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We used RNA-Seq on the Illumina platform to analyze transcriptome profiles of BHK-21 cells acutely and persistently infected with FMDV. In total, we obtained more than 342.8 million raw sequencing reads. Approximately 336.5 million clean reads (98.14%) were acquired by filtering out the adapters and trimming ambiguous reads. The transcriptome profiles of acutely or persistently infected BHK-21 cells were each compared with that of mockinfected BHK-21 cells. The numbers and distribution of DEGs varied noticeably for acutely and persistently infected BHK-21 cells compared with mock-infected BHK-21 cells. A histogram was plotted to compare numbers of DEGs (Fig. 1A). The numbers of up-regulated and down-regulated genes were similar in both cell lines. However, the amount of DEGs relative to mock-infected BHK-21 cells was greater in persistently infected than in acutely infected cells. DEGs were classified according to log2-fold changes (Fig. 1B). The distribution of log2-fold changes for DEGs in acutely infected BHK-21 cells covered a relatively narrower range (from - 1 to 1) than that of DEGs in persistently infected BHK-21 cells (from - 12 to 10).
Figure 1. Analysis of differentially expressed genes (DEGs) in serotypeO FMDV acutely (BHKOa) and persistently (BHKOp) infected BHK- 21 cells. A Histogram of DEG numbers in acutely and persistently infected BHK-21 cells. Up-regulated and down-regulated genes are compared in acutely and persistently infected BHK-21 cells. B Distribution of DEGs according to log2-fold change. The horizontal axis represents fold change of DEGs and vertical axis represents percentage of DEGs.
DEGs were compared indirectly between acutely and persistently infected cells using mock-infected BHK-21 cells as a reference. Volcano plots of magnitude (fold change) and significance (q value) demonstrate the presence of DEGs (Fig. 2A). The red and green spots in the volcano plot represent DEGs that are up-regulated and down-regulated, respectively. Compared with persistently infected BHK-21 cells, 8378 DEGs were identified in acutely infected BHK-21 cells, of which 4298 were upregulated and 4080 were down-regulated (Fig. 2A, Supplementary Table S2).
Figure 2. Analysis of GO and KEGG enrichment in acutely infected BHK-21 cells (BHKOa) compared with persistently infected BHK-21 cells (BHKOp). A Volcano plot showing distribution and quantity of DEGs in acutely infected BHK-21 cells compared with persistently infected BHK-21 cells. The horizontal and vertical axes represent log2-fold changes and significance of DEGs, respectively. The red, green, and blue spots represent genes that are up-regulated, downregulated, and not significantly different, respectively. B Analysis of GO enrichment. Thirty GO terms with the most significant enrichment are shown in the histogram. The vertical axis represents the enriched GO term; the horizontal axis represents the number of DEGs. The significantly enriched term is marked "*". C Analysis of KEGG enrichment. Twenty pathways with the most significant enrichment are shown in a scatter plot. The vertical axis represents enriched pathways; the horizontal axis represents enrichment factor, which is the ratio of DEG number to background gene number. The size of the spot is proportional to the number of DEGs. Q value is plotted in a color map.
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GO enrichment analysis was performed to understand the function of the 8378 DEGs. The significantly enriched GO terms, classified into three categories (biological process, cellular component, and molecular function), are presented in Fig. 2B. In the biological process category, terms related to gene expression, metabolic process, and biosynthetic process (GO:0044237, GO:0008152, GO:0034641, GO:0044260, GO:0044238, GO:1901576, GO:0009058, GO:0071704, GO:0006807, GO:0044249, GO:0034645, GO:0009059, GO:0010467, GO:0044271, GO:0006139, GO:0044710, GO:1901360 and GO:0043170) were significantly enriched. In the cellular component category, terms related to nucleus, intracellular, and organelle (GO:0005622, GO:0005623, GO:0044464, GO:0043226, GO:0044424, GO:0043229, GO:0005737, GO:0005634, GO:0043231 and GO:0043227) were significantly enriched. In the molecular function category, significant enrichment was found for binding (GO:0005488) and structural constituent of ribosome (GO:0003735).
KEGG enrichment analysis was performed to identify the metabolic pathways and signaling pathways in which DEGs were involved. A scatter plot was created to display significantly enriched pathway terms. Ribosome (114 DEGs) and endocytosis (138 DEGs) were the most significantly enriched terms in acutely infected BHK-21 cells compared with persistently infected BHK-21 cells (Fig. 2C).
