Nanofluidic Devices for Studying Assembly of Single Virus Particles

描述(申请人提供)：我们将建立一般方法来确定病毒和自组装纳米颗粒的组装路径。我们将使用乙肝病毒(乙肝)衣壳组装来演示这些方法。电阻脉冲传感通过识别单个颗粒和组装中间体，实现了对组装的实时监控。了解病毒组装的机制不仅需要了解前体和最终产品结构，还需要获得中间体。在涉及许多稀有中间体的地方，集合方法使它们变得模糊，因此病毒组装类似于两种状态的反应。乙肝病毒已急性感染了超过2B人；约3.6亿人患有慢性乙肝；每年有近100万人死于与乙肝病毒相关的肝病。乙肝病毒二十面体衣壳的组装已被确定为抗病毒治疗的新靶点。乙肝病毒组装和抗病毒组装效应器的行为(和发育)相对较好，主要源于Zlotnick实验室的工作，但还没有直接观察到和表征溶液中关键的早期中间体。组装的计算模型表明，观察到的动力学反映了支持衣壳形成所需的中间体星座的早期建立。核化步骤和早期中间产物被认为在体内招募病毒成分方面发挥了作用。抗病毒组装效应器过度刺激核化，扭曲中间产物的分布，经常扭曲它们的结构。在我们以前的工作中，我们建立了一个定义明确的纳米流体实验系统，现在有了询问组装和抗病毒组装效应器的基础。与微流体设备集成的纳米流体组件为回答这些悬而未决的问题提供了一个独特的平台。这些设备上的电阻脉冲传感允许实时、无标签的方法来监测生物相关浓度(NM到Mm)下的组装。更具体地说，我们将开发设备和方法来研究粒子通过纳米通道网络的传输特性。这些耦合的纳米通道几乎可以以任何二维形式排列，并以适度的应用潜力操作。粒子传输如何受到影响很大程度上取决于纳米管道的尺寸和几何形状、施加的波形、管道的表面性质、传输介质的组成以及粒子的形状和组成。我们将优化这些设备参数，以便对衣壳的形成有一个基本的了解。这一应用的具体目标是：(1)表征不同反应条件下的衣壳组装；(2)制备和测试单孔和多孔串联的面内纳米通道，以改进阻性脉冲传感；(3)计算模拟(I)组装和(Ii)粒子在纳米流体器件中的传输；以及(4)开发耦合纳米通道来感测不同尺寸的颗粒并与单个衣壳进行反应。 与公共卫生相关：在这个项目中，我们将建立确定病毒组装途径的一般方法，特别强调核化和中间形成，以及抗病毒组装效应物如何影响这些过程。组装的早期步骤被认为在体内招募病毒成分方面起着至关重要的作用，而病毒衣壳的组装，如乙肝病毒(HBV)，已被确定为抗病毒治疗的新靶点。乙肝病毒已急性感染超过2B人~约3.6亿慢性乙肝患者~每年将有近100万人死于乙肝相关肝病。

DESCRIPTION (provided by applicant): We will establish general methods to determine the assembly pathways of viruses and self- assembling nanoparticles. We will demonstrate these methods using Hepatitis B virus (HBV) capsid assembly. Resistive-pulse sensing allows real time monitoring of assembly by identifying single particles and assembly intermediates. Understanding the mechanism of virus assembly requires not only knowledge of precursors and final product structures, but also access to intermediates. Where many rare intermediates are involved, ensemble methods obscure them so that virus assembly resembles a two-state reaction. HBV has acutely infected more than 2B people; about 360M people have chronic HBV; every year nearly 1M will die of HBV-related liver disease. Assembly of HBV's icosahedral capsid has been identified as a new target for antiviral therapies. HBV assembly and the behavior (and development) of antiviral assembly effectors are relatively well understood, largely stemming from work in the Zlotnick lab, but there has been no direct observation and characterization of critical early intermediates in solution. Computational models of assembly suggest that the observed kinetics reflect early establishment of a constellation of intermediates needed to support capsid formation. The nucleation step and early intermediates are believed to play a role in recruiting viral components in vivo. Antiviral assembly effectors over-stimulate nucleation, distorting the distribution of intermediates and very often their structure. In our previous work, we established HBV assembly as a well-defined experimental system for nanofluidics and now have the foundation to interrogate assembly and antiviral assembly effectors. Nanofluidic components integrated with microfluidic devices offer a unique platform for answering these outstanding questions. Resistive-pulse sensing on these devices permits a real time, label-free approach to monitoring assembly at biologically relevant concentrations (nM to mM). More specifically, we will develop devices and methods to study particle transport properties through nanochannel networks. These coupled nanochannels can be arranged in virtually any two-dimensional format and operated with modest applied potentials. How particle transport is influenced depends strongly on the dimensions and geometries of the nanoscale conduits, applied waveforms, surface properties of the conduit, and composition of the transport medium, and particle shape and composition. We will optimize these device parameters in order to develop a fundamental understanding of capsid formation. The Specific Aims for this application are to: (1) characterize capsid assembly under various reaction conditions; (2) fabricate and test in-plane nanochannels with single pores and multiple pores in series for improved resistive-pulse sensing; (3) computationally simulate (i) assembly and (ii) particle transport in nanofluidic devices; and (4) develop coupled nanochannels to sense particles of different sizes and to perform reactions with single capsids. PUBLIC HEALTH RELEVANCE: In this project, we will establish general methods to determine the assembly pathways of viruses with particular emphasis placed on nucleation and intermediate formation and how antiviral assembly effectors impact these processes. The early steps in assembly are believed to play a crucial role in recruiting viral components in vivo, and assembly of virus capsids, such as Hepatitis B virus (HBV), has been identified as a new target for antiviral therapies. HBV has acutely infected more than 2B people~ about 360M people have chronic HBV~ every year nearly 1M will die of HBV-related liver disease.