids expressing Myc-PCV3-Cap (A), or Myc-PCV4-Cap (B), respectively. Among them, Myc-PCV3-Cap (A), Myc-PCV4-Cap (B), or FLAG-NPM1 (A,B) co-transfected with an empty vector served as negative controls, while Myc-PCV3-Cap (A) or Myc-PCV4-Cap (B) co-transfected with FLAG-NPM1 served as positive controls. Cell lysates were immunoprecipitated with FLAG beads or anti-Myc mAbs, separated via SDS-PAGE, and then subject to Western blotting with the corresponding primary and secondary antibodies. β-actin served as the internal loading control. (C) Whole-cell lysates of PCV3 or PCV4 Cap were separately added to the GST, GST-NCL, GST-hnRNPA2/B1, or GST-YBX1 proteins; subjected to GST pull-down assays; and then immunoblotted with the corresponding primary and secondary antibodies. (D) The PCV3 and PCV4 Cap-host interaction network. The interaction map of PCV3 and PCV4-Cap and the corresponding host proteins was constructed using Cytoscape software, version 3.7.1. Proteins were…