The Biophysics of Virus Capsid Assembly

病毒衣壳组装的生物物理学

• 批准号: 8076332
• 负责人: Adam Zlotnick
• 金额: $ 29.21万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-10 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8076332
• 关键词: Address; Affect; Allosteric Regulation; Antiviral Agents; Behavior; Binding; Biophysics; Capsid; Capsid Proteins; Cells; Cessation of life; Chronic; Chronic Hepatitis B; Cirrhosis; Complement; Complex; Core Protein; Cultured Cells; Cytoplasm; DNA Viruses; Data; Enzymes; Escherichia coli; Family; Frequencies; Future; Future Generations; Genome; Hepatitis B Virus; Homologous Gene; In Vitro; Infection; Integration Host Factors; Kinetics; Lead; Length; Liver Cirrhosis; Molecular Machines; Mutation; Nucleic Acid Binding; Nucleic Acids; Phenotype; Phosphorylation; Physical Examination; Play; Primary carcinoma of the liver cells; Process; Proteins; RNA; RNA Binding; RNA Recognition Motif; RNA-Directed DNA Polymerase; Reaction; Regulation; Regulatory Element; Resistance; Reverse Transcription; Risk Factors; Role; Series; Site; Specificity; Structure; Testing; Theoretical Studies; Therapeutic; Thermodynamics; Time; Tube; Viral; Viral Packaging; Virus; Virus Assembly; Virus Replication; base; chaperonin; design; dimer; human APEX1 protein; in vivo; mutant; prevent; protein protein interaction; research study; small molecule; trafficking; viral RNA; virus core

DESCRIPTION (provided by applicant): In an infected cell, Hepatitis B virus (HBV) cores assemble in the cytoplasm. These cores are comprised of an icosahedral capsid of core protein (Cp) containing a reverse transcriptase-pregenomic RNA complex and associated chaperonins. We show that the dimeric Cp has a strong propensity to self-assemble in vitro and will bind any nucleic acid. Assembly at the wrong time or place, encapsidating the wrong nucleic acid or complement of proteins, would lead to non-productive infections. Preliminary data support our hypothesis that assembly can be allosterically triggered. We will use biophysical and structural approaches to examine assembly in vitro. In aim 1, we will examine the intra-dimer interface (within a Cp dimer) for evidence of allosteric transitions. Mutations at this interface have dramatic effects on assembly though they are not involved in the inter-dimer contacts that hold a capsid together. In aim 2, we will dissect that inter-dimer contact. Theoretical studies indicate that tight binding will lead to kinetic traps and thus interfere with capsid assembly. We will examine mutants that enhance and inhibit assembly, quantifying assembly in vitro and observing phenotype in cultured cells. In aim 3, we will examine how regulation of assembly plays a role in the formation of RNA-filled capsids. When over-expressed in E. coli, HBV Cp packages random bacterial nucleic acid. By examining mechanisms of regulating assembly and assembly on random and viral nucleic, we address whether there is an intrinsic mechanism for specificity in vivo. These experiments will lead to examination of the physical and structural basis of Cp assembly. By considering Cp as a molecular machine, we will be able to identify regulatory elements in the structure that may be targets for interfering with assembly and thus the HBV lifecycle.

描述（由申请人提供）：在受感染的细胞中，乙型肝炎病毒（HBV）核心在细胞质中组装。这些核心由核心蛋白 (Cp) 的二十面体衣壳组成，其中含有逆转录酶-前基因组 RNA 复合物和相关的伴侣蛋白。我们发现二聚体 Cp 具有很强的体外自组装倾向，并且可以结合任何核酸。在错误的时间或地点组装，封装错误的核酸或蛋白质补体，会导致非生产性感染。初步数据支持我们的假设，即组装可以被变构触发。我们将使用生物物理和结构方法来检查体外组装。在目标 1 中，我们将检查二聚体内界面（Cp 二聚体内）以获取变构转变的证据。该界面上的突变对组装具有显着影响，尽管它们不涉及将衣壳固定在一起的二聚体间接触。在目标 2 中，我们将剖析二聚体间的接触。理论研究表明，紧密结合将导致动力学陷阱，从而干扰衣壳组装。我们将检查增强和抑制组装的突变体，量化体外组装并观察培养细胞中的表型。在目标 3 中，我们将研究组装调控如何在 RNA 填充衣壳的形成中发挥作用。当在大肠杆菌中过度表达时，HBV Cp 会包装随机细菌核酸。通过检查随机和病毒核酸上调节组装和组装的机制，我们解决了体内是否存在特异性的内在机制。这些实验将导致对 Cp 组装的物理和结构基础的检查。通过将 Cp 视为分子机器，我们将能够识别结构中的调控元件，这些元件可能是干扰组装的目标，从而干扰 HBV 生命周期。