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

项目摘要 一个典型的病毒衣壳由数百个衣壳蛋白拷贝组成，它们起着保护性包装的作用 病毒基因组。因此，典型的衣壳组装反应将具有数百个步骤，每一个步骤 这可能会出错然而，即使在体外，病毒衣壳的自组装也可以自发地发生，并且具有很高的生物学活性。 忠诚这种反应引起了病毒学家的根本兴趣，是抗病毒药物开发的焦点，也是一般的 自组装模型与利用纳米和生物技术的病毒的影响。 了解病毒组装的机制不仅需要了解前体和最终的 产品，而且还可以获得中间体。在涉及许多稀有中间体的情况下， 模糊它们，使病毒组装类似于两态反应，即，只有亚基和衣壳 观察组装的计算模型表明，观察到的动力学反映了早期组装的建立。 支持衣壳形成所需的中间体。成核步骤和这些早期的中间体是 被认为在体内募集病毒成分中起作用。在我们之前的工作中，我们建立了B型肝炎 病毒（HBV）组装作为一个明确的和强大的实验系统，询问组装反应。 HBV衣壳由120个同源二聚体组成，在缓冲液条件下自组装。令人惊讶的是，我们 在组装和分解过程中发现了亚稳态中间体。这些结果是及时的， 组装已经成为开发抗病毒组装效应物的重要靶点， 刺激成核，扭曲中间体的分布，往往是它们的结构。 平面内纳米流体装置上的电阻脉冲感测是一种独特的平台， 单粒子方法，以生物相关浓度（nM至µM）真实的时间监测组装。 我们的连续脉冲测量为其他最先进的 技术，例如，时间分辨小角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 浓度比连续脉冲感测低，因此模糊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，并监测了它们的 在低于、接近和高于伪临界二聚体浓度的情况下组装。因为我们的探测能力 单粒子在真实的时间和一系列的组装条件，我们现在准备解决一些 以前认为无法回答的问题。本申请的具体目标是：（1）整合- 装置混合和多重检测以探测组装的早期时间点;（2）比较 （3）评估病毒衣壳的装配和拆卸， 离液剂的存在;（4）监测不完整颗粒的演变;和（5）制造纳米压印在- 平面纳米流体装置的组装和拆卸实验。

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.