外体（exosomes）介导干扰素抗乙肝病毒活性的细胞间传递机制-进入肝细胞的机制

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%. Our recent study showed that IFN-α induced the transfer of antiviral molecules from Liver nonparenchymal cells (LNPCs) to hepatocytes through the release and internalization of exosomes. This intercellular transfer of exosomes bypassed the viral inhibition of interferon-induced expression of genes encoding antiviral molecules and resulted in restoration of the antiviral state in virus-infected cells. However, how the exosomes from LNPCs enter into hepatocytes, thereby transmitting anti-HBV activity between cells remains unclear. The entry of exosomes into target cells might takes place predominatly via receptor-mediated endocytosis, which is similar to that of virus evading target cells. In Specific Aim 1, we will identify the ligands exoposed on the surface of the exosomes, which are responsible for the internalizaiton of exosomes and transmission of IFN-α-induced antiviral effect. In Specific Aim 2, we will define the hepatocyte receptors that bind to exosomes and mediate their uptake. In Specific Aim 3, we will study the intracellular trafficking pathway of exosomes in hepatocytes after endocytosis. The proposed studies will expand our understanding of the mechanism of exosomes "envading" target cells and may lead to the development of novel therapeutics for CHB and other infectious diseases.

干扰素是机体抵御病毒入侵的关键防线，其中干扰素-α（interferon-α, IFN-α）已被广泛用于临床治疗乙型肝炎，但是其抗病毒机制仍未完全阐明。申请人前期研究表明IFN-α诱导的抗乙型肝炎病毒（Hepatitis B virus，HBV）活性可通过外体（exosomes）从肝非实质细胞传递到肝细胞，但是尚不明确肝非实质细胞分泌的外体怎样进入肝细胞以传递IFN-α的抗HBV活性。本项目根据外体的生化特性和前期的研究结果提出外体进入肝细胞可能主要通过配体-受体介导的内吞作用的假设。为此，将首先确定外体表面暴露的介导外体进入肝细胞的配体，然后鉴定与配体结合的肝细胞表面的受体分子，并研究他们在外体进入肝细胞和传递IFN-α的抗HBV活性过程中的作用，最后将研究外体与肝细胞结合后通过何种内吞方式入胞以完成IFN-α抗HBV活性的传递。本项目的开展将为开发基于外体的抗HBV药物打下良好基础。

目前干扰素-α（Interferon-α, IFN-α）为临床上乙肝治疗的主要手段之一。本课题组前期的研究阐明了受制于HBV的IFN-α在肝脏微环境中发挥抗病毒作用的机制，包括巨噬细胞在内的肝非实质细胞（nonpermissive liver nonparenchymal cells，LNPCs）可通过外泌体（exosome）传递IFN-α诱导的抗病毒效应至HBV复制的肝细胞中，反拮抗病毒对IFN-α抗病毒活性的干扰，从而有效抑制HBV在肝细胞中的复制。然而，肝非实质细胞来源的外泌体是依赖何种机制进入肝细胞以传递IFN-α诱导的抗HBV效应有待深入阐明。本课题从受体、内吞至潜在的内体逃逸等多个环节，对涉及巨噬细胞外泌体传递IFN-α抗HBV效应进入肝细胞的通路进行了深入研究，发现巨噬细胞外泌体可以利用HAV受体TIM-1及后续CME和大胞饮进入肝细胞，传递IFN-α的抗HBV活性。进入肝细胞的巨噬细胞外泌体可以在LE/sMVBs中发生LBPA介导的膜融合以释放其内容物至膜结构外。本研究深化了对IFN-α利用肝非实质细胞外泌体传递抗病毒活性分子机制的理解，以上研究还揭示了外泌体与病毒在入胞机制上的相似点，为改造外泌体成运输抗病毒分子的高效治疗性载体提供了理论基础。