Mapping the assembly pathways for viral capsids by direct single-particle measurements

项目摘要 许多RNA病毒的衣壳在细胞质中自发地围绕病毒RNA组装， 被感染的宿主细胞绘制出组装路径--也就是说， 最后的感染结构-是开发抗病毒药物的第一步， 致病病毒的传播。现有的实验技术，测量在一个整体中发生了什么 衣壳的溶液而不是单个衣壳，模糊了基本的动力学。因此，我们缺乏答案， 关于衣壳如何形成的基本问题，例如速率限制步骤是成核还是生长，或者 外壳蛋白在最初无序附着于RNA后的重排。 纳米科学的新工具可以应对这些挑战。该提案描述了一项衡量和 了解衣壳组装体外单衣壳规模：一个强大的纳米成像技术，称为 应用干涉散射（iSCAT）显微镜来监测单个病毒衣壳在 单个RNA链。这些测量的数据可用于构建装配的新模型 这条途径以前所未有的细节描述了这一过程。特别关注的是病毒RNA的作用， 引导这些路径。新的基于DNA的技术被开发出来，以解开复杂的折叠模式， 病毒RNA和选择性地探测特定的RNA-蛋白质相互作用，已经提出发挥作用， 在衣壳装配中。快速纳米成像和基于DNA的方法的组合方法， 操纵特定的分子相互作用可以揭示阻断衣壳组装的意想不到的途径， 对抗病毒性疾病。

Project Summary The capsids of many RNA viruses spontaneously assemble around the viral RNA in the cytoplasm of an infected host cell. Mapping out the assembly pathway—that is, the set of molecular rearrangements that produce the final infectious structure—is the first step toward developing antiviral drugs that block the process and halt the spread of pathogenic viruses. Available experimental techniques, which measure what happens in a bulk solution of capsids and not in individual capsids, obscure the essential kinetics. As a result, we lack answers to basic questions about how capsids form, such as whether the rate limiting step is nucleation, or growth, or the rearrangement of coat proteins following an initial disordered attachment to the RNA. New tools from nanoscience can tackle these challenges. This proposal describes a plan to measure and understand capsid assembly in vitro at the single-capsid scale: a powerful nanoscale imaging technique called interferometric scattering (iSCAT) microscopy is applied to monitor the growth of individual viral capsids on individual strands of RNA. The data from these measurements can be used to build new models of the assembly pathway that describe the process in unprecedented detail. Special focus is paid to the role of the viral RNA in directing these pathways. New DNA-based techniques are developed to unravel the complex folding patterns of viral RNA and to selectively probe the specific RNA-protein interactions that have been proposed to play a role in capsid assembly. The combined approach of fast nanoscale imaging and DNA-based methods for manipulating specific molecular interactions may reveal unexpected routes for blocking capsid assembly and combating viral disease.