Wang Jie, Liu Qiuju, Zhang Kehui, Zhao Shurui, Shao Qi, Fu Feiyu, Ma Jingjiao, Wang Zhaofei, Yan Ya-Xian, Wang Hengan, Sun Jianhe, Cheng Yuqiang
Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China.
J Virol. 2025 Mar 18;99(3):e0200824. doi: 10.1128/jvi.02008-24. Epub 2025 Feb 25.
RIG-I and MDA5, known as the RIG-I-like receptors (RLRs), play a pivotal role in inducing antiviral responses to RNA viral infections. While chickens lack RIG-I, they possess a functionally enhanced MDA5 that recognizes pathogens and regulates immunity, underscoring the critical role of MDA5 in maintaining immune homeostasis in chickens. However, the precise mechanisms governing the expression and optimal activation of MDA5 remain unclear. Here, we reveal that the chicken E3 ubiquitin ligase RNF20 is essential for modulating MDA5-mediated innate immune homeostasis. Transcriptome sequencing analysis revealed that RNA viral infection of DF-1 cells significantly upregulated the expression of chicken RNF20. Overexpression of RNF20 markedly suppresses the expression of chicken innate immunity-related genes, while RNF20 knockout leads to immune deficiency both and . Mechanistically, RNF20 is located in the nucleus, where it maintains the basic expression and regulates the inducible expression of MDA5 to establish immune defense during the early infection phase. In the late phase, RNF20 translocates to the cytoplasm, where it facilitates the K27- and K48-linked polyubiquitination and subsequent degradation of MDA5, thereby preventing immune overstimulation. Overall, this study establishes RNF20 as an important E3 ubiquitin ligase that maintains chicken innate immune homeostasis.
Chicken MDA5 is an important RNA viral sensor for initiating the antiviral innate immune response. The protein level of MDA5 must be tightly regulated to maintain antiviral innate immune homeostasis. In this study, we demonstrate that the E3 ubiquitin ligase RNF20 precisely regulates MDA5 protein stabilization through nucleoplasmic translocation. Specifically, in uninfected and during early infection, RNF20 regulates MDA5 transcription in the nucleus. While in the late stages of infection, RNF20 translocates out of the nucleus and catalyzes the ubiquitinated degradation of MDA5. Thus, RNF20 is important in regulating chicken antiviral innate immune homeostasis.
维甲酸诱导基因I（RIG-I）和黑色素瘤分化相关基因5（MDA5），即RIG-I样受体（RLRs），在诱导针对RNA病毒感染的抗病毒反应中起关键作用。鸡虽缺乏RIG-I，但拥有功能增强的MDA5，可识别病原体并调节免疫，这突出了MDA5在维持鸡免疫稳态中的关键作用。然而，调控MDA5表达和最佳激活的精确机制仍不清楚。在此，我们揭示鸡E3泛素连接酶RNF20对调节MDA5介导的先天性免疫稳态至关重要。转录组测序分析显示，DF-1细胞的RNA病毒感染显著上调了鸡RNF20的表达。RNF20的过表达显著抑制鸡先天性免疫相关基因的表达，而RNF20基因敲除在体内和体外均导致免疫缺陷。机制上，RNF20定位于细胞核，在早期感染阶段维持MDA5的基础表达并调节其诱导性表达以建立免疫防御。在后期，RNF20转移至细胞质，促进K27和K48连接的多聚泛素化以及随后MDA5的降解，从而防止免疫过度刺激。总体而言，本研究确立了RNF20作为维持鸡先天性免疫稳态的重要E3泛素连接酶。
鸡MDA5是启动抗病毒先天性免疫反应的重要RNA病毒传感器。MDA5的蛋白水平必须受到严格调控以维持抗病毒先天性免疫稳态。在本研究中，我们证明E3泛素连接酶RNF20通过核质转运精确调节MDA5蛋白稳定性。具体而言，在未感染和早期感染期间，RNF20在细胞核中调节MDA5转录。而在感染后期，RNF20从细胞核中转移出来并催化MDA5的泛素化降解。因此，RNF20在调节鸡抗病毒先天性免疫稳态中很重要。
