Proteome Analysis Reveals Syndecan 1 Regulates Porcine Sapelovirus Replication

Figures

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  Figure 1. Sapelovirus A (PSV) infection in pig kidney (PK)-15 cells. (A) PK-15 cells were mock-treated or treated with PSV at a multiplicity of infection (MOI) of 2, and the cytopathogenic effects at indicated time were observed. Bars, 50 μm. (B) One-step growth curve of PSV in PK-15 cells. (C) Western blotting assay of PSV virus protein (VP)1 expression in PK-15 cells at the indicated time points (2–12 h). The α-tubulin was used as a sample loading control. Data are expressed as mean ± SD for two independently experiments.

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  Figure 2. Volcano plot and the hierarchical clustering involved in the differentially expressed proteins (DEPs). (A) Volcano plot showed the levels of DEPs detected in mock-treated or PSV-infected groups. X axis, mean log2(ratio of fold change); y axis, log10(p-value); red balls, DEPs between the two groups (p < 0.05 and ratio > 2 or <0.5). (B) Hierarchical cluster of 181 DEPs with 1.2-fold up-regulation and 0.85-fold down-regulation. The proteins were classified into two categories with opposite directional variation, suggesting the screening rationality of DEPs. Different color indicated different fold change. Red: Highly expressed, blue: Lowly expressed, and white: No change.

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  Figure 3. Top 20 enriched, differentially expressed proteins processed of gene ontology (GO) terms and enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in PSV-infected PK-15 cells. (A) GO annotation of the DEPs. BP: Biological process; MF: Molecular function; CC: Cellular component. (B) Enriched KEGG pathways. The number on the top of each column represents enrichment factor. The color of the bar chart indicates the p value based on Fisher’s exact test.

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  Figure 4. Subcellular locations of the DEPs in PK-15 cells infected with PSV.

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  Figure 5. Protein-protein interaction networks were constructed based on the DEPs. The yellow nodes indicate the DEPs, and the lines are protein-protein interactions.

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  Figure 6. DEPs including induced protein with tetratricopeptide repeats 5 (A, IFIT5), vinculin (B, VCL), nucleoporin (C, NUP153), and syndecan-1 (D, SDC1) were verified using PRM and the virus/control (V/C) ratio was compared with the TMT result. Data are expressed as mean ± SD for three replicates.

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  Figure 7. The expression of the indicated genes and kinetics in PK-15 cells infected with PSV for 4 h and 8 h was measured by RT-qPCR. Data are shown as mean ± SD for two experiments; * p < 0.05, ** p < 0.01.

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  Figure 8. The effect of syndecan-1 (SDC1) on PSV replication. (A) After 24 h transfection with pcDNA-HA empty or HA-SDC1, PK-15 cells were infected with PSV (MOI = 2), cells were lysed, and virus VP1 proteins were analyzed by Western blot. (B) The effect of siRNA against SDC1 was determined by RT-qPCR. (C) PK-15 cells were untreated or transfected with siNC, 20 pmol, 30 pmol, or 40 pmol siSDC1. At indicated time after transfection, cells were infected with PSV and processed for Western blot. (D) Virus titers were determined by 50% tissue culture infective doses (TCID50) of samples prepared at 8 h post-infection from PK-15 cells infected with PSV after transfection with pcDNA-HA empty, HA-SDC1, siNC, or 30 pmol siSDC1. Two independent experiments were performed. The ** p < 0.01 compared with siNC or pcDNA-HA group. The # p < 0.05, ## p < 0.01 compared with 20 pmol group.