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

项目摘要 许多RNA病毒的衣壳自发地聚集在细胞质中的病毒RNA周围 被感染的宿主细胞。绘制出组装路径--也就是产生 最终的感染结构-是开发抗病毒药物的第一步，这种药物可以阻止这一过程并阻止 致病病毒的传播。可用的实验技术，它测量大量发生的事情 衣壳的溶解，而不是在单个衣壳中，模糊了基本的动力学。因此，我们对以下问题缺乏答案 有关衣壳如何形成的基本问题，例如限速步骤是成核、生长还是 在最初无序地附着在RNA上之后，外壳蛋白的重排。 来自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 了解衣壳在单衣壳尺度上的体外组装：一种名为 干涉散射(ISCAT)显微镜被应用于监测单个病毒衣壳的生长 核糖核酸的单链。来自这些测量的数据可以用来建立新的装配模型 以前所未有的细节描述这一过程的途径。特别关注病毒RNA在病毒感染中的作用 指引着这些道路。基于DNA的新技术被开发来解开复杂的折叠模式 病毒RNA，并选择性地探测特定的RNA-蛋白质相互作用，这些作用已被提出 在衣冠楚楚的集合中。快速纳米级成像和基于DNA的方法相结合的方法 操纵特定的分子相互作用可能揭示阻止衣壳组装和 与病毒性疾病作斗争。

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.