Multimode Observation of Virus Capsid Assembly

病毒衣壳组装的多模式观察

• 批准号: 9900731
• 负责人: Adam Zlotnick
• 金额: $ 38.29万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-14 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9900731
• 关键词: Antiviral Agents; Biochemical; Biochemistry; Biology; Buffers; Capsid; Capsid Proteins; Cell Culture Techniques; Cessation of life; Charge; Chronic Hepatitis B; Complex; Computer Models; Cryoelectron Microscopy; Custom; DNA; Data; Detection; Development; Electron Microscopy; Evolution; Family; Fluorescence; Genes; Genome; Hepatitis B Virus; In Vitro; Indiana; Infection; Interruption; Intervention; Ions; Kinetics; Lead; Maps; Mass Spectrum Analysis; Modeling; Monitor; Nanotechnology; Nucleic Acids; Pathway interactions; Pharmacologic Substance; Population; Process; Reaction; Research; Research Personnel; Resolution; Structure; Study models; Techniques; Technology; Testing; Therapeutic; Time; Universities; Virus; Virus Assembly; Work; base; combinatorial; computerized tools; dimer; experimental study; human pathogen; in vivo; inhibitor/antagonist; instrument; light scattering; mathematical model; millisecond; mutant; particle; predictive modeling; public health relevance; reconstruction; self assembly; small molecule; theories; viral RNA

 DESCRIPTION (provided by applicant): Self-assembling icosahedral capsids are found in about half of known virus families. Yet, the mechanisms of assembly remain a black box, even though assembly is fundamental to biology. For example, assembly is critical to, cellular localization, nucleic acid packaging, and new classes of assembly-directed antivirals (sometimes described as assembly inhibitors though they actually promote mis-assembly). To date, almost all observations of assembly have depended on ensembles of structurally ill-defined complexes. Similarly, almost all models of assembly have depended on ad hoc assumptions regarding the state of subunits in solution, the state of subunits in incomplete capsids, and the list of intermediates participating in assembly. We propose a detailed analysis of the assembly of Hepatitis B Virus (HBV). In vitro experiments, involving mutants and combinations of mutants will be validated by comparison to assembly in cell culture. Based on modeling studies we hypothesize that assembly of the 120-dimer T=4 HBV capsid will involve a relatively small number of intermediates. If this is the case it has immediate implications for the organization of viral RNA, which in vivo is packaged during the assembly process. In this proposal we will characterize HBV capsid self-assembly using biochemical techniques and electron microscopy (Zlotnick, Wang labs), time resolved SAXS (Raviv Lab), and single particle observations by charge detection mass spectrometry (Jarrold Lab). Preliminary data show the feasibility of experiments and indicate the presence of some favored intermediates. These results will contribute to the development of mathematical models that faithfully replicate assembly and can help identify salient points for interfering with normal assembly. HBV is one the smallest human pathogens (3200 bp DNA genome) and one of the most widespread. About 350 million people have chronic HBV leading to about 600,000 deaths each year. Current therapeutics suppress but do not cure infection. Several pharmaceutical companies are investigating HBV capsid protein as an antiviral target with potential to achieve a functional cure

 描述（由申请人提供）：在大约一半的已知病毒家族中发现了自组装二十面体衣壳。然而，尽管组装是生物学的基础，但组装机制仍然是一个黑匣子。例如，组装对于细胞定位、核酸包装和新型组装导向抗病毒药物（有时被描述为组装抑制剂，尽管它们实际上会促进错误组装）至关重要。迄今为止，几乎所有对组装的观察都依赖于结构不明确的复合体的集合。同样，几乎所有组装模型都依赖于关于溶液中亚基状态、不完整衣壳中亚基状态以及参与组装的中间体列表的临时假设。我们建议对乙型肝炎病毒（HBV）的组装进行详细分析。涉及突变体和突变体组合的体外实验将通过与细胞培养中的组装进行比较来验证。基于模型研究，我们假设 120-二聚体 T=4 HBV 衣壳的组装将涉及相对少量的中间体。如果情况属实，将会直接影响 病毒RNA的组织，在体内组装过程中被包装。在本提案中，我们将使用生化技术和电子显微镜（Zlotnick、Wang 实验室）、时间分辨 SAXS（Raviv 实验室）以及通过电荷检测质谱法（Jarrold 实验室）进行单粒子观察来表征 HBV 衣壳自组装。初步数据显示了实验的可行性，并表明存在一些受欢迎的中间体。这些结果将有助于开发忠实地复制装配的数学模型，并有助于识别干扰正常装配的显着点。 HBV 是最小的人类病原体之一（DNA 基因组为 3200 bp），也是最广泛的病原体之一。大约 3.5 亿人患有慢性乙型肝炎，每年导致大约 60 万人死亡。目前的治疗方法可以抑制但不能治愈感染。多家制药公司正在研究 HBV 衣壳蛋白作为抗病毒靶点，有可能实现功能性治愈