Hepatitis B virus cccDNA

项目概要 乙型肝炎病毒长期感染全球约 3 亿人。全部都显着增加 发生肝细胞癌的风险。尽管核苷治疗可以阻止病毒复制 类似物，受感染的肝细胞无法治愈，因为共价闭合环状DNA（cccDNA）是 病毒RNA的转录在受感染的细胞中持续存在。迄今为止，预防cccDNA形成的治疗策略 或缺乏功能性失活甚至破坏受感染肝细胞内的 cccDNA。这样做的目的 该应用程序旨在研究与 cccDNA 生物学相关的四个问题： 鉴定所需的酶 cccDNA 形成，阐明 cccDNA 形成的确切机制，研究 cccDNA 是如何形成的 在肝细胞再生过程中分配给子细胞并确定其物理组织 受感染肝细胞中的 cccDNA。对cccDNA形成机制的研究可以揭示cccDNA的身份 细胞 DNA 修复酶反过来可能成为新型抗病毒疗法的靶点。调查 cccDNA 在受感染细胞核中的物理位置可以揭示 cccDNA 如何维持和 细胞分裂后分配给子细胞，以及它最终如何在自发恢复过程中丢失 来自天然 HBV 感染。更好地理解这些过程对于开发至关重要 治疗慢性乙型肝炎（CHB）的新策略。 目标 1. cccDNA 合成机制。我们将确定仍然难以捉摸的细胞酶，这些酶负责 rc 到 cccDNA 的转换。候选者是处理所需的 DNA 连接酶和核酸内切酶 以及连接两条 rcDNA 链的 5' 和 3' 端。基因敲除细胞将用于确定如何 rcDNA 转化为 cccDNA。此外，我们建议开发一种新的筛选方法 鉴定 HBV 感染和 cccDNA 形成所需的宿主基因。 目标 2. 细胞分裂和核定位过程中 cccDNA 的命运。这个目标建立在我们可视化的能力之上 通过荧光原位杂交 (FISH) 检测中期和间期细胞核中的 cccDNA。目标是 确定 cccDNA 在 DHBV 和 HBV 产生细胞中的分布并研究 cccDNA 的分布 在正常条件下和细胞因子存在的情况下分布到子细胞。实验设计 研究功能性和转录沉默 cccDNA 的核定位。 为了实现这些目标，我们将依靠基于我们的研究工作的实验方法 过去两年，细胞培养平台允许 HepG2 细胞与 HBV 感染相结合 CRISPR/Cas9基因敲除技术。此外，这些目标建立在我们最近成功开发 允许通过 FISH 检测 cccDNA 的方法。

PROJECT SUMMARY Hepatitis B virus chronically infects approximately 300 million people worldwide. All are at significantly increased risk of developing hepatocellular carcinoma. Although virus replication can be blocked by therapy with nucleoside analogs, infected hepatocytes are not cured, because covalently closed circular DNA (cccDNA), the template for transcription of viral RNAs persists in infected cells. So far, therapeutic strategies to prevent cccDNA formation or functionally inactivate or even destroy cccDNA within infected hepatocytes are lacking. The purpose of this application is to investigate four problems pertinent to cccDNA biology: identification of the enzymes required for cccDNA formation, elucidation of the exact mechanism for cccDNA formation, investigating how cccDNA is distributed to daughter cells during regeneration of hepatocytes and determining the physical organization of cccDNA in infected hepatocytes. Research on the mechanism of cccDNA formation could reveal the identity of cellular DNA repair enzymes that in turn, could be become targets for novel antiviral therapies. Investigations on the physical location of cccDNA in nuclei of infected cells could shed light on how cccDNA is maintained and distributed to daughter cells following cell division, and how it is eventually lost during spontaneous recovery from natural HBV infections. A better understanding of these processes is deemed essential for the development of novel strategies to cure chronic hepatitis B (CHB). Aim 1. Mechanism for cccDNA synthesis. We will identify still elusive cellular enzymes responsible for the conversion of rc to cccDNA. Candidates are DNA ligase(s) and endonuclease(s), required for the processing and joining of the 5’ and 3’ ends of the two rcDNA strands. Gene knockout cells will be used to determine how rcDNA is converted into cccDNA. In addition, we propose the development of a novel screening assay for identification of host genes required for HBV infection and cccDNA formation. Aim 2. cccDNA fate during cell division and nuclear localization. This aim builds on our ability to visualize cccDNA in metaphase and interphase nuclei by fluorescence in situ hybridization (FISH). The goal is to determine the distribution of cccDNA in DHBV and HBV producing cells and investigate how cccDNA is distributed to daughter cells under normal conditions and in the presence of cytokines. Experiments are designed to investigate the nuclear localization of functional and transcriptionally silenced cccDNA. To address these aims, we will rely on an experimental approach that builds on our research efforts during the past two years leading to cell culture platforms permitting HBV infections of HepG2 cells in combination with CRISPR/Cas9 gene knockout technology. In addition, the aims build on our recent success in developing methods permitting detection of cccDNA by FISH.