Hepatitis B virus cccDNA

乙型肝炎病毒cccDNA

基本信息

批准号：
9764249
负责人：
Christoph Seeger
金额：
$ 45.75万
依托单位：
RESEARCH INST OF FOX CHASE CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-15 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9764249
关键词：
Address Antiviral Therapy Biological Assay Biology CRISPR/Cas technology Cell Culture Techniques Cell Nucleus Cell division Cells Chronic Chronic Hepatitis B Circular DNA Clinical DNA DNA Ligases DNA Repair Enzymes DNA biosynthesis Detection Development Duck Hepatitis B Virus Enzymes Fluorescent in Situ Hybridization Future Gene Silencing Genes Genetic Transcription Goals HepG2 Hepatitis B Virus Hepatocyte Image In Situ Hybridization Infection Interphase Investigation Knock-out Life Light Location Metaphase Methods Natural regeneration Nuclear Patients Play Primary carcinoma of the liver cells Process Proteins Public Health Recovery Research Risk Role System Technology Therapeutic Virus Replication cytokine daughter cell design endonuclease experimental study knockout gene novel novel strategies nucleoside analog prevent screening success viral RNA

项目摘要

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.

项目概要乙型肝炎病毒长期感染全球约 3 亿人，且感染人数均显着增加。尽管核苷治疗可以阻止病毒复制，但仍存在发生肝细胞癌的风险。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，目前尚有阻止 cccDNA 形成的治疗策略。或功能性失活甚至破坏受感染肝细胞内的 cccDNA 的目的是缺乏的。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研究目标。 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更好地了解这些过程对于发展至关重要。治疗慢性乙型肝炎（CHB）的新策略。目标 1. cccDNA 合成机制我们将鉴定仍然难以捉摸的负责 cccDNA 合成的细胞酶。 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。两条 rcDNA 链的 5' 和 3' 末端的连接将用于确定如何连接。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的 cccDNA 命运该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。确定 cccDNA 在 DHBV 和 HBV 产生细胞中的分布并研究 cccDNA 的分布设计了在正常条件下和存在细胞因子的情况下分配给子细胞的实验。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在研究期间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 感染相结合此外，这些目标建立在我们最近成功开发的基础上。允许通过 FISH 检测 cccDNA 的方法。