Multimode Observation of Virus Capsid Assembly

病毒衣壳组装的多模式观察

基本信息

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
项目类别：
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

项目摘要

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，但组装机制仍然是一个黑匣子，尽管组装对于生物学来说至关重要。定位、核酸包装和新型装配导向抗病毒药物（有时被描述为装配抑制剂，尽管它们实际上促进了错误装配）迄今为止，几乎所有装配观察都依赖于整体。同样，几乎所有的组装模型都依赖于关于溶液中的亚基状态、不完整衣壳中的亚基状态以及参与组装的中间体列表的临时假设。涉及突变体和突变体组合的乙型肝炎病毒 (HBV) 组装的体外实验将通过与细胞培养物中的组装进行比较来验证。 T=4 HBV 衣壳将涉及相对少量的中间体，如果是这种情况，则会立即产生影响。病毒 RNA 的组织，在体内组装过程中进行包装。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）和单颗粒来表征 HBV 衣壳自组装。电荷检测质谱（Jarrold 实验室）的观察结果表明了实验的可行性，并表明了一些受青睐的中间体的存在。这些结果将有助于开发忠实地复制组装和组装的数学模型。 HBV 是最小的人类病原体（3200 bp DNA 基因组），也是传播最广泛的病原体之一，每年约有 3.5 亿人患有慢性 HBV，导致约 60 万人死亡。一些制药公司正在研究 HBV 衣壳蛋白作为抗病毒靶标，有可能实现功能性治愈。