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Given the significant differences identified in ribosomes and biosynthesis related genes via the above GO and KEGG enrichment, DEGs related to ribosomes and translation were further analyzed in these two cell lines. Mockinfected BHK-21 cells served as a negative control. The amount of DEGs related to ribosomes and translation was compared in Table 1. Twelve DEGs related to ribosomes and translation were found in acutely infected BHK-21 cells, including four large subunit ribosomal protein genes (RPLs), two small subunit ribosomal protein genes (RPSs), five eukaryotic translation initiation factor genes (EIFs), and one eukaryotic translation elongation factor genes (EEFs). The number of DEGs related to ribosomes and translation was greater in persistently infected BHK-21 cells (39 DEGs) and included 18 RPLs, 12 RPSs, 6 EIFs, and 3 EEFs. In addition, the number of down-regulated genes was greater than the number of up-regulated genes in both cell lines. The fold changes of the above genes were shown in Fig. 3A. The log2-fold changes in the levels of genes in persistently infected BHK-21 cells were notably lower than those in acutely infected BHK-21 cells, indicating that ribosome- and translation- related genes were expressed at lower levels. To verify the RNA-Seq results, RT-qPCR was performed to quantify mRNA expression levels of nine of the above genes. Variation tendencies in gene expression were identical for both methods (correlation coefficient = 0.94, sig. = 5.55 9 10-9) (Fig. 3B). These data reveal that the expression levels of genes related to ribosomes and translation are lower in persistently infected BHK-21 cells than in acutely infected cells.
Gene categories Number of DEGs in BHKOa Number of DEGs in BHKOp Up-regulated Down-regulated Up-regulated Down-regulated RPL 0 4 0 18 RPS 0 2 0 12 EIF 2 3 2 4 EEF 0 1 2 1 Total 2 10 4 35 RPL large subunit ribosomal protein genes; RPS small subunit ribosomal protein genes; EIF eukaryotic translation initiation factor genes; EEF eukaryotic translation elongation factor genes Table 1. Comparison of the number of DEGs related to ribosome and translation.
Figure 3. Analysis of ribosomeand translation- related DEGs in serotype-O FMDV acutely (BHKOa) and persistently (BHKOp) infected BHK-21 cells. A Fold changes of genes related to ribosomes and translation in acutely and persistently infected BHK-21 cells. A Rader map was plotted to compare the differential expression levels of genes related to ribosomes and translation. The horizontal and vertical axes represent the name and log2-fold changes of genes, respectively. The black and red lines represent acutely and persistently infected BHK-21 cells, respectively. B Comparison of fold changes in DEGs related to the ribosome and translation between RNASeq and RT-qPCR. The horizontal and vertical axes represent the name and log2- fold changes of DEGs, respectively.
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The immune response is critical for protecting cells against viral infection. DEGs related to the innate and adaptive immune responses were analyzed in BHK-21 cells acutely and persistently infected with FMDV compared with mock-infected BHK-21 cells. Table and scatter diagrams were created to show the quantity and magnitude (fold change) of DEGs, respectively. Thirty-four DEGs related to the innate immune response were identified in acutely infected BHK-21 cells, with 18 up-regulated and 16 downregulated (Table 2). The corresponding distribution of log2-fold changes in these DEGs covered a relatively narrow range from - 0.5 to 1 (Fig. 4A, left panel). By contrast, 93 DEGs were identified in persistently infected BHK-21 cells, with 52 up-regulated and 41 down-regulated (Table 2). The corresponding distribution of log2-fold changes in these DEGs covered a much wider range, from - 8 to 5 (Fig. 4A, right panel). Similar observations were made for the adaptive immune response. Twenty-one DEGs were identified in acutely infected BHK-21 cells, with 19 up-regulated and 2 down-regulated (Table 2) and a corresponding distribution of log2-fold changes from - 0.5 to 1 (Fig. 4B, left panel). Persistently infected BHK-21 cells showed 67 DEGs, of which 39 were up-regulated and 28 were down-regulated (Table 2), with a corresponding distribution of log2-fold changes from - 9 to 6 (Fig. 4B, right panel). To verify the reliability of the RNA-Seq results, mRNA expression levels of nine of the above genes were quantified via RT-qPCR. The log2-fold changes of these genes in acutely and persistently infected cells are shown using these two methods (Fig. 4C). Variation tendencies in mRNA expression were identical using the two methods (correlation coefficient = 0.97, sig. = 5.23 × 10-11), supporting the reliability of RNA-Seq. These results indicate that a higher number of immune-related genes are activated during persistent infection than during acute infection.
DEGs categories Number of DEGs in BHKOa Number of DEGs in BHKOp Up-regulated Down-regulated Up-regulated Down-regulated Innate immune response 18 16 52 41 Adaptive immune response 19 2 39 28 Table 2. Comparison of the number of DEGs related to immune response.
