Function And Molecular Mechanism Of Duck Rig-I Gene In Immune Regulation

In last few years, frequent outbreaks of highly pathogenic avian influenza occurred, and the poultry industry suffered from huge losses. Furthermore, it created significant public health and safety problems. Chicken and duck had different susceptibility against the influenza virus that was associated with expression and regulation of the genes for disease-resistant and immunity. Retinoic acid inducible gene Ⅰ (RIG-I) initiate innate immunity in ducks, but this gene was absent in chickens and in this case made chicken much more susceptible to avian influenza. It was not clear how duRIG-I produce antiviral immune response and its molecular mechanism.This study aimed to interpret the expression patterns of duRIG-I from various aspects and reveal the cell signal transmission pathway in antiviral innate immunity and the compensatory mechanisms of duRIG-I. Transcriptomic analysis was carried out using the stably transfected cells. The results would further refine the fundamental studies of chicken signal transmission pathway in antiviral innate immunity and immune system of poultry. This research was carried out as follows.1. To study the duRIG-I gene function, the duRIG-Igene was cloned and the sequence was deposited in GenBank as two alternative transcript variants (GenBank accession nos. JQ946323.2 and KC869660.1). The duRIG-I fusion protein was distributed throughout the cell, mostly in the cytoplasm and nucleus by indirect immunofluorescence. Semi-quantitative RT-PCR was used to detect duRIG-I mRNA levels in various tissues, including heart, liver, kidney, glandular stomach, large intestine, small intestine, muscle, thymus, and hypothalamus. The results showed that mRNA levels of duRIG-I in most tissues were very low and only a little amount in spleen and lung. Further, synthetic double-stranded RNA (poly[I:C]) was used to mimic viral infection in vivo and the mRNA expression levels of RIG-I in spleen and liver during 96 h were detected by RT-PCR.The expression levels increased significantly at 8 h (P<0.05) in both spleen and liver.2. Based on its expression profile, the study on the regulatory mechanism was carried out. We cloned the duRIG-Ipromoter region and predicted its DNA methylation level. However, level of duRIG-I detected by bisulfite sequencing PCR remained unmethylated in spleen. The transcriptional activity was detected by Luciferase reporter assay. There was a positive regulation in the region+14～-301 bp. Combined with the prediction of the promoter site and transcription factor binding sites of this region showed that cis-regulatory elements IRF1 may play an important role in regulation of the duRIG-I expression.3. To confirm that the activation mechanism of the duRIG-I activation, we predicted conserved domains of duRIG-I using online software and found that duRIG-I had the characterizations of the RLR family (including CARDs, RNA helicases, and so on). Based on the prediction, the eukaryotic expression vectors of deletion mutants were constructed and the expression was detected via RT-PCR, indirect immunofluorescent assay and Western blot, respectively. The results of RT-qPCR indicated that CARDs significantly up-regulated the mRNA level of IFN-β, Mx1 and PKR genes. Further, NF-κB luciferase reporter assay and IFN-P enzyme-linked immunosorbent assay detected the duRIG-I significantly activated NF-κB and induced the expression of IFN-P when poly [I:C] challenge DF-1 cells and CARD domain could induce the chicken IFN-β by activating NF-κB.4. To do further research on the function analysis of duRIG-Ⅰ in antiviral innate immunity, stable overexpression of duRIG-I in DF-1 cells was carried out by lentiviral transfection technique. The stably transfected cells, DF-1/LV5-RIG-I and DF-1/LV5, mRNA and protein expression level were detected using RT-PCR and Western Blot. The obtained cells can be used as good tools for further research.5. To reveal the cell signal transmission pathway in antiviral innate immunity and the compensatory mechanisms of duRIG-I.5’ppp-dsRNA was used to mimic viral infection in vivo and RNA-seq was carried out using those cells samples. There were 278 differently expressed genes as RIG-I responsive genes.120 genes of these genes were annotated in KEGG databases. KEGG pathway enrichment analysis showed that RIG-I-like receptor signaling pathway was the most reliable. Furthermore, GO enrichment analysis was carried out to find out type Ⅰ interferon signaling pathway, positive regulation of T cell mediated cytotoxicity, cellular response to interferon-beta and positive regulation of RIG-I signaling pathway, and so on. Based on the common genes in those pathways, the network between RIG-I-like receptor signaling pathway and other signaling pathways was constructed using Cytoscape software. It was speculated that there was a crosstalk between RIG-I-like receptor signaling pathway and those signaling pathways in antiviral signal transmission, including Jak-STAT signaling pathway, Toll-like receptor signaling pathway, Cytokine-cytokine receptor interaction, Wnt signaling pathway, Ubiquitin mediated proteolysis and MAPK signaling pathway. There were key responsive genes in RIG-I mediated signal pathway, such as ISG12-2, OSA*A, Mxl, IFIT5, TRIM25, USP18, STAT1, STAT2, IRF1, IRF3 and IRF8. The results of real-time PCR corresponded with that of RNA sequencing. Based on the results of the corresponding experiments and the report, phantom drawing of RIG-I mediated signaling pathway transduction in chicken was proposed.