基于活细胞成像的乙型肝炎病毒共价闭合环状DNA储存库维持及其转录调控的分子机制研究

作为一种严重危害人类健康的病原体，学术界对乙型肝炎病毒（HBV）的复制模式已有了较深入的了解。然而，目前对HBV持续感染、复制的最关键分子cccDNA（共价闭合环状DNA）储存库的维持及其转录调控的分子机制，仍然缺乏深入认识。 课题组前期通过基因组改造，构建基CRISPR/Cas9 DNA成像和基于MS2-MBP的RNA活细胞成像的重组cccDNA载体， 在不影响病毒正常生活周期的前提下，成功在活细胞中观察到病毒cccDNA及其转录本。 本研究拟基于此技术，研究病毒微小染色体在宿主细胞内的代谢动态，特别是在有丝分裂中的命运；在单分子水平研究cccDNA的mRNA转录动力学特性，并探讨抗病毒药物（如干扰素α）对这一过程的影响。上述研究获得的活细胞观察数据将有助于阐明HBV储存库的维持与mRNA合成的动力学特性，为寻找新型沉默/清除病毒cccDNA的治疗策略提供理论基础和潜在靶点。

As an infectious agent causing great health care burden, a relatively thorough understanding of the life cycle of hepatitis B virus (HBV) has been garnered. However, the maintenance and transcriptional control of the covalently closed circular DNA (cccDNA), the essential molecule responsible for the persistent infection and replication of HBV, remains unclear. Previously, by genome engineering, we constructed recombinant cccDNA vectors which harbor genetic elements for CRISPR/Cas9 DNA imaging and MS2-MBP RNA imaging without interfering its life cycle and successfully visualized the cccDNA and its transcription in live cells using fluorescent microscopy. Based on this technique, we plan to study the dynamics and turnover of cccDNA, particularly during cell mitosis. In addition, the kinetics of cccDNA transcription at single-molecule level, with or without antiviral treatment such as interferon-α, can also be interrogated. The data generated in this project would help clarify the basic dynamics of cccDNA turn-over and its mRNA synthesis, which would potentially provide novel clues for therapeutic solutions.