RNA结合蛋白RBM24通过ε元件调控HBV pgRNA翻译和逆转录的分子机制

Hepatitis B virus (HBV) is one of the most important pathogens which can cause chronic hepatitis. HBV pregenomic RNA (pgRNA) plays essential roles during virus replication, which acts either as mRNA for translation or as a template for reverse transcription. However, it remains unclear how pgRNA translation and reverse transcription are coordinated. Our preliminary data has shown that RBM24, one of RNA binding proteins, binds to ε, the essential element of pgRNA to negatively regulate pgRNA translation while positively regulate pgRNA reverse transcription. Therefore, RBM24 may be a novel host factor to orchest the function of pgRNA. In the present project, the impact of RBM24 on the ribosome assembly on ε element will be analyzed. Moreover, the key domains in deciding RBM24-ε binding will be identified and their role in the ribosome assembly will be clarified to reveal the mechanism by which RBM24 negatively modulates pgRNA translation. The influence of RBM24 on pgRNA encapsidation, protein priming and reverse transcription into minus strand DNA will be investigated to clarify the step by which RBM24 positively regulates pgRNA reverse transcription. The key domains mediating RBM24-ε-Pol interaction will be identified and their role in pgRNA reverse transcription will be analyzed to uncover the mechanism by which RBM24 positively regulates pgRNA reverse transcription. Furthermore, it will also be revealed that the mechanism by which RBM24 modulates switch of pgRNA from translation to reverse transcription. Taken together, this project could not only shed light on the new molecular mechanism by which host factors ochest HBV translation and replication, but also provide new clues for antiviral drug screening.

乙肝病毒（HBV）是引起慢性肝炎的重要病原体。其前基因组RNA （pgRNA）作为关键的复制中间体，承担着翻译模板和逆转录模板的双重功能，但其间的调控机制尚不清晰。前期研究提示RNA结合蛋白RBM24与pgRNA的关键元件ε结合负调控pgRNA翻译、但正调控pgRNA逆转录，可能是一个全新的pgRNA功能调控蛋白。本项目拟通过具体分析RBM24对核糖体在ε元件上组装的影响，及RBM24-ε结合的关键结构域在其中的功能，阐释RBM24负调控pgRNA翻译的机制；通过分析RBM24对pgRNA包装、蛋白引发及逆转录成负链DNA的影响，及RBM24-ε-Pol互作的关键结构域在其中所起的作用，解析RBM24正调控pgRNA逆转录的环节和机制；进而揭示RBM24对pgRNA翻译—逆转录的动态调控机理。研究结果有望揭示新型宿主因子调控HBV翻译及复制的分子新机制，为抗HBV新药的研发提供全新的线索。

乙肝病毒（HBV）是引起慢性肝炎的重要病原体。其前基因组RNA （pgRNA）作为关键的复制中间体，承担着翻译模板和逆转录模板的双重功能，但其间的调控机制尚不清晰。前期研究提示RNA结合蛋白RBM24与pgRNA的关键元件ε结合负调控pgRNA翻译、但正调控pgRNA逆转录，可能是一个全新的pgRNA功能调控蛋白。本项目围绕RBM24通过ε元件调控HBV pgRNA翻译和逆转录的分子机制开展以下研究：①分析RBM24调控pgRNA翻译及其机制： 明确RBM24对pgRNA翻译过程的影响；验证ε元件在RBM24负调控pgRNA翻译中的作用；鉴定调控pgRNA翻译的RBM24-ε互作关键结构域；分析RBM24对核糖体在ε元件上组装的影响。②分析RBM24在pgRNA包装中的功能和机制：明确RBM24对pgRNA包装的影响；分析RBM24在Pol-ε互作中的功能；明确RBM24-ε-Pol互作在pgRNA包装过程中的作用。③分析RBM24在pgRNA逆转录中的功能和机制：分析RBM24是否被包装到核衣壳；明确RBM24对pgRNA逆转录过程的影响并鉴定出相互作用位点。获得的研究结果如下：Southern blot实验表明RBM24为HBV复制所必需；RNA免疫沉淀实验和生物素pull-down实验证实RBM24通过其RNA结合结构域RNP1和RNP2与pgRNA的末端冗余序列结合参与HBV复制；RNA稳定性实验证实RBM24通过与pgRNA 3’末端的冗余序列结合促进HBV RNA的稳定，核糖体组装实验证实RBM24通过与pgRNA 5’末端的冗余序列结合，阻止80S核糖体组装到pgRNA上，从而抑制HBV核心蛋白（core）的翻译（已发表在Journal of Virology杂志）。pgRNA序列上的ε信号是RBM24结合的主要位点；RBM24不依赖于RNA/ε信号与HBV pol蛋白结合，相互作用的位点分别位于RBM24的丙氨酸富集区和Pol的逆转录酶区；RBM24介导Pol-ε互作并与之形成Pol-RBM24-ε复合物，从而参与pgRNA包装和逆转录，并进一步被包装到病毒核衣壳中。研究成果揭示了RNA结合蛋白在HBV复制生活周期中的重要作用，回答了项目提出的关键科学问题，完成了预期目标，为深入了解HBV复制机制和抗HBV药物的研发等提供了新的参考。