Regulation of Hepatitis B Virus Capsid Assembly

乙型肝炎病毒衣壳组装的调控

• 批准号: 9213611
• 负责人: Jianming Hu
• 金额: $ 37.88万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 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/9213611
• 关键词: Antiviral Agents; Arginine; Bacteria; Binding; C-Peptide; C-terminal; CDK2 gene; Capsid; Capsid Proteins; Cell Extracts; Cell-Free System; Cells; Chronic viral hepatitis; Cirrhosis; Complex; Cyclins; DNA; DNA Binding; DNA Viruses; Development; Genome; Hepatitis; Hepatitis B Virus; 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; Staging; System; Viral; Viral Packaging; Viral Proteins; Viral Reverse Transcription; Virion; Virus; Virus Assembly; Virus Diseases; Virus Replication; abstracting; base; chronic liver disease; insight; novel; overexpression; pathogen; protein protein interaction; virus core

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).

摘要 乙肝病毒(乙肝病毒)是慢性病毒性肝炎的主要原因，它极大地增加了患肝脏的风险 癌症和其他终末期肝病，如肝硬变。乙肝病毒是一种小的DNA病毒，可以复制它的DNA 通过逆转录一种称为前基因组RNA(PgRNA)的RNA中间体来获得基因组。病毒复制 关键依赖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)的每个副本，逆转录酶(RT)转换RNA 前基因组到NC内的DNA基因组。病毒衣壳还包裹依赖于细胞周期蛋白的宿主 激酶2(CDK2)通过未知的机制被激活。挑战当前的教条，即船体组装依赖于 仅在HBC的N-末端结构域(NTD)上，我们最近发现在生理条件下 条件，衣壳组装关键取决于HBC的C-末端结构域(CTD)。此外，我们还拥有 开发了一种哺乳动物无细胞系统，它概括了依赖CTD的衣壳组装，并进一步 通过宿主介导的CTD磷酸化和去磷酸化来调节衣壳组装。无细胞的 衣壳组装系统还概括了CDK2的特异性包裹。建立在这些基础上 发展，我们建议剖析ctd的作用，以及它受细胞调控的磷酸化状态。 衣壳组装中的蛋白激酶和磷酸酶(特定目标1)。我们还计划澄清HBC和 对CDK2封装的要求(具体目标2)。此外，我们最近开发了一种 方法分离病毒pgRNA与RT的复合体，RT是NC过程中HBC识别的底物 组装以实现pgRNA和RT的特异性封装。我们现在建议将隔离 PgRNA-RT复合体与无细胞衣壳组装系统共同开发无细胞系统 将pgRNA(和RT)特定包装成NCS(特定目标3)。