嗜肝DNA病毒聚合酶新功能区的结构和功能研究

Polymerase is the only replicase encoded by hepadnavirus. Currently, due to notorious low solubility and expression amount, hepadnaviral polymerase is not yet be crystallized and structurally analyzed. When hepadnavirus replicates, polymerase should set up and maintain its active conformations by interacting with nucleic acids and intramolecular interactions occurring among its various domains.Previous work done by this project group demonstrated that helix clamp and primer grip motifs are two novel functional regions of hepadnaviral polymerase. By structural predictions, these two novel functional regions could bind DNA primer strand when polymerase replicates progeny DNA. In this project, unnatural amino acid mutagenesis system and other methods of molecular biology such as natural amnino acid mutagenesis will be applied to verify and dissect the key interactions occurring between polymerase and viral nucleic acids or among intramolecular domains. This project will provide in-depth knowledge of structures and functions of hepadnaviral polymerases as well as replication mechanisms of hepadnavirus, which will pave the basis for the design of novel antiviral reagents.

聚合酶是嗜肝DNA病毒编码的唯一复制酶。目前，嗜肝DNA病毒聚合酶的不溶性和低表达量使得无法进行晶体结构分析。在病毒复制DNA时，聚合酶需要通过与核酸作用以及聚合酶分子内结构域间相互作用建立并维持活性构型。本项目组已有的工作发现helix clamp和primer grip基序是嗜肝DNA病毒聚合酶的新功能区。通过结构预测发现，这两个新功能区在聚合酶复制DNA时都可能与DNA引物链结合。本项目将采用非天然氨基酸突变系统并结合常规氨基酸点突变等其他分子生物学方法验证和研究嗜肝DNA病毒复制时聚合酶-核酸、聚合酶分子内不同结构域间的关键相互作用。本项目将有助于深入了解嗜肝DNA病毒聚合酶的结构和功能以及病毒复制机理，为后续抗病毒制剂的设计提供理论依据。

聚合酶是嗜肝DNA病毒编码的唯一复制酶。目前，嗜肝DNA病毒聚合酶的不溶性和低表达量使得无法进行晶体结构分析。本项目组已有的工作发现helix clamp和primer grip基序是嗜肝DNA病毒聚合酶的新功能区。本项目建立了原核和真核细胞的非天然氨基酸定点突变系统,将具有光交联属性的非天然氨基酸定点掺入到helix clamp和primer grip基序,通过对病毒突变体复制和聚合酶活性研究进一步证明了这两个基序在病毒复制中起重要作用。此外，在该项目的支持下，我们还系统地揭示了干扰素诱导的效应蛋白Mx2抑制乙肝病毒复制的机理。Mx2很可能通过抑制松弛环状DNA(relaxed circular DNA, rcDNA)转化成共价闭合环状DNA(covalently closed circular DNA, cccDNA)而显著降低细胞核内cccDNA水平，此外还通过减慢病毒RNA合成而加速病毒RNA降解下调病毒RNA稳态水平进一步降低病毒复制。目前cccDNA清除是乙肝病毒病毒学治愈的唯一指标。因此深入了解Mx2如何抑制rcDNA转化成cccDNA将有助于达到这一目标。