Single-Particle Analysis of Virus Capsid Assembly and Disassembly by Resistive-Pulse Sensing

通过电阻脉冲传感对病毒衣壳组装和拆卸进行单粒子分析

• 批准号: 9751353
• 负责人: Stephen C Jacobson
• 金额: $ 30.09万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9751353
• 关键词: Address; Antiviral Agents; Biological; Buffers; Capsid; Capsid Proteins; Charge; Complex; Computer Simulation; Data; Detection; Development; Devices; Dissociation; Evolution; Feedback; Geometry; Glass; Hepatitis B Virus; In Vitro; Individual; Ions; Kinetics; Knowledge; Label; Liver diseases; Mass Spectrum Analysis; Measurement; Measures; Methods; Modeling; Molecular; Monitor; Nanotechnology; Nanovirus; Pathway interactions; Physiologic pulse; Play; Polymers; Process; Proteins; Reaction; Replication-Associated Process; Resolution; Roentgen Rays; Role; Sampling; Sodium Chloride; Structure; System; Techniques; Time; Transmission Electron Microscopy; Viral; Viral Genome; Virus; Virus Assembly; Work; base; cost; dimer; experimental study; in vivo; instrument; instrumentation; interest; light scattering; multiplex detection; nanobiotechnology; nanofluidic; nanoimprinting; particle; recruit; response; self assembly; small molecule

Project Summary A typical virus capsid consists of hundreds of copies of capsid protein that act as the protective package of the viral genome. Therefore, the typical capsid assembly reaction will have hundreds of steps, each one of which can go wrong. Yet, even in vitro, self-assembly of virus capsids can occur spontaneously and with high fidelity. This reaction is of fundamental interest to virologists, a focus of antiviral development, and a general model of self-assembly with implications for harnessing viruses for nano- and biotechnology. Understanding the mechanism of virus assembly requires not only knowledge of precursors and final products, but also access to intermediates. Where many rare intermediates are involved, ensemble methods obscure them so that virus assembly resembles a two-state reaction, i.e., only subunits and capsids are observed. Computational models of assembly suggest that the observed kinetics reflect establishment of early intermediates needed to support capsid formation. The nucleation step and these early intermediates are believed to play a role in recruiting viral components in vivo. In our previous work, we established hepatitis B virus (HBV) assembly as a well-defined and robust experimental system for interrogating assembly reactions. HBV capsids, composed of 120 homodimers, self-assemble in response to buffer conditions. Surprisingly, we found metastable intermediates in assembly and disassembly. These results are timely as HBV capsid assembly has become an important target for development of antiviral assembly effectors which over- stimulate nucleation, distorting the distribution of intermediates and often their structure. Resistive-pulse sensing on in-plane nanofluidic devices is a unique platform and permits a label-free, single-particle approach to monitor assembly in real time at biologically relevant concentrations (nM to µM). Our resistive-pulse measurements have provided highly complementary data to other state-of-the-art techniques, e.g., time-resolved small angle x-ray scattering, light scattering, charge detection mass spectrometry, and transmission electron microscopy. All of these approaches require much higher protein concentrations than resistive-pulse sensing and, thus, obscure many features of these complex reactions. We have developed needed fabrication methods, characterized individual HBV capsids, and monitored their assembly below, near, and above the pseudo-critical dimer concentration. Because of our ability to probe single particles in real time and over a range of assembly conditions, we are now poised to address a number of questions, previously thought unanswerable. The specific aims for this application are to: (1) integrate on- device mixing and multiplexed detection to probe early time points of assembly; (2) compare assembly of virus capsids with and without assembly effectors; (3) evaluate capsid assembly and disassembly in the presence of chaotropes; (4) monitor the evolution of incomplete particles; and (5) fabricate nanoimprinted in- plane nanofluidic devices for assembly and disassembly experiments.

项目摘要 典型的病毒capsID由数百份的衣壳蛋白组成，可作为保护包装 病毒基因组。因此，典型的衣壳装配反应将具有数百个步骤，每个步骤 可能会出错。然而，即使在体外，病毒capsids的自组装也可能会发起，并且很高 忠诚。这种反应是病毒学家的基本兴趣，抗病毒发育的重点以及一般 自组装模型，对利用病毒对纳米和生物技术的影响有影响。 了解病毒装配的机制不仅需要前体知识和最终知识 产品，但也可以使用中间体。涉及许多稀有中间体的地方，合奏方法 掩盖它们，使病毒组装类似于两态反应，即仅亚基和衣壳是 观察到。组装的计算模型表明，观察到的动力学反映了早期的建立 支撑衣壳形成所需的中间体。核步骤和这些早期中间体是 被认为在体内招募病毒成分中发挥作用。在我们以前的工作中，我们建立了乙型肝炎 病毒（HBV）组装是一种质疑组装反应的明确且健壮的实验系统。 HBV衣壳由120个同型二聚体组成，响应缓冲液条件而自组装。令人惊讶的是，我们 在组装和拆卸中发现了亚稳态中间体。这些结果及时为HBV CAPSID 组装已成为开发抗病毒装配效应的重要目标，该效应过度 刺激成核，扭曲中间体的分布及其结构。 平面纳米流体设备上的电阻脉冲传感是一个独特的平台，允许无标签， 单粒子在生物学相关浓度（NM至µM）以实时监测组装的方法。 我们的抵抗性脉冲测量为其他最先进的数据提供了高度完善的数据 技术，例如时间分辨的小角度X射线散射，光散射，电荷检测质量 光谱法和透射电子显微镜。所有这些方法都需要更高的蛋白质 浓度比电阻脉冲敏感性​​，因此掩盖了这些复杂反应的许多特征。 我们已经开发了所需的制造方法，表征了单个HBV衣壳，并监视了它们 组装下面，近距离和高于伪二聚体浓度。因为我们有能力证明 单个粒子是实时的，在各种装配条件下，我们现在被毒死以解决一个数字 问题，以前认为无法回答。该应用程序的具体目的是：（1）集成在 - 设备混合和多路复用检测，以探测组装的早期时间点； （2）比较组件 具有和没有组装作用的病毒衣壳； （3）评估CAPSID组装和拆卸 存在交际； （4）监测不完全颗粒的演变； （5）制造纳米膜印刷 用于组装和拆卸实验的平面纳米流体设备。