The Biophysics of Virus Capsid Assembly

病毒衣壳组装的生物物理学

• 批准号: 8277332
• 负责人: Adam Zlotnick
• 金额: $ 29.16万
• 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/8277332
• 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

6. Summary 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.

6.总结 在受感染的细胞中，B型肝炎病毒（HBV）核心聚集在细胞质中。这些核心由 含有逆转录酶-前基因组RNA复合物的核心蛋白（Cp）的二十面体衣壳，和 相关的伴侣蛋白。我们表明，二聚Cp具有很强的倾向，在体外自组装， 将结合任何核酸。在错误的时间或地点组装，扩增错误的核酸或 补充蛋白质，会导致非生产性感染。初步数据支持我们的假设 这种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。在目的1中，我们将检查二聚体内界面（Cp二聚体内），以获得以下证据： 变构转变在这个界面上的突变对组装有戏剧性的影响，尽管它们不是 参与将衣壳结合在一起的二聚体间接触。在aim 2中，我们将分析 contact.理论研究表明，紧密结合将导致动力学陷阱，从而干扰衣壳 组装件.我们将研究增强和抑制组装的突变体，在体外定量组装， 观察培养细胞的表型。在目标3中，我们将研究组装的调节如何在以下方面发挥作用： RNA填充的衣壳的形成。当在E.大肠杆菌、HBV Cp包装随机细菌 核酸通过研究随机和病毒核酸上的调节组装和组装的机制， 我们研究是否存在体内特异性的内在机制。 这些实验将导致检查的物理和结构基础的Cp组装。通过 考虑到Cp是一个分子机器，我们将能够识别结构中的调节元件， 可能是干扰组装的靶点，从而干扰HBV的生命周期。