Hepatitis B virus cccDNA

项目摘要 B型肝炎病毒慢性感染全世界约3亿人。所有人都在显著增加 患肝细胞癌的风险。虽然病毒复制可以被阻断治疗核苷 类似地，感染的肝细胞不能治愈，因为共价闭合环状DNA（cccDNA）， 病毒RNA的转录持续存在于受感染的细胞中。到目前为止，预防cccDNA形成的治疗策略 或功能性地阻断或甚至破坏感染的肝细胞内的cccDNA。这样做的目的 本申请旨在研究与cccDNA生物学相关的四个问题： cccDNA形成，阐明cccDNA形成的确切机制，研究cccDNA是如何 在肝细胞再生过程中分布到子细胞，并决定肝细胞的物理组织。 感染肝细胞中的cccDNA。对cccDNA形成机制的研究可以揭示 细胞DNA修复酶，反过来，可以成为新的抗病毒治疗的目标。调查 cccDNA在受感染细胞核中的物理位置可以阐明cccDNA是如何维持的， 在细胞分裂后分布到子细胞，以及在自发恢复过程中如何最终丢失 自然感染HBV。更好地了解这些过程被认为是发展的关键 治疗慢性B型肝炎（CH B）的新策略。 目标1. cccDNA合成的机制。我们将确定仍然难以捉摸的细胞酶负责 将rc转化为cccDNA。候选物是DNA连接酶和核酸内切酶，其是加工所需的 以及连接两条rcDNA链的5 ′和3 ′末端。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. cccDNA在细胞分裂和核定位期间的命运。这一目标建立在我们的能力， 通过荧光原位杂交（FISH）检测中期和间期核中的cccDNA。目标是 确定cccDNA在DHBV和HBV产生细胞中的分布，并研究cccDNA是如何在DHBV和HBV产生细胞中表达的。 在正常条件下和在细胞因子存在下分布到子细胞。实验设计 研究功能性和转录沉默的cccDNA的核定位。 为了实现这些目标，我们将依靠一种实验性的方法，这种方法建立在我们在2008年期间的研究工作的基础上。 在过去的两年中，细胞培养平台允许HBV感染HepG 2细胞， 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.