Figure 4. Analysis of immunerelated DEGs in serotype-O FMDV acutely (BHKOa) and persistently (BHKOp) infected BHK-21 cells. The scatter plot shows the fold changes of DEGs related to the innate immune response (A) and adaptive immune response (B). C Comparison of fold changes in DEGs related to the immune response between RNA-Seq and RT-qPCR. The horizontal and vertical axes represent the name and log2-fold changes of DEGs, respectively.
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Further analysis of DEGs revealed that a small portion of genes showed differential expression in both acutely and persistently infected BHK-21 cells compared with mockinfected BHK-21 cells. These genes were sorted into four categories: 58 genes were up-regulated in both lines; 59 were down-regulated in both lines; 56 genes were upregulated in acutely infected cells but down-regulated in persistently infected cells; 101 genes were down-regulated in acutely infected cells but up-regulated in persistently infected cells (Fig. 5A). To verify the reliability of the RNA-Seq results, mRNA expression levels of 20 genes were quantified by RT-qPCR. The log2-fold changes of these genes in acutely and persistently infected cells using these two methods were compared (Fig. 5B). Variation tendencies in mRNA expression were identical using the two methods (correlation coefficient = 0.86, sig. = 1.37 × 10-12), verifying the reliability of RNA-Seq.
Figure 5. Analysis and verification of DEGs in both acutely and persistently infected BHK-21 cells. For simplicity, BHK-21 cells acutely and persistently infected with serotype-O FMDV are abbreviated as BHKOa and BHKOp, respectively. A Venn diagram with four sets: BHKOa-up, BHKOa-down, BHKOp-up, and BHKOp-down and their intersections, with "up" and "down" representing upregulation and down-regulation, respectively. The number of DEGs is shown in each section. B Comparison of fold changes of DEGs between RNA-Seq and RT-qPCR. The horizontal and vertical axes represent the name and log2-fold changes of DEGs, respectively.
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We selected the host factor Hspb1, which was up-regulated in acutely infected cells and down-regulated in persistently infected cells, for additional study. The protein expression level of Hspb1 was analyzed by Western blotting in acutely and persistently infected cells. Consistent with mRNA expression analyses, the protein expression level of Hspb1 was down-regulated in persistently infected BHK-21 cells (Fig. 6A). No significant difference was observed in Hspb1 protein expression level between acutely infected and mock-infected cells. To further investigate the role Hspb1 plays during FMDV infection, three shRNA sequences were designed to knock down Hspb1 expression. The mRNA and protein expression levels were examined via RT-qPCR and Western blotting, respectively, after silencing Hspb1 expression. The mRNA and protein expression levels were knocked down by all three shRNAs (Fig. 6B, 6C), and shHspb1 #1 was used in further studies. After transfection with shHspb1#1 for 24 h, BHK-21 cells were infected with FMDV at 10-3 TCID50/cell. The mRNA and protein expression levels of viral non-structural protein 3D were examined 24 h post infection (Fig. 6D–6F). The mRNA expression levels of 3D were significantly reduced by 65% when Hspb1 was knocked down. Similarly, the protein expression levels of 3D were reduced by 30% when Hspb1 was knocked down. To further reveal the role of Hspb1 in FMDV replication, we examined the negativestrand RNA of 3D. The negative-strand RNA of 3D was significantly reduced by 65% upon knockdown of Hspb1 (Fig. 6G). These results suggest that Hspb1 plays a significant role in FMDV replication.
Figure 6. Hspb1 is beneficial for FMDV replication. For simplicity, BHK-21 cells acutely and persistently infected with serotype-O FMDV are abbreviated as BHKOa and BHKOp, respectively. A Protein expression levels of Hspb1 in mock, acutely, and persistently infected BHK-21 cells. B, C BHK-21 cells were transfected with pSUPER-NC or pSUPER-shHspb1 #1, #2, or #3 for 48 h. The mRNA and protein expression levels of Hspb1 were examined by RT-qPCR and Western blot analysis, respectively. D, E BHK-21 cells were transfected with pSUPER-shHspb1 #1 and pSUPER-NC for 24 h and then infected with FMDV at 10-3 TCID50/cell. The mRNA and protein expression levels of 3D and Hspb1 were examined 24 h post infection. F Relative densitometry quantification of 3D protein expression level was quantified by Image Lab software. G BHK-21 cells were transfected with pSUPER-shHspb1 and pSUPER-NC for 24 h and then infected with FMDV at 10-3 TCID50/cell. The relative expression levels of negative stand RNA of 3D were examined 24 h post infection. Relative expression levels were normalized against GAPDH. Three independent replicates were conducted for each sample. Data are expressed as the mean ± SE (n = 3). **P < 0.01.