TMEM101调控RIG-I介导的抗病毒天然免疫信号通路的分子机制研究

Host cells have the ability to recognize virus infection and build up a powerful antiviral response. Retinoic acid-inducible gene-I (RIG-I) plays a critical role in activating host innate immune pathways in response to RNA virus infections. The RIG-I can specifically recognize virus-derived RNA species as a molecular feature discriminating the pathogen from the host. The recognition of foreign RNA by the RIG-I activates antiviral signaling cascades, resulting in the production of antiviral cytokines and the establishment of a broadly effective cellular antiviral status. By taking the approach of luciferase reporter assay, we identified that transmembrane protein TMEM101 inhibited the activation of IFNβ, NF-κB and ISRE promoter induced by MAVS.We also found that TMEM101 can interact with RIG-I and MAVS. In order to address the mechanism of TMEM101 in regulating RIG-I signaling pathway, we proposed the following specific aims: (1) To confirm the inhibitory effect of TMEM101 on RIG-I mediated signal pathway. (2) To examine whether TMEM101 expression and localization were regulated during virus infection or as RIG-I pathway activated. (3) To identify the functional domain of TMEM101 exerting the inhibitory effect on RIG-I pathway. We expect to reveal the mechanism of TMEM101 in negatively regulating the RIG-I mediated innate immune signaling during RNA virus infection.

RIG-I介导的信号通路是宿主抗病毒天然免疫反应的重要途径。我们前期通过IFNβ荧光素酶报告基因筛选，发现跨膜蛋白TMEM101能显著抑制MAVS和polyI:C诱导的RIG-I信号通路的激活，还发现它可以结合RIG-I和MAVS等相关蛋白。为了揭示TMEM101调控RIG-I介导的信号通路的分子机制，我们将从以下几个方面进行研究：（1）通过仙台病毒感染激活RIG-I信号通路，利用实时定量RCR等方法，进一步验证TMEM101对RIG-I通路的抑制作用；（2）通过不同方式激活RIG-I信号通路，检测TMEM101表达水平及在细胞中定位的变化，分析TMEM101与RIG-I介导的天然免疫的相关性；（3）研究TMEM101如何通过与RIG-I和MAVS等相关蛋白相互作用抑制RIG-I信号通路，明确TMEM101发挥抑制功能的结构域。拟通过上述研究阐明TMEM101负调控天然免疫信号通路的机制。

RIG-I介导的信号通路是宿主抗病毒天然免疫反应的重要途径。我们前期研究发现TMEM101和CK1γ1都参与了RIG-I天然免疫通路的调控，并对其调控机制进行了深入的研究，经研究发现CK1γ1对RIG-I信号通路起更明显的抑制作用，过表达CK1γ1能抑制RIG-I信号通路介导的IFN-β的激活，相反，敲低CK1γ1可以增强IFN-β和NF-κB的活性；进一步的实验发现CK1γ1能与RLR通路下游分子NF-κB亚基p65相互作用并使其发生磷酸化修饰；通过蛋白激酶的体外实验，我们发现蛋白激酶CK1γ1能够磷酸化NF-κB亚基p65的Ser536，促进E3泛素连接酶CUL2和COMMD1介导的p65的泛素化降解；同时，失去蛋白激酶活性的CK1γ1K73A和CK1γ1N169A突变体，不能抑制仙台病毒（SeV）介导的IFN-β与NF-κB报告基因的激活, 揭示出CK1γ1的激酶活性对于它抑制RIG-I信号通路是非常重要的；同时我们发现，CK1γ1对TLR信号通路也有抑制作用。上述研究揭示了CK1γ1负调控RIG-I天然免疫信号通路的分子机制。