Foot-and-mouth disease virus VP1 promotes viral replication by regulating the expression of chemokines and GBP1

Figures

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  Figure 1. Construction and expression of pCAGGS-HA-VP1 Plasmid. (A) Model of a cloned fragment. (B) Target fragment VP1 amplified by PCR (lane 2), respectively. (C) The product of recombinant plasmid pCAGGS-HA-VP1 verified by colony PCR (lanes 2 and 3 on the left panel) and digested with EcoR1 and Xho1 (lanes 1 and 2 on the right panel) was observed with agarose gel electrophoresis. (D) The pCAGGS-HA vectors (control) or pCAGGS-HA-VP1 plasmids were transfected in PK-15 cells for 36 h, respectively, and detected by western blot analysis.

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  Figure 2. Quality control of samples and quantitative analysis of differentially expressed genes (DEGs). (A) Sample correlation heat map. The shades of color and numbers represent Pearson's correlation coefficient (R) values, indicating sample repeatability in the same group. (B) Volcano plots of the distribution of DEGs. The x-axis represents the fold change (log2Fold Change) of gene expression between the VP1 and control group, and the y-axis indicates the significance level (-log10padj) of gene expression difference. The dot diameter represents expression levels of DEGs which are either up-regulated (red) or down-regulated (green). And blue dots represent non-significantly regulated genes. (C) Differential gene cluster map. The x-axis in the figure is the sample name, and the y-axis is the value after the normalization of differential gene FPKM. The color scale represents gene expression abundance, with red representing more pronounced up-regulation and green representing more pronounced down-regulation.

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  Figure 3. GO and KEGG annotation of differentially expressed genes. (A) GO functional classification of the up-regulated DEGs. The x-axis indicates the number of DEGs and the y-axis represents GO terms. From the GO enrichment analysis results, significant term plotted histograms were selected for presentation. (B) GO functional classification of the down-regulated DEGs. The x-axis indicates the number of DEGs and the y-axis represents GO terms. (C) Analysis of KEGG enrichment. The x-axis is the ratio of the number of differential genes to the total number of differential genes, the y-axis is the KEGG pathway, The dot diameter represents the number of genes annotated to the KEGG pathway, and the color represents the significant size of the enrichment.

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  Figure 4. Comparison of fold changes of DEGs between RNA-Seq and qRT-PCR. PK-15 cells were transfected with pCAGGS-HA and pCAGGS-HA-VP1 for 36 h, then qRT-PCR was performed to detect the relative expression of selected DEGs. The horizontal axe represents the name and the vertical axe indicates log2-fold changes of DEGs.

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  Figure 5. The relationship between chemokines and FMDV replication. (A) FMDV induces expression of chemokines in PK-15 cells. The PK-15 cells were collected at 4, 8, and 12 h after infection with FMDV at an MOI = 0.1 and then subjected to qRT-PCR assays. The mRNA levels of CCL5, CXCL8, and CXCL10 were tested. (B) The overexpression of chemokines in PK-15 cells. The pCAGGS-HA-CCL5/CXCL8/CXCL10 plasmids were transfected in PK-15 cells, respectively, and the expression levels of these chemokines were detected by qRT-PCR at 24 h after transfection. (C) Chemokines promotes FMDV replication. The pCAGGS-HA-CCL5/CXCL8/CXCL10 plasmids were transfected in PK-15 cells, respectively. At 24 h post-transfection, the cells were collected at 4, 8, and 12 h after infection with FMDV at an MOI = 0.1 and then subjected to qRT-PCR assays. The relative 3D gene mRNA levels were tested. The significant difference is marked by “*” (P < 0.05), “**” (P < 0.01), and “***” (P < 0.001). (D) PK-15 cells were transfected with pCAGGS-HA- CCL5/CXCL8/CXCL10 plasmid or co-transfected with these three plasmids for 24 h, followed by infection with FMDV for 12 h. The viral 3D protein was detected by Western blot.

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  Figure 6. The relationship between GBP1 and viral replication. (A) FMDV infection downregulates GBP1 mRNA expression. After infection of PK-15 cells with FMDV for 4, 8, and 12 h, the mRNA levels of GBP1 and 3D in the samples were detected by qRT-PCR. (B,C) GBP1 inhibits FMDV replication. PK-15 cells were transfected with pCAGGS-HA-GBP1 plasmid for 24 h, followed by infection with FMDV for 4, 8, or 12 h. The viral RNA was determined by qPCR assay (C, upper panel), the GBP1 (B) and viral 3D protein (C, lower panel) was detected by Western blot. (D,E) Influence of GBP1 knockdown on FMDV replication. PK-15 cells were transfected with siGBP1 (siGBP1-597) in indicated concentration or siNC for 36 h, followed by infection with FMDV for 12 h. Knockdown of GBP1 mRNA was determined by qRT-PCR (D), the viral 3D protein was detected by Western blot (E).