Dbf2-相关激酶NDR1分子对抗病毒固有免疫应答的调控作用及机制探讨

Type I Interferon plays key role in anti-viral innate immune response. To counteract the cellular defense and secure viral replication, viruses have evolved effective strategies to evade immune surveillance pathways including impairing IFN induction or blocking signaling downstream of the IFNα/β receptor. To our interesting, NDR1 mRNA is significantly reduced in peripheral blood samples from patients infected with respiratory syncytial virus (RSV) comparing to those from the healthy control. The downregulated mRNA and protein levels of NDR1 were further observed in mouse peritoneal macrophages (PMs) infected with RNA virus including RSV and vesicular stomatitis virus (VSV) and DNA viruses including herpes simplex virus 1(HSV-1) and murine herpesvirus 68 (MHV68). Moreover, our data showed that NDR1 knockdown or knockdown inhibits the virus infection induced- proinflammatory cytokines, type I interferon and antiviral ISGs production and promotes the viral infection and replication in macrophage. These preliminary results not only indicate the importance of NDR1 in antiviral innate immunity, but also indicate that NDR1 may be regulated by virus infections and involved in immune response escaping. We will further clarify the role of NDR1 in the anti-viral immune response and the related molecular mechanisms. Also, we will explore the underlined mechanisms by which virus infection inhibits the expression of NDR1. All these data will reveal a novel immune evasion pathway exploited by viruses and provide novel targets for the therapy of infection disease.

I型干扰素在机体抗病毒固有免疫应答中发挥关键作用，而病毒可通过调控I型干扰素的产生及其应答过程完成免疫逃逸。我们初步实验发现，与正常人群相比，感染RSV病毒的病人外周血Dbf2p-相关激酶NDR1表达明显下调，RNA病毒(RSV和VSV)或DNA病毒(HSV-1和MHV68)感染显著下调巨噬细胞中NDR1的表达。而NDR1表达降低或者缺失显著抑制进VSV感染的的I型干扰素、炎症细胞因子及干扰素诱导蛋白ISGs的生成，促进VSV病毒的复制，提示病毒感染可能通过下调NDR1的表达产生免疫逃逸。本项目拟在此基础上利用NDR1基因敲除小鼠，免疫共沉淀及质谱分析等技术，明确NDR1对抗病毒天然免疫反应的调控作用及分子机制，丰富NDR1分子的生理、病理功能。同时探讨病毒诱导 NDR1表达下调的分子机制，可为阐明病毒逃逸免疫系统的监控提供新的理论基础，并为感染性疾病防治提供新靶点。

在病毒感染机体时，机体模式识别受体通过识别病毒中病原相关模式分子启动抗病毒天然免疫应答反应，上调I型干扰素及炎性细胞因子。I型干扰素与干扰素受体结合，诱导直接发挥抗病毒效应的干扰诱导基因，同时正向反馈干扰素产生信号通路。我们研究发现丝苏氨酸蛋白激酶NDR1（Nuclear Dbf2p-related kinase 1）正向调控抗病毒天然免疫应答。干扰或者敲除小鼠腹腔巨噬细胞中NDR1，显著抑制了病毒感染引起的I型干扰素、炎性细胞因子及抗病毒相关的干扰素诱导基因ISGs的产生。小鼠巨噬细胞系RAW264.7中过表达NDR1及其激酶活性突变体均可促进病毒感染诱导的I型干扰素、炎性细胞因子及抗病毒相关的干扰素诱导基因ISGs的产生，表明NDR1 发挥抗病毒功能不依赖其蛋白激酶活性。VSV感染小鼠后，NDR1缺失小鼠组与野生对照组相比，其生存率明显降低，外周血中I型干扰素及炎性细胞因子表达量下降，病毒感染导致的组织损伤更加严重。机制研究发现，NDR1促进STAT1蛋白质翻译，进而促进干扰素信号通路，发挥抗病毒功能。进一步研究发现NDR1通过抑制miRNA146a的表达，促进STAT1的翻译。NDR1可以作为转录调控因子结合于miRNA146a启动子区，通过与NF-kB相互作用，抑制miRNA146a转录，进而解除miRNA146a对STAT1的翻译抑制，最终在病毒感染机体时通过正向调控干扰素信号通路，发挥抗病毒功能。 我们的研究结果首次揭示了NDR1以激酶非依赖形式促进STAT1翻译过程，为深入了解抗病毒反应的分子机制提供新思路。此外，我们的研究发现NDR1正向调控机体抗病毒天然免疫反应的新功能，提出一种新的机体抵抗病毒感染和防止病毒免疫逃逸的新机制。