A Comparative Transcriptomic Analysis Reveals That HSP90AB1 Is Involved in the Immune and Inflammatory Responses to Porcine Deltacoronavirus Infection

Abstract

PDCoV is an emerging enteropathogenic coronavirus that mainly causes acute diarrhea in piglets, seriously affecting pig breeding industries worldwide. To date, the molecular mechanisms of PDCoV-induced immune and inflammatory responses or host responses in LLC-PK cells in vitro are not well understood. HSP90 plays important roles in various viral infections. In this study, HSP90AB1 knockout cells (HSP90AB1^KO) were constructed and a comparative transcriptomic analysis between PDCoV-infected HSP90AB1^WT and HSP90AB1^KO cells was conducted using RNA sequencing to explore the effect of HSP90AB1 on PDCoV infection. A total of 1295 and 3746 differentially expressed genes (DEGs) were identified in PDCoV-infected HSP90AB1^WT and HSP90AB1^KO cells, respectively. Moreover, most of the significantly enriched pathways were related to immune and inflammatory response-associated pathways upon PDCoV infection. The DEGs enriched in NF-κB pathways were specifically detected in HSP90AB1^WT cells, and NF-κB inhibitors JSH-23, SC75741 and QNZ treatment reduced PDCoV infection. Further research revealed most cytokines associated with immune and inflammatory responses were upregulated during PDCoV infection. Knockout of HSP90AB1 altered the upregulated levels of some cytokines. Taken together, our findings provide new insights into the host response to PDCoV infection from the transcriptome perspective, which will contribute to illustrating the molecular basis of the interaction between PDCoV and HSP90AB1.

Keywords: HSP90AB1; PDCoV; immune and inflammatory response; transcriptomic analysis.

Figure 1

HSP90AB1 knockout reduces PDCoV infection. (A) The mRNA level of HSP90AB1 in HSP90AB1^WT and HSP90AB1^KO cells was detected using qRT-PCR. (B) CCK-8 assay of the viability of HSP90AB1^WT and HSP90AB1^KO cells. (C) The HSP90AB1 protein expression level in HSP90AB1^WT and HSP90AB1^KO cells was analyzed using western blotting. ACTB was used as the sample loading control. (D) Detection of the mRNA level of the PDCoV M gene using qRT-PCR. (E) The virus titer in the culture supernatants was determined using TCID₅₀ assay. (F) Detection of the PDCoV N protein level using western blotting. ACTB was used as the sample loading control. (G) The band density of the protein was quantified using ImageJ software, and the PDCoV-N to ACTB ratios were normalized to the control. ** means p ≤ 0.01, *** means p ≤ 0.001.

Figure 2

HSP90AB1 knockout reduces HSP70 expression level. (A) The mRNA level of HSP70 in HSP90AB1^WT and HSP90AB1^KO cells was detected using qRT-PCR. (B) Detection of the HSP70 protein level using western blotting. ACTB was used as the sample loading control. (C) The band density of the protein was quantified using ImageJ software, and the HSP70 to ACTB ratios were normalized to the control. * means p ≤ 0.05, ** means p ≤ 0.01.

Figure 3

Differentially expressed genes (DEGs) in HSP90AB1^WT and HSP90AB1^KO cells were analyzed following mock and PDCoV infection. (A) Violin plot of gene expression patterns for each sample, with the origin representing the median. (B) Principal component analysis (PCA) of eight samples of mock- or PDCoV-infected HSP90AB1^WT or HSP90AB1^KO cells.

Figure 4

DEGs in HSP90AB1^WT and HSP90AB1^KO cells were analyzed following mock and PDCoV infection. (A,B) The numbers of upregulated and downregulated DEGs in HSP90AB1^WT (A) and HSP90AB1^KO (B) cells were evaluated. (C,D) Volcano plot showing the DEGs in HSP90AB1^WT (C) and HSP90AB1^KO (D) cells. The upregulated DEGs are represented by red dots, downregulated DEGs are represented by blue dots, and genes with no significant differences in expression are represented by gray dots.

Figure 5

Bubble map of the top 20 most enriched GO terms. (A,B) The top 20 significantly enriched GO terms in HSP90AB1^WT (A) and HSP90AB1^KO (B) cells in the BP category. (C,D) The top 20 significantly enriched GO terms in HSP90AB1^WT (C) and HSP90AB1^KO (D) cells in the CC category. (E,F) The top 20 significantly enriched GO terms in HSP90AB1^WT (E) and HSP90AB1^KO (F) cells in the MF category. Enriched terms were selected with a corrected p-value ≤ 0.05. The dot size indicates the number of enriched DEGs, and the dot color indicates the Q-value.

Figure 6

Histograms of the top 20 most enriched signaling pathways. (A) The top 20 significantly enriched pathways were identified in HSP90AB1^WT cells. (B) The top 20 significantly enriched pathways identified in HSP90AB1^KO cells. Enriched pathways were selected with a corrected p-value ≤ 0.05. The length indicates the percentage of genes, and the color indicates the Q-value.

Figure 7

Immune and inflammatory responses in PDCoV-infected HSP90AB1^WT and HSP90AB1^KO cells. (A,B) KEGG analysis of immune- and inflammation-related signaling pathways in HSP90AB1^WT (A) and HSP90AB1^KO (B) cells following PDCoV infection. (C) The interferon-related genes. (D) The chemokine and chemokine receptor related genes. (E) The interleukin-related genes. (F) Tumor necrosis factor-related genes.

Figure 8

Confirmation of the transcriptome sequencing data by qRT-PCR. (A–N) DEGs related to immune and inflammatory responses were selected randomly for qRT-PCR analysis, and the expression levels of those DEGs were estimated using the 2^{− ∆∆CT} method. (O) The correlation of the DEG expression levels between RNA sequencing and qRT-PCR was analyzed using Pearson’s correlation analysis. * means p ≤ 0.05, ** means p ≤ 0.01, **** means p ≤ 0.0001.

Figure 9

HSP90AB1 knockout alters NF-κB activity. (A) The expression patterns of DEGs involved in regulating NF-κB activity were analyzed using RNA sequencing. (B) qRT-PCR analysis of the expression of NFKB1, NFKBIZ, NFKBIA, NFKBIB and NKAP in HSP90AB1^WT and HSP90AB1^KO cells. (C) The correlation of the DEGs between RNA sequencing and qRT-PCR data was analyzed by calculating Pearson’s correlation coefficient. (D) The mRNA level of NFKB1, NFKBIZ, NFKBIA, NFKBIB and NKAP in PDCoV uninfected HSP90AB1^WT and HSP90AB1^KO cells was analyzed by qRT-PCR. * means p ≤ 0.05.

Figure 10

The NF-κB inhibitor reduced PDCoV infection. (A,D,G) qRT-PCR analysis of the mRNA level of the PDCoV M gene after JSH-23 (A), SC75741 (D) and QNZ (G) treatment. (B,E,H) The viral titer in the culture supernatants was determined using TCID₅₀ assay. (C,F,I) Western blotting detection of the PDCoV N protein level after JSH-23 (C), SC75741 (F) and QNZ (I) treatment. ACTB was used as the sample loading control. * means p ≤ 0.05, ** means p ≤ 0.01, *** means p ≤ 0.001.