Hepatitis B virus cccDNA

项目总结 全球约有3亿人慢性感染乙肝病毒。所有这些都显著增加了 患上肝细胞癌的风险。虽然病毒复制可以通过核苷治疗来阻止 类似地，感染的肝细胞不能治愈，因为共价闭合环状DNA(CccDNA)，模板为 病毒RNA的转录在受感染的细胞中持续存在。到目前为止，预防cccDNA形成的治疗策略 或者在感染的肝细胞内缺乏功能失活甚至破坏的cccDNA。这样做的目的是 应用是研究与cccDNA生物学有关的四个问题：鉴定所需的酶 CccDNA的形成，阐明cccDNA形成的确切机制，研究cccDNA是如何 肝细胞再生过程中对子细胞的分布及决定肝细胞的物理组织 在感染的肝细胞中发现ccDNA。对cccDNA形成机制的研究可以揭示其同源性。 细胞DNA修复酶反过来可能成为新的抗病毒疗法的靶点。关于调查的 CccDNA在感染细胞细胞核中的物理位置可能有助于阐明cccDNA是如何维持和 在细胞分裂后分配到子细胞，以及在自发恢复过程中最终如何丢失 不受自然的乙肝病毒感染。对这些过程的更好理解被认为是发展的关键 治疗慢性乙型肝炎(CHB)的新策略。 目的1.cccDNA的合成机制。我们将确定仍然难以捉摸的细胞酶负责 RC向cccDNA的转化。候选的是DNA连接酶(S)和内切酶(S)，这是处理所需的 以及两条rcDNA链的5‘和3’末端的连接。基因敲除细胞将被用来确定 将rcDNA转化为cccDNA。此外，我们还建议开发一种新的筛查方法 乙肝病毒感染和cccDNA形成所需宿主基因的鉴定。 目的2.cccDNA在细胞分裂和核定位过程中的去向。这个目标建立在我们视觉化能力的基础上 用荧光原位杂交技术(FISH)检测中期和间期细胞核中的CCDNA。我们的目标是 检测cccDNA在DHBVDNA和乙肝病毒产生细胞中的分布，并研究cccDNA是如何 在正常情况下和在细胞因子存在的情况下分配给子细胞。设计了实验 目的：研究转录沉默和功能性cccDNA的核定位。 为了实现这些目标，我们将依靠一种实验方法，这种方法建立在我们在 过去两年导致细胞培养平台允许乙肝病毒感染HepG2细胞与 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的基础上 方法采用FISH技术检测cccDNA。

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.