Multimode Observation of Virus Capsid Assembly

病毒衣壳组装的多模式观察

• 批准号: 9116986
• 负责人: Adam Zlotnick
• 金额: $ 45.28万
• 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 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9116986
• 关键词: Antiviral Agents; Biochemical; Biochemistry; Biology; Boxing; Buffers; Capsid; Capsid Proteins; Cell Culture Techniques; Cessation of life; Charge; Chronic Hepatitis B; Complex; Computer Simulation; Cryoelectron Microscopy; Custom; DNA; Data; Detection; Development; Electron Microscopy; Evolution; Family; Fluorescence; Genes; Genome; Health; Hepatitis B Virus; Human; In Vitro; Indiana; Infection; Intervention; Ions; Kinetics; Lead; Maps; Mass Spectrum Analysis; Microscopy; Modeling; Monitor; Nanotechnology; 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; computerized tools; dimer; in vivo; inhibitor/antagonist; instrument; light scattering; mathematical model; millisecond; mutant; nucleic acid localization; particle; pathogen; reconstruction; research study; 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（由适用提供）：在大约一半已知病毒家族中发现自组装二十面体capsids。然而，即使组装是生物学的基础，装配机制仍然是黑匣子。例如，组装对细胞定位，核酸包装和新的组装抗病毒药物至关重要（有时将其描述为装配抑制剂，尽管它们实际上促进了错误组装）。日期，几乎所有的组装观察都取决于结构不确定的复合物的集合。同样，几乎所有的组装模型都取决于有关解决方案中亚基状态，不完整的衣壳中的亚基状态以及参与汇编的中间体列表的临时假设。我们提出了丙型肝炎病毒（HBV）组装的详细分析。在体外实验中，通过与细胞培养中的组装相比，将涉及突变体和突变体的组合。基于建模研究，我们假设120二聚体T = 4 HBV衣壳的组装将涉及相对较少的中间体。如果是这种情况，它对 病毒RNA的组织，体内在组装过程中包装。在此提案中，我们将使用生化技术和电子显微镜（Zlotnick，Wang Labs），时间分辨的SAXS（Raviv Lab）以及通过电荷检测质谱法（Jarrold Lab）（Jarrold Lab）来表征HBV CAPSID自组装（Zlotnick，Wang Labs），时间分辨率的SAXS（RAVIV LAB）和单个粒子观察。初步数据显示了实验的可行性，并表明存在一些有利的中间体。这些结果将有助于忠实地复制组装的数学模型的发展，并可以帮助识别干扰正常组装的显着点。 HBV是最小的人类病原体（3200 bp DNA基因组），也是最广泛的。大约3.5亿人患有慢性HBV，每年约60万人死亡。当前的疗法抑制但不能治愈感染。几家制药公司正在调查HBV胶囊蛋白作为一种抗病毒靶标的，具有实现功能性治疗的潜力