Transcriptome Sequencing Reveals That Intact Expression of the Chicken Endogenous Retrovirus chERV3 In Vitro Can Possibly Block the Key Innate Immune Pathway

Abstract

Endogenous retroviruses (ERVs) are viral sequences that have integrated into the genomes of vertebrates. Our preliminary transcriptome sequencing analysis revealed that chERV3 is active and is located on chromosome 1:32602284-32615631. We hypothesized that chERV3 may have a role in the host innate immune response to viral infection. In this study, using reverse genetics, we constructed the puc57-chERV3 full-length reverse cloning plasmid in vitro. We measured the p27 content in culture supernatant by enzyme-linked immunosorbent assay (ELISA). Finally, transcriptome analysis was performed to analyze the function of chERV3 in innate immunity. The results showed that chERV3 may generate p27 viral particles. We found that compared to the negative control (NC) group (transfected with pMD18T-EGFP), the chERV3 group exhibited 2538 up-regulated differentially expressed genes (DEGs) and 1828 down-regulated DEGs at 24 hours (h) and 1752 up-regulated DEGs and 1282 down-regulated DEGs at 48 h. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, the down-regulated DEGs were enriched mainly in immune-related processes such as the inflammatory response, innate immune response, and Toll-like receptor signaling pathway. GSEA showed that the Toll-like receptor signaling pathway was suppressed by chERV3 at both time points. We hypothesized that chERV3 can influence the activation of the innate immune pathway by blocking the Toll-like receptor signaling pathway to achieve immune evasion.

Keywords: RNA-seq; Toll-like receptor signaling pathway; chicken; endogenous retroviruses; innate immune.

Figure 1

Construction of the full-length reverse cloning plasmid of puc57-chERV3. Note: (A) digestion analysis of the puc57-chERV3 plasmid by EcoRV (lane 1); (B) full-length sequence of the puc57-chERV3 plasmid.

Figure 2

The chERV3 viral capsid protein (p27) level was measured by ELISA. The Y-axis is the value of SP, and the X-axis is the time of chERV3 transfection in CEFs.

Figure 3

DEGs in CEFs transfected with chERV3 at 24 and 48 h. Note: (A) statistical chart of all DEGs in CEFs transfected with chERV3 at 24 h; (B) statistical chart of all DEGs in CEFs transfected with chERV3 at 48 h. The Y-axis is the value of −log₁₀ (p value), and the X-axis is the value of log₂ (Fold Change); (C) Venn diagrams of up-regulated and down-regulated DEGs in CEFs transfected with chERV3 at 24 and 48 h.

Figure 4

GO term analysis of down-regulated DEGs in CEFs transfected with chERV3 at 24 and 48 h; (A) up-regulated DEGs at 24 h; (B) down-regulated DEGs at 24 h; (C) up-regulated DEGs at 48 h; and (D) down-regulated DEGs at 48 h.

Figure 5

Validation of RNA-seq data by qPCR. (A) chERV3 transfected in CEFs at 24 h, (B) chERV3 transfected in CEFs at 48 h. Blue represents NC, and purple represents chERV3 (* p < 0.05; ** p < 0.01; **** p < 0.0001; ns: Non-significant).

Figure 6

KEGG pathways of up-regulated and down-regulated DEGs expressed in CEFs transfected with chERV3 at 24 and 48 h; (A) up-regulated DEGs at 24 h; (B) down-regulated DEGS at 24 h; (C) up-regulated DEGS at 48 h; and (D) down-regulated DEGs at 48 h.

Figure 7

Expression validation of the Toll-like receptor signaling pathway. (A,B) Gene set enrichment analysis of the Toll-like receptor signaling pathway in CEFs transfected with chERV3 at 24 h and 48 h.

Figure 8

Identification of the expression of key genes of the TLR signaling pathway. Note: (A,B) Toll-like receptor signaling pathway (24 and 48 h), we have circled seven immune-related genes in red; (C,D) qPCR verification of DEGs at 24 and 48 h. Blue represents NC and purple represents chERV3 (* p < 0.05; ** p < 0.01; *** p < 0.001).