SMARCB1介导HBV cccDNA微染色体染色质重塑调控cccDNA转录的机制研究

The template of hepatitis B virus (HBV) transcription, the covalently closed circular DNA (cccDNA) plays a key role in the life cycle of the virus. cccDNA accumulates in the nucleus of infected cells as a stable episome organized minichromosomes by histones and non-histone viral and celluar proteins. The modification of chromatin structure of cccDNA minichromosome is an important determinant of cccDNA function. We performed a screen using an siRNA library targeting host chromatin remodeling genes in order to identify new host factors that contribute to cccDNA transcription. SMARCB1 is identified in the screen and subsequently validated in follow-up experiments. We propose a novel hypothesis that cccDNA minichromosome use SMARCB1 to modify its chromatin structure and regulate the cccDNA activity. By gene deletion mutation, co-immunoprecipitation and chromatin immunoprecipitation, the mechanism of SMARCB1 mediated chromatin remodeling of cccDNA minichromosome will be examined. The proposed studies focus on how SMARCB1 and chromatin remodelling regulate cccDNA function, and lead to the development of novel therapeutics to control hbv infections.

HBV共价闭合环状DNA（cccDNA）转录是HBV复制的首要环节。cccDNA在体内以微染色体形式存在，染色质活化对cccDNA转录非常关键。在对染色质重塑相关基因的siRNA文库筛选中，我们发现ATP依赖的染色质改构复合物SWI/SNF的核心组分SMARCB1是cccDNA转录中的关键分子，SMARCB1在cccDNA微染色体募集水平与cccDNA转录活性密切相关，推测SMARCB1介导cccDNA微染色体染色质重塑调控cccDNA转录。本项目将采用缺失突变分析，染色质免疫共沉淀等技术在已建立的cccDNA研究模型中鉴定SMARCB1介导cccDNA微染色体染色质重塑的关键结构域，阐述SMARCB1在cccDNA微染色体的募集机制，明确SMARCB1与cccDNA组蛋白修饰的相互作用及其在cccDNA转录中的意义，揭示SMARCB1调控cccDNA转录的机制，为抗HBV治疗提供新思路。

乙型肝炎共价闭合环状DNA(HBV cccDNA)是HBV生活周期的核心，项目紧密围绕研究目标HBV cccDNA转录这一过程，针对SMARCB1在HBV cccDNA转录中的作用进行了系列完整的研究。项目从细胞实验和临床样本研究两方面解析了SMARCB1在cccDNA转录中的作用。结合该研究领域论文发表情况和项目研究结果，课题组纳入HBV感染相关宿主miRNA（miR-6762，miR-3188，miR-378等），以及MRE11、Trex1、IFI16等免疫因子，对其在HBV cccDNA的生活周期中的作用进行了系列研究，系统阐述了宿主关键因子、免疫等多因素在HBV cccDNA转录等中的作用。依据本项目，课题组建成了完善的HBV cccDNA研究平台体系，获国家发明专利授权1项，发表SCI论文2篇，中文核心期刊1篇，申请获得上海市科委基金资助1项，培养研究生1名，组织学术会议三项，基本实现研究目标。