宿主蛋白Staufen1促进传染性法氏囊病病毒复制的分子机理研究

Infectious bursal disease virus (IBDV), the causative agent of infectious bursal disease (IBD), mainly destroys the immature B lymphocyte in the bursa of Fabricius resulting in severe immunosuppression. During the process of IBDV infection, the antiviral innate immunity which was mediated by the induction of type I Interferon (IFN) was not activated. Previously, viral protein VP3 and VP4 were identified to be the main factors to block the IFN-β induction. However, it remains elusive that whether there are host factors involving in the process of IBDV escaping from the antiviral innate immunity. In this study, the IBDV dsRNA binding proteins profile was achieved by the liquid chromatography tandem-mass spectrometry analysis of the precipitate from the Biotin labeled IBDV genomic dsRNA incubating with the whole DF-1 cell lysate, and a host factor, Staufen1 (STAU1) was identified to be a specific binding partner of IBDV genomic dsRNA firstly. Furthermore, we aim to explore the difference of viral proteins expression and IFN-β induction between the normal and STAU1-downregulated DF-1 cells with the infection of IBDV. Moreover, on the basis of that the STAU1 was negatively regulated by the treatment of IFN-β, we will further investigate the function of STAU1 during the process of the dsRNA-induced IFN-β expression and the relationship between STAU1 and the intracellular pattern recognition receptor MDA5 to analyze the mechanism by which STAU1 regulates the MDA5-mediated IFN-β expression, and finally, reveal the molecular mechanism of STAU1 used by IBDV in escaping from the antiviral innate immunity and promoting its intracellular replication. Together, this study will for the first time provide new insights into the how IBDV escapes the host antiviral innate immunity, and pave the way for the development of pharmacological interventions and clinical strategies for treatment of IBDV infection.

传染性法氏囊病病毒（IBDV）感染主要导致雏鸡发生严重的免疫抑制，在其感染过程中，I型干扰素（IFN）介导的宿主抗病毒先天免疫并未被激活。病毒蛋白VP3和VP4被认为是IBDV用来执行逃逸宿主抗病毒先天免疫的主要分子，目前仍不清楚是否有宿主分子参与此逃逸过程。本研究首先利用高通量质谱技术鉴定出与IBDV基因组dsRNA结合的宿主蛋白Staufen1（STAU1）；其次，探讨敲低STAU1对病毒蛋白表达的影响，同时分析IBDV感染诱导IFN-β表达水平的变化；最后，在IFN-β负调控STAU1表达的基础上，探索STAU1在IFN-β表达过程中的作用及其与胞内模式识别受体MDA5的关系，分析STAU1对MDA5信号通路的调控机制，解析IBDV利用宿主STAU1逃逸抗病毒先天免疫并促进自身复制的分子机理。这将为揭示IBDV逃逸宿主抗病毒先天免疫提供新机制，也为开发新型IBDV防制方法奠定基础。

传染性法氏囊病病毒（Infectious bursal disease virus，IBDV）是一种具有双节段双链RNA（double-stranded RNA，dsRNA）基因组的无囊膜病毒，IBDV感染会促发由胞内模式识别受体MDA5（Melanoma differentiation‐associated protein 5）介导的I型干扰素（Interferon，IFN）的诱导，但是IBDV逃逸I型IFN抗病毒效应的分子机制依然不清楚。本研究中，我们首先通过IBDV基因组dsRNA介导的pulldown实验和串联质谱技术鉴定特异结合IBDV基因组dsRNA的宿主蛋白Staufen1（STAU1），通过Western blot分析、免疫荧光实验和凝胶迁移阻滞实验证实了此种结合，并且利用构建的各种STAU1表达突变体，成功将其结合IBDV基因组dsRNA的区域锁定在N端468个氨基酸；利用RNA干扰介导的敲低和瞬时过表达技术证明STAU1促进IBDV的胞内复制而不影响其侵入；通过分析在敲低和过表达STAU1表达时宿主细胞对IBDV基因组dsRNA诱导的IFN-β水平，发现STAU1促进IBDV胞内复制与其抑制IBDV基因组dsRNA诱导的IFN-β表达水平有关；通过比较STAU1、MDA5和病毒蛋白VP3对IBDV基因组dsRNA的结合能力，证明STAU1具有比MDA5更高结合IBDV基因组dsRNA的能力是其抑制IBDV基因组dsRNA诱导IFN-β表达的原因，并且这种抑制作用是通过MDA5识别通路发挥功能的；最后，病毒蛋白VP3对STAU1抑制IBDV基因组dsRNA诱导的IFN-β表达具有叠加效应，并且VP3不足以补偿因STAU1敲低而导致的抑制减弱。综上，本研究为深入理解IBDV与宿主间相互作用积累了理论资料，为阐明IBDV引起免疫抑制的机理提供了新的研究方向，也为开发抗IBDV提供了新的潜在靶点。