Chen Yang, Xu Qi, Li Yang, Liu Ran, Huang Zhengyang, Wang Bin, Chen Guohong
The Key Laboratory of Animal Genetics and Breeding and Molecular Design of Jiangsu Province, Yangzhou University, Yangzhou 225009, PR China.
The Key Laboratory of Animal Genetics and Breeding and Molecular Design of Jiangsu Province, Yangzhou University, Yangzhou 225009, PR China.
Dev Comp Immunol. 2016 Dec;65:191-200. doi: 10.1016/j.dci.2016.07.009. Epub 2016 Jul 20.
Retinoic acid inducible gene I (RIG-I) can recognize influenza viruses and evoke the innate immune response. RIG-I is absent in the chicken genome, but is conserved in the genome of ducks. Lack of RIG-I renders chickens more susceptible to avian influenza infection, and the clinical symptoms are more prominent than in other poultry. It is unknown whether introduction of duck RIG-I into chicken cells can establish the immunity as is seen in ducks and the role of RIG-I in established immunity is unknown. In this study, a chicken cell strain with stable expression of duRIG-I was established by lentiviral infection, giving DF1/LV5-RIG-I, and a control strain DF1/LV5 was established in parallel. To verify stable, high level expression of duRIG-I in DF1 cells, the levels of duRIG-I mRNA and protein were determined by real-time RT-PCR and Western blot, respectively. Further, 5'triphosphate double stranded RNA (5'ppp-dsRNA) was used to mimic an RNA virus infection and the infected DF1/LV5-RIG-I and DF1/LV5 cells were subjected to high-throughput RNA-sequencing, which yielded 193.46 M reads and 39.07 G bases. A total of 278 differentially expressed genes (DEGs), i.e., duRIG-I-mediated responsive genes, were identified by RNA-seq. Among the 278 genes, 120 DEGs are annotated in the KEGG database, and the most reliable KEGG pathways are likely to be the signaling pathways of RIG-I like receptors. Functional analysis by Gene ontology (GO) indicates that the functions of these DEGs are primarily related to Type I interferon (IFN) signaling, IFN-β-mediated cellular responses and up-regulation of the RIG-I signaling pathway. Based on the shared genes among different pathways, a network representing crosstalk between RIG-I and other signaling pathways was constructed using Cytoscape software. The network suggests that RIG-mediated pathway may crosstalk with the Jak-STAT signaling pathway, Toll-like receptor signaling pathway, Wnt signaling pathway, ubiquitin-mediated proteolysis and MAPK signaling pathway during the transduction of antiviral signals. After screening, a group of key responsive genes in RIG-I-mediated signaling pathways, such as ISG12-2, Mx1, IFIT5, TRIM25, USP18, STAT1, STAT2, IRF1, IRF7 and IRF8, were tested for differential expression by real-time RT-PCR. In summary, by combining our results and the current literature, we propose a RIG-I-mediated signaling network in chickens.
维甲酸诱导基因I（RIG-I）能够识别流感病毒并引发先天性免疫反应。鸡基因组中不存在RIG-I，但在鸭基因组中保守存在。缺乏RIG-I使鸡对禽流感感染更易感，且临床症状比其他家禽更明显。将鸭RIG-I导入鸡细胞是否能建立如鸭所见的免疫力以及RIG-I在已建立的免疫中的作用尚不清楚。在本研究中，通过慢病毒感染建立了稳定表达鸭RIG-I的鸡细胞系，命名为DF1/LV5-RIG-I，并同时建立了对照细胞系DF1/LV5。为验证鸭RIG-I在DF1细胞中稳定、高水平表达，分别通过实时RT-PCR和蛋白质印迹法测定鸭RIG-I的mRNA和蛋白质水平。此外，使用5'三磷酸双链RNA（5'ppp-dsRNA）模拟RNA病毒感染，并对感染的DF1/LV5-RIG-I和DF1/LV5细胞进行高通量RNA测序，共产生193.46 M条读数和39.07 G碱基。通过RNA测序鉴定出总共278个差异表达基因（DEGs），即鸭RIG-I介导的反应性基因。在这278个基因中，120个DEGs在KEGG数据库中有注释，最可靠的KEGG途径可能是RIG-I样受体的信号通路。通过基因本体论（GO）进行的功能分析表明，这些DEGs的功能主要与I型干扰素（IFN）信号传导、IFN-β介导的细胞反应以及RIG-I信号通路的上调有关。基于不同途径之间的共享基因，使用Cytoscape软件构建了一个代表RIG-I与其他信号通路之间相互作用的网络。该网络表明，在抗病毒信号转导过程中，RIG介导的途径可能与Jak-STAT信号通路、Toll样受体信号通路、Wnt信号通路、泛素介导的蛋白水解和MAPK信号通路相互作用。筛选后，通过实时RT-PCR检测了RIG-I介导的信号通路中的一组关键反应性基因，如ISG12-2、Mx1、IFIT5、TRIM25、USP18、STAT1、STAT2、IRF1、IRF7和IRF8的差异表达。总之，结合我们的结果和当前文献，我们提出了鸡中RIG-I介导的信号网络。
