DNA感受器在抗HBV免疫应答中的功能研究

Hepatitis B virus (HBV) is a Hepatotropic DNA virus. The molecular mechanism how the viral infection is cleared by the immune system or develop to a persistent infection is not yet clear. Recently, a new family of pathogen pattern recognition receptors, named DNA sensor, was identified. Owing to DNA sensors are able to recognize DNA molecular, it warrant to study whether DNA sensors can recognize the HBV DNA genome and initiate the anit-HBV immune response. The applicant's previous studies have primarily proved that DNA sensor IFI16 is able to inhibit HBV replication in cell culture system. In this proposal, we plan to perform a comprehensive study of DNA sensors, In which six newly discovered DNA sensors, including proteins DAI，AIM2，IFI16,DHX9，DHX36，Ku70, will be explored for their roles in the anti-HBV immune system. We plan to construct the expressing and knock-down systems for the DNA sensor genes, and detect their function of recognition of HBV DNA and initiation of immune response basing on the HBV transfection or infection cell culture system, as well as HBV transgenic mouse model. We also will adopt the PBMC and liver tissue from chronic HBV infection to study whether the persistent HBV replication inhibits the expression and function of DNA sensors signaling pathways. We will also observe the effect of intferon treatment on the DNA sensor pathways. This project will help to elucidate the molecular mechanisms of immune clearance or viral escape during HBV infection, and contribute to the development of new antiviral strategies.

乙型肝炎病毒(HBV)是嗜肝DNA病毒，HBV感染后可为急性自限性过程或发展为慢性感染，但分子机制尚不明确。近年发现了一类可识别DNA分子的病原模式识别受体：DNA感受器(Sensor)，其在抗HBV的免疫识别和清除中的作用尚有待研究。申请人既往研究已证明DNA感受器IFI16具抑制HBV复制的功能。拟通过本项目系统研究6人新近发现的DNA感受器（DAI，AIM2，IFI16,DHX9，DHX36，Ku70）在抗HBV免疫中的作用。拟构建上述DNA感受器的表达及敲除体系，应用HBV转染或感染细胞模型、转基因小鼠模型，以及慢性HBV感染者的PBMC及肝组织样本；研究HBV DNA对DNA感受器及其信号通路的激活，以及慢性感染中HBV对DNA感受器信号通路的可能抑制，并观察干扰素治疗对DNA感受器通路的影响。本项目研究将有助于阐明HBV感染后免疫清除或逃逸的分子机制，有助于发展新型抗病毒策略。

乙型肝炎病毒感染的慢性化机制仍尚未明确。本项目主要聚焦于近年所发现的天然免疫分子DNA受体对HBV基因组的识别，免疫应答的启动及免疫逃逸机制。研究重点在于核DNA受体IFI16与HBV cccDNA的相互作用。我们的研究显示IFI16能识别肝细胞核内的HBV cccDNA，结合后通过乙酰化相关的修饰促使IFI16转位进入细胞浆，在胞浆内IFI16被降解成片段，这些片段可抑制其它DNA受体如cGAS、DAI、AIM2启动STING信号通路和产生IFN的能力。研究还显示IFI16的表达水平受细胞分化影响，DMSO等细胞分化诱导促使IFI16的表达及重新核定位。此外，胞浆的IFI16可进一步分泌进入血清及外泌体，外泌体中亦可检测到IFI16片段。对慢乙肝患者样本的分析显示血清中IFI16水平和干扰素及核苷类似物抗病毒治疗的疗效相关。外周血单个核细胞中的IFI16同样也存在降解。我们的研究显示IFI16可通过对HBV cccDNA的识别启动干扰素信号通路，但其在胞浆中的降解产物则又抑制了干扰素信号通路的进一步激活。IFI16在分化细胞中表达，及HBV感染中对干扰素信号通路的双向调控作用，为婴幼儿感染易于慢性化，以及乙肝感染慢性化的持续机制提供了解释。