干扰素通过肝非实质细胞分泌的外体（exosomes）抑制乙型肝炎病毒在肝细胞中复制的机制研究

The hepatitis B virus (HBV) belongs to the family Hepadnaviridae, which exclusively infects hepatocytes,and approximately 400 million people are chronically infected worldwide. The overall goal of this research project is to understand the mechanisms by which interferon alpha (IFN-α) inhibits HBV replication. Type I interferons, predominantly IFN-α and IFN-β, play an important role in controlling the replication of a virus during the initial stages of infection. IFN-α is currently approved for the treatment of chronic hepatitis B (CHB) with a response rate of 30-40%. To evade the IFN-α-mediated antiviral effect, HBV has evolved many mechanisms to block IFN-α activities in virus-infected hepatocytes, which raises the question of why the IFN-induced antiviral response is still capable of controlling HBV replication in vivo. The liver is an organ with predominant innate immunity and is composed of liver parenchymal (hepatocytes) and nonparenchymal cells. Liver nonparenchymal cells (LNPCs), including Kupffer cells (the resident macrophages of the liver), sinusoidal endothelial cells (LSECs), and lymphocytes constitute approximately one third of the total liver cell population. Exosomes are membrane vesicles 40 to 100 nm in diameter that originate in the late endosomal compartment by the inward budding of endosomal membranes, thereby generating intracellular multivesicular bodies (MVBs). Pools of exosomes are packed in MVBs and are released into the extracellular environment after the fusion of MVBs with plasma membranes. These vesicles can horizontally transfer functional proteins, mRNAs, and microRNAs (miRNAs) to neighboring cells as mediators of intercellular communication. We hypothesize that IFN-α treatment induces the transfer of antiviral molecules from LNPCs to HBV-replicating hepatocytes to restore the antiviral state in hepatocytes and result in the inhibition of HBV relication. In Specific Aim 1, we will investigate the role of LNPCs and exosomes from LNPCs in the IFN-α-induced antiviral effect in cell co-cluture model. In Specific Aim 2, we will define the exact step(s) in the HBV life cycle hindered by the exosomes from IFN-α-stimulated LNPCs.In Specific Aim 3, we will identify differentially expressed molecues, including proteins, mRNA, and microRNA, between exosomes from IFN-α-treated and untreated LNPCs. In Specific Aim 4, we will examine whether these differntially expressed exosomal molecules have anti-HBV activity and are associated with early virological response to IFN-α therapy in CHB patients. The proposed studies will expand our understanding of the strategy of the host-virus 'arms race' and may lead to the development of novel therapeutics for CHB and other infectious diseases.

乙型肝炎病毒（Hepatitis B virus，HBV）持续性感染是影响人类健康的重大问题。I型干扰素（interferon， IFN）包括IFN-alpha (IFN-α)和 IFN-beta (IFN-β)是宿主抵御病原生物入侵的第一道防线，IFN-α是目前临床治疗乙型肝炎的主要药物之一。HBV在肝细胞中能通过多种机制对抗IFN-α的抗病毒作用，作为肝脏微环境中的肝非实质细胞是否在IFN-α抑制HBV复制中发挥作用尚不清楚。外体（exosomes）是由细胞分泌至细胞外的膜性小囊泡，通过在细胞之间水平转移功能性分子在细胞之间的通讯过程中发挥重要作用。本项目拟证实IFN-α通过肝非实质细胞分泌的外体抑制HBV复制这一假设，同时将深入研究其分子机制，包括 IFN-α处理的肝非实质细胞分泌的外体抑制HBV复制的关键环节、是否被细胞内化以及所携带的效应分子等。

由乙型肝炎病毒（Hepatitis B virus，HBV）持续性感染引起的慢性乙型肝炎是严重危害人类健康的疾病。HBV在机体细胞内持续性存在是病毒和机体细胞之间复杂的相互作用的结果。临床上针对HBV感染的抗病毒药物主要为核苷类似物和干扰素-α（Interferon-α, IFN-α）。IFN-α临床治疗乙型肝炎的应答率为30-40%。与此同时HBV可通过多种机制对抗IFN-α的抗病毒作用。本研究发现肝非实质细胞（包括巨噬细胞和肝窦内皮细胞）能促进IFN-α在肝细胞中的抗HBV作用。这种作用不依赖于IFN-α对肝细胞中Jak-STAT信号通路的激活，但是依赖于IFN-α对肝非实质细胞中Jak-STAT信号通路的激活。进一步发现，通过化学抑制剂GW4869和nSMase2 shRNA抑制肝非实质细胞外体的释放能极大地削弱肝非实质细胞介导的IFN-α的抗病毒活性；与此同时经超速离心纯化的、IFN-α处理的肝非实质细胞分泌的外体能通过下调病毒的RNA和DNA水平直接抑制HBV的复制。进一步通过Wenstern blot和microArray发现IFN-α处理和不处理的肝非实质细胞释放的外体包含不同的内容，发现抗病毒蛋白去氨基酶APOBEC3G、618个mRNA和3个microRNA在IFN-α处理的肝非实质细胞释放的外体中上调表达。通过在肝非实质细胞中抑制APOBEC3G的表达、在肝细胞中利用mRNA翻译抑制剂Cycloheximide (CHX)阻断外体携带的mRNA的翻译和利用microRNA抑制剂抑制外体携带的microRNA的功能显示差异表达的APOBEC3G、mRNA和microRNA均在外体抑制HBV复制中发挥一定作用。此外，发现鼠肝炎病毒A56诱导产生的内源性的IFN-α亦通过外体发挥抗病毒作用。.综上所述，本研究探讨了IFN-α抑制乙肝病毒复制的作用和机制，以及HBV与自噬过程的相互调控作用，对这些机制的阐明不仅有助于揭示HBV持续感染的分子机制，并可能为优化临床治疗方案、研制新型抗HBV药物提供理论依据。