磷酸酶Cdc25A抑制RIG-I介导的抗病毒天然免疫信号通路的机制

RIG-I (Retinoic acid-inducible gene-I) mediated innate immune signaling plays a critical role in activating host response to RNA virus infections. RIG-I pathway is controlled by both positive and negative regulators. We identified that phosphatase Cdc25A (cell division cycle 25 A) can inhibit the activation of IFN? promoter induced by Sendai virus infection. Further analysis indicated that Cdc25A can interact with RIG-I, MAVS and TRAF6. We also found that over-expression of Cdc25A interferes with the interaction between MAVS and TBK1. To further investigate the molecular mechanisms of how Cdc25A regulates RIG-I pathway, we propose to address the following issues:（1）To confirm the inhibitory effect of Cdc25A on RIG-I signaling pathway by luciferase reporter assay and real-time PCR.（2）To examine whether Cdc25A phosphartase activity is required for regulating the RIG-I pathway and to analyze which domain of Cdc25A is critical for its function; (3) To investigate the molecular mechanism of how Cdc25A affects the formation of MAVS signalsome. (4) To analyze the dynamic correlation between the protein level, localization and the activity of phosphatase of Cdc25A and RNA virus infection or the activation of RIG-I pathway. We expect to reveal the molecular mechanisms of Cdc25A in negatively regulating the RIG-I mediated innate immune signaling during RNA virus infection.

RIG-I介导的天然免疫反应是宿主抗病毒的重要防御机制。我们发现磷酸酶Cdc25A能显著下调RIG-I介导的信号通路的激活；进一步的研究表明，Cdc25A能够干扰RIG-I下游MAVS信号复合体的形成。为了揭示Cdc25A负调控RIG-I信号通路的机制，我们拟开展以下研究：（1）利用过表达和RNA干扰等方法结合实时定量PCR，进一步验证Cdc25A对RIG-I信号通路的抑制作用；（2）构建Cdc25A点突变或缺失突变体，分析Cdc25A磷酸酶活性对RIG-I信号通路的影响，定位其发挥抑制功能的结构域；（3）研究Cdc25A在RIG-I信号通路中的作用节点及影响MAVS信号复合体形成的机制；（4）分析病毒感染对Cdc25A表达水平、定位以及磷酸酶活性的影响，探究Cdc25A与病毒激活的天然免疫之间的相关性。拟通过上述研究阐明Cdc25A负调控RIG-I信号通路的机制及其在抗病毒免疫中的作用。

RIG-I信号通路是细胞内重要的抗病毒天然免疫信号通路之一。RIG-I信号通路的适度激活对细胞和机体有很重要的作用。我们发现Cdc25A可能是RIG-I信号通路的一个负调控因子。我们的早期结果显示，Cdc25A可以抑制仙台病毒和流感病毒RNA诱导的I型干扰素IFNb的启动子活性，也与RIG-I信号通路重要组分RIG-I和VISA有相互作用。这些结果提示我们Cdc25A可能是潜在的RIG-I信号通路的负调控因子。我们发现Cdc25A可以直接抑制TBK1的磷酸化激活，且此抑制作用依赖其磷酸酶活性。免疫印迹结果显示，过表达Cdc25A能够抑制仙台病毒诱导的TBK1 第127位丝氨酸的磷酸化， Cdc25A的酶活突变体C431S却没有此抑制作用。此外，Cdc25A也可以抑制过表达的Flag-TBK1的127位的丝氨酸磷酸化，且此抑制作用依赖其磷酸酶活性。进一步的免疫共沉淀实验显示外源CDC25A可以与TBK1相互作用。我们的结果说明Cdc25A可以直接抑制TBK1的磷酸化激活，且此抑制作用依赖其磷酸酶活性，进而抑制IFN-b的激活。该研究阐明了Cdc25A负调控抗病毒天然免疫信号通路的机制。