Regulation of Hepatitis B Virus Capsid Assembly

乙型肝炎病毒衣壳组装的调控

基本信息

批准号：
9357504
负责人：
Jianming Hu
金额：
$ 37.85万
依托单位：
PENNSYLVANIA STATE UNIV HERSHEY MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-23 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9357504
关键词：
Alpha Cell Antiviral Agents Arginine Bacteria Binding C-Peptide C-terminal CDK2 gene Capsid Capsid Proteins Cell Extracts Cell-Free System Cells Chronic Chronic viral hepatitis Cirrhosis Complex Core Protein Cyclins DNA DNA Binding DNA Viruses Development Genome Hepatitis Hepatitis B In Vitro Insecta Integration Host Factors Life Cycle Stages Liver Cirrhosis Liver diseases Malignant neoplasm of liver Mammalian Cell Mediating Methods N-terminal Nucleocapsid Oryctolagus cuniculus Peptides Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Physiological Play Process Protamines Protein Dephosphorylation Protein Kinase Protein phosphatase Proteins RNA RNA Binding RNA-Directed DNA Polymerase Reaction Regulation Reticulocytes Reverse Transcription Risk Role System Viral Viral Core Proteins Viral Packaging Viral Proteins Viral Reverse Transcription Virion Virus Virus Assembly Virus Diseases Virus Replication base chronic liver disease insight novel overexpression pathogen protein protein interaction targeted agent virus envelope

项目摘要

Abstract Hepatitis B virus (HBV) is a major cause of chronic viral hepatitis that increases dramatically the risk of liver cancer and other end-stage liver diseases such as cirrhosis. HBV is a small DNA virus and replicates its DNA genome via reverse transcription of a RNA intermediate called the pregenomic RNA (pgRNA). Viral replication depends critically on the assembly of a nucleocapsid (NC) that is composed of the viral core or capsids protein (HBc) and encapsidates a copy each of pgRNA and the reverse transcriptase (RT), which converts the RNA pregenome to the DNA genome within the NC. Viral capsids also encapsidate the host cyclin-dependent kinase 2 (CDK2) via unknown mechanisms. Challenging the current dogma that capsid assembly depends solely on the N-terminal domain (NTD) of HBc, we have recently discovered that under physiological conditions, capsid assembly critically depends on the C-terminal domain (CTD) of HBc. Furthermore, we have developed a mammalian cell-free system that recapitulates CTD-dependent capsid assembly and further regulates capsid assembly through host-mediated CTD phosphorylation and dephosphorylation. The cell-free capsid assembly system also recapitulates the specific encapsidation of CDK2. Building on these developments, we propose to dissect the role of CTD, and its state of phosphorylation as regulated by cellular protein kinases and phosphatases, in capsid assembly (Specific Aim 1). We also plan to elucidate the HBc and CDK2 requirements for CDK2 encapsidation (Specific Aim 2). Furthermore, we have recently developed methods to isolate the viral pgRNA in complex with RT, which is the substrate recognized by HBc during NC assembly to achieve specific encapsidation of pgRNA and RT. We now propose to combine the isolation of the pgRNA-RT complex together with the cell-free capsid assembly system to develop cell-free systems for the specific packaging of pgRNA (and RT) into NCs (Specific Aim 3).

抽象的乙型肝炎病毒 (HBV) 是慢性病毒性肝炎的主要原因，可显着增加肝病风险癌症和其他终末期肝脏疾病，例如肝硬化。 HBV 是一种小型 DNA 病毒，可以复制其 DNA 基因组通过称为前基因组 RNA (pgRNA) 的 RNA 中间体的逆转录来实现。病毒复制关键取决于由病毒核心或衣壳蛋白组成的核衣壳 (NC) 的组装 (HBc) 并封装 pgRNA 和逆转录酶 (RT) 各一个副本，该酶将 RNA 转化为 NC 内的前基因组到 DNA 基因组。病毒衣壳也依赖于细胞周期蛋白来包裹宿主激酶 2 (CDK2) 通过未知机制。挑战当前衣壳组装所依赖的教条仅在HBc的N端结构域（NTD）上，我们最近发现在生理条件下在条件下，衣壳组装关键取决于 HBc 的 C 端结构域 (CTD)。此外，我们还有开发了一种哺乳动物无细胞系统，可概括 CTD 依赖性衣壳组装，并进一步通过宿主介导的 CTD 磷酸化和去磷酸化调节衣壳组装。无细胞的衣壳组装系统也概括了CDK2的特定衣壳化。在此基础上随着进展，我们建议剖析 CTD 的作用及其受细胞调节的磷酸化状态衣壳组装中的蛋白激酶和磷酸酶（具体目标 1）。我们还计划阐明 HBc 和 CDK2 封装的 CDK2 要求（具体目标 2）。此外，我们最近还开发了分离病毒 pgRNA 与 RT 复合物的方法，RT 是 NC 期间 HBc 识别的底物组装以实现 pgRNA 和 RT 的特异性衣壳化。我们现在建议将隔离结合起来 pgRNA-RT 复合物与无细胞衣壳组装系统一起开发无细胞系统将 pgRNA（和 RT）特定包装到 NC 中（具体目标 3）。