MxA蛋白通过与乙型肝炎病毒的核心抗原相互作用抑制病毒复制

发布时间：2019-07-10 12:29

【摘要】：乙型肝炎病毒(HBV)慢性感染是导致肝硬化和诱发肝癌的重要因素。我国有超过一亿人的乙肝病毒携带者,50%以上的原发性肝癌来自慢性乙肝患者。世界范围内,人们对乙肝病毒的感染和其所致病理发展还缺乏有效的预防和干预手段。黏病毒抗性蛋白A(MxA)是由Ⅰ型干扰素诱导表达的人体天然免疫蛋白,主要分布在细胞质中,属于动力蛋白超家族,具有鸟苷三磷酸酶活性,能够自我组装发生寡聚化。既往研究表明,MxA具有广谱的抗RNA病毒活性,如抗流感病毒、水疱性口炎病毒、索戈托病毒和麻疹病毒等。近期的研究显示MxA对属DNA病毒的乙肝病毒的复制也有抑制作用,但具体的机制尚不明确。本课题旨在研究MxA蛋白对乙肝病毒的抑制作用并解析其分子细胞学机制,为探索乙肝治疗的新途径提供理论依据。我们广泛运用了分子手段、生化手段、细胞共聚焦成像、活细胞实时跟踪和光漂白技术,深入研究了相关蛋白的相互作用、细胞内动力学、HBV病毒复制周期的各个环节。我们发现,在HepG2.2.15细胞模型中,MxA能够显著下调细胞外乙肝病毒表面抗原HBsAg的量、细胞内外乙肝病毒DNA的水平和核衣壳pgRNA的水平,但并不影响核心抗原HBcAg的合成和乙肝病毒mRNA的转录和核质分布,而且MxA对乙肝病毒的这些作用并不依赖其鸟苷三磷酸酶活性。进一步的研究表明,MxA通过其中心互作区CID结构域直接跟HBV的核心抗原HBcAg相互作用,二者形成蛋白复合物聚集在肝细胞核周,限制了HBcAg的细胞内运动,从而阻碍了病毒核衣壳在肝细胞内的组装。我们还鉴定出核周MxA-HBcAg蛋白聚合物的形成和维持依赖于胞内微管,而与内质网和高尔基体不存在共定位。本研究揭示了MxA抗乙肝病毒作用的分子机制：MxA通过与乙肝病毒的核心抗原HBcAg相互作用,干扰了核衣壳的组装,进而抑制了乙肝病毒的复制。该研究结果为抗乙肝小分子药物的研发提供了新思路和理论依据。
[Abstract]:Chronic infection of hepatitis B virus (HBV) is an important factor leading to liver cirrhosis and inducing liver cancer. There are more than 100 million hepatitis B virus carriers in China, and more than 50% of primary liver cancer comes from chronic hepatitis B. Worldwide, there is still a lack of effective prevention and intervention methods for hepatitis B virus infection and its pathological development. Myxovirus resistant protein A (MxA) is a human innate immune protein induced by interferon I. It is mainly distributed in the cytoplasm and belongs to the dynamic protein superfamily. It has the activity of guanidine triphosphatase and can self-assemble and oligomerize. Previous studies have shown that MxA has broad-spectrum anti-RNA virus activities, such as anti-influenza virus, vesicular stomatitis virus, Sogotto virus and measles virus. Recent studies have shown that MxA can also inhibit the replication of hepatitis B virus belonging to DNA virus, but the specific mechanism is not clear. The purpose of this study was to study the inhibitory effect of MxA protein on hepatitis B virus and analyze its molecular cellular mechanism, so as to provide theoretical basis for exploring a new way of hepatitis B treatment. We have widely used molecular, biochemical, cell confocal imaging, live cell real-time tracking and photobleaching techniques to study the interaction of related proteins, intracellular kinetics and the replication cycle of HBV virus. We found that in HepG2.2.15 cell model, MxA could significantly down-regulate the amount of extracellular hepatitis B virus surface antigen HBsAg, the level of extracellular hepatitis B virus DNA and the level of nucleocapsid pgRNA, but did not affect the synthesis of core antigen HBcAg and the transcription and nucleoplasmic distribution of hepatitis B virus mRNA, and the effects of MxA on hepatitis B virus did not depend on the activity of guanosine triphosphatase. Further studies show that MxA interacts directly with the core antigen HBcAg of HBV through the CID domain of its central interaction region, and the protein complexes form protein complexes accumulate around the nucleus of hepatocytes, which limits the intracellular movement of HBcAg, thus hindering the assembly of virus nucleocapsid in hepatocytes. We also identified that the formation and maintenance of perinuclear MxA-HBcAg protein polymers depended on intracellular microtubules, but did not co-localize with endoplasmic reticulum and Golgi apparatus. This study reveals the molecular mechanism of anti-hepatitis B virus action of MxA: MxA interferes with the assembly of nucleocapsid by interacting with HBcAg, the core antigen of hepatitis B virus, and then suppresses the replication of hepatitis B virus. The results of this study provide a new idea and theoretical basis for the research and development of anti-hepatitis B small molecule drugs.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.62

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