Interaction network of African swine fever virus structural protein p30 with host proteins

Figures

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  Figure 1. Identification of the interaction of ASFV p30 with host cellular proteins by DUALmembrane yeast two-hybrid system. (A) Co-transform bait and prey plasmid into NMY51, coat TDO/X/3′AT 5 mM plate, pick blue colonies, and coat QDO/X/3′AT 5 mM plate to further screen positive clones. (B) Agarose gel electrophoresis of PCR products of positive clone colonies. 1–53: PCR product; +: positive control (PCR using PPR3-N as template), −: negative control (PCR using water as template). (C) pBT3-N-CP204L (bait plasmid) was verified point-to-point with pPR3-N-Dab2 and pPR3-N-OAS1 (prey plasmid). 1: pBT3-N-CP204L and pOst1-NubI (functional verification); 2: pBT3-N-CP204L and pPR3-N (self-activation); 3: pBT3-N-CP204L and pPR3-N-Dab2 (experimental group); 4: pBT3-N-CP204L and pPR3-N-OAS1 (experimental group); +: pTSU2-APP and pNubG-Fe65 (positive control group); −: pTSU2-APP and PPR3-N (negative control group).

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  Figure 2. Colocalization of ASFV p30 with ARPC5, CAPG, DAB2, OAS1, PARP9, RPSA, and VBP1. (A) PK-15 cells were co-transfected with plasmids expressing HA-Dab2, HA-RPSA, HA-OAS1, HA-VBP1, HA-PARP9, HA-CAPG, or HA-ARPC5, and plasmids expressing EGFP-p30. Co-transfection of pEGFP-C1 and pCAGGS-HA as negative control. After 24 h, the cells were fixed with 4% PFA, stained with mouse anti-HA, and then examined by confocal microscopy. Scale bar = 10 μm. (B) PAM or MA104 cells were inoculated with ASFV (MOI = 0.5). After 24 h, the cells were fixed with 4% PFA, stained with mouse anti-p30 and rabbit anti-OAS1, and then examined by confocal microscopy. Scale bar = 5 μm.

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  Figure 3. Verification of ASFV p30-cell protein interaction using Co-IP. HEK-293T cells were co-transfected with plasmids expressing HA-Dab2, HA-RPSA, HA-OAS1, HA-VBP1, HA-PARP9, HA-CAPG, or HA-ARPC5, and plasmids expressing Flag-p30. Cell lysates were immunoprecipitated with IgG and anti-Flag antibodies, followed by immunoblotting with anti-HA antibodies. The first lane indicates the input group, the second lane is the IgG control group, and the third lane is the experimental group.

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  Figure 4. Verification of ASFV p30-cell protein interaction using GST-Pulldown assay. ASFV p30 bound to the recombinant Dab2, RPSA, OAS1, VBP1, PARP9, CAPG, or ARPC5. The GST-p30 recombinant proteins were expressed in prokaryotic cells, purified with GST beads, then incubated with lysate of HEK-293 T cells expressing HA-Dab2, HA-RPSA HA-OAS1, HA-VBP1, HA-PARP9, HA-CAPG, or HA-ARPC5. After washing with cold-PBS, the eluted complexes were determined by immunoblotting and detected with specific antibodies.

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  Figure 5. Network of interactions between ASFV p30 and host cellular proteins. Using the STRING database, a protein–protein interaction network was constructed and analyzed. Protein interactions with a total score of >0.7 from the STRING database were chosen for network development. The network analyzer tool Cytoscape v.3.8.1 was used to create and plot the map of ASFV p30-interacting proteins that interacted with the other proteins in our dataset. Straight lines between the nodes represent the interactions between proteins. Matching symbols are used to indicate distinct protein classes. NCBI gene names are used to represent proteins.

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  Figure 6. GO and KEGG pathway0 enrichment analysis. (A) The biological process of GO enrichment; (B) molecular function of GO enrichment; (C) cellular components of GO enrichment; (D) KEGG pathway enrichment. The enriched databases targeted by ASFV p30-interacting proteins. The terms that were significantly enriched (p < 0.05) were shown.

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  Figure 7. Predicted schematic model depicting the interaction between ASFV p30 and host cells. 1. After ASFV attaches to cells, it may bind to Dab2 and RPSA on the cell membrane through the structural protein p30, and then Dab2 recruits clathrin to mediate ASFV endocytosis. 2. p30 binds ARPC5 and CAPG, causing cellular actin rearrangement to trigger macropinocytosis. 3. After viral endocytosis, p30 protein may inhibit the entry of TLR4 into endosomes by recruiting Dab2 and clathrin, and the interaction between PARP9 and p30 may also inhibit the binding of PARP9 to PI3K and AKT3 activation. They ultimately inhibit IRF3 phosphorylation and reduce type I interferon production. 4. p30 may promote viral replication by interacting with RPSA to eliminate the inhibitory effect of RPSA on the MAPK pathway; p30 may prevent RNase L-dependent or independent antiviral responses by interacting with OAS1. 5. p30 can also interact with VBP1, which may enhance the activation of NF-κB and regulate the occurrence of apoptosis.