The Network of Interactions Between Classical Swine Fever Virus Nonstructural Protein p7 and Host Proteins

Figures

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  FIGURE 1. Identification of the interaction of CSFV p7 with host cellular proteins by the Yeast two-hybrid assay. To confirm the interaction between CSFV p7 and host cellular protein, the yeast strain Y2HGold was co-transformed with pGBKT7-p7 (BD-p7)/pGADT7-cellular protein (AD-cellular protein), pGBKT7 (BD)/AD-cellular protein (negative control), pGBKT7-p53 (BD-53)/pGADT7-T (AD-T) (positive control), pGBKT7-Lamin (BD-Lamin)/AD-T (negative control), and BD/pGADT7 (AD) (negative control). Host cellular proteins included microtubule-associated protein RP/EB family member 1 (MAPRE1), voltage-dependent anion channel 1 (VDAC1), proteasome maturation protein (POMP), protein inhibitor of activated STAT 1 (PIAS1), gametogenetin binding protein 2 (GGNBP2), COP9 signalosome subunit 2 (COPS2), and contactin 1 (CNTN1). Yeast cells co-transformed with AD-cellular protein and BD-p7 could grow on three kinds of synthetically defined media and form blue colonies on QDO/X/A medium, which indicated that the host cellular protein interacted with CSFV p7 protein in yeast cells.

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  FIGURE 2. Identification of the interaction of CSFV p7 with MAPRE1 (A) and VDAC1 (B) by co-IP assay, respectively. Co-immunoprecipitation assay demonstrated that CSFV p7 bonded MAPRE1 and VDAC1 in HEK 293T cells, respectively. HEK 293T cells were co-transfected with EGFP-p7 and p3 × Flag plasmids and harvested at 24 h post-transfection. Cell lysates were immunoprecipitated with antibodies against EGFP or antibody against p3 × Flag followed by Western blot analysis.

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  FIGURE 3. Identification of the interaction of CSFV p7 with MAPRE1 (A) and VDAC1 (B) by GST-pulldown assay, respectively. Glutathione beads conjugated to GST or the GST-p7 protein were incubated with recombinant Flag-MAPRE1 or Flag-VDAC1 protein. After washing, proteins were eluted from the beads and SDS-PAGE was performed. The expression of MAPRE1 or VDAC1 was determined by immunoblotting with anti-Flag mAb. GST and GST-p7 protein expression were confirmed by immunoblotting.

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  FIGURE 4. The network of interactions between CSFV p7 and host cellular proteins. The p7-cellular protein interaction network was constructed based on the interactions of proteins including CSFV p7, the identified proteins by Y2H screening, and other host proteins interacting with the identified proteins. In brief, the seven identified proteins screened by Y2H assay were inputted into the STRING database, while the specie was set to “Sus scrofa” and other parameters were the default settings. The total list of proteins directly and indirectly related to the seven identified proteins based on protein-protein interactions was obtained from the STRING database. Protein interactions from the STRING database with a combined score >0.7 were selected for network construction. Based on the experimental results screened by the Y2H system and the correlation between the proteins in the STRING database, the p7-cellular protein interaction network was built using the Cytoscape v3.7.2 software. In the network, the core protein p7 was marked as a hexagon. The orange squares indicated proteins identified by Y2H assay to interact with p7. The other proteins, which were identified as potentially…

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  FIGURE 5. GO and KEGG enrichment of host cellular proteins in the p7-cellular protein interaction network. (A) Biological process of GO enrichment; (B) Molecular function of GO enrichment; (C) Cellular components of GO enrichment; (D) KEGG pathway enrichment. The abscissa represents the “RichFactor.” RichFactor = “count”/“pop hits.” The “count” is the number of hub genes enriched in a certain term. The “pop hits” is the number of all genes enriched in a certain term. The size of the bubbles indicates the “gene count.” The color of the bubbles indicates the “-log10(p-value).” A p-value < 0.05 is regarded as statistically significant for the correlations.

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  FIGURE 6. Predicted pathways might be involved in the interaction between CSFV p7 and host cells. 1 JAK-STAT pathway. Our previous study revealed that the JAK-STAT pathway might play a significant role in CSFV infection (Chen et al., 2020). In the present study, PIAS1 (protein inhibitor of activated STAT 1) was found to be a potential binding partner of the CSFV p7, which suggested that p7 might regulate the JAK-STAT pathway by interacting with PIAS1. 2 The ubiquitin-proteasome system (UPS). It was reported that p7 was a short-lived protein as it could be easily degraded by the proteasome (Lin et al., 2014). PIAS1 was involved in ubiquitin-mediated proteolysis based on the results of the KEGG pathway analysis. POMP (proteasome maturation protein) was related to the proteasome assembly. COPS2 (COP9 signalosome subunit 2) might be related to the UPS as the COP9 signalosome could regulate the UPS (Rao et al., 2020). Thus, PIAS1, POMP, and COPS2 might be involved in the UPS and CSFV p7 might have a close relationship with the UPS. 3 The calcium signaling pathway. The calcium signaling pathway was also enriched based on the KEGG pathway analysis and VDAC1 (voltage-dependent anion channel 1) was…