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

通过直接单颗粒测量绘制病毒衣壳的组装途径

• 批准号: 10359241
• 负责人: Rees F Garmann
• 金额: $ 24.89万
• 依托单位: SAN DIEGO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10359241
• 关键词: Acylation; Address; Antiviral Agents; Bacteria; Binding; Biological Models; Buffers; Capsid; Capsid Proteins; Cells; Collaborations; Complex; Computer Simulation; Cytoplasm; DNA; Data; Disease; Distant; Enterobacteria phage MS2; Environment; Eye; Genetic Transcription; Genomics; Growth; Human; Hydroxyl Radical; Image; Imaging Techniques; In Vitro; Indiana; Individual; Infection; Kinetics; Knowledge; Label; Maps; Measurement; Measures; Methods; Microscopy; Modeling; Monitor; Nucleotides; Oligonucleotides; Organism; Pathway interactions; Pattern; Phylogenetic Analysis; Pisum sativum; Plants; Play; Primer Extension; Process; Production; Proteins; Protocols documentation; RNA; RNA Viruses; RNA-Protein Interaction; Role; Ross river virus; Route; Shapes; Signal Transduction; Structural Models; Structure; System; Techniques; Therapeutic; Translations; Universities; Viral; Virus; Virus Assembly; Virus Diseases; Work; base; biophysical properties; computerized tools; cowpea chlorotic mottle virus; design; insight; molecular rearrangement; nanoscale; nanoscience; particle; pathogenic virus; tool; viral RNA

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.

项目摘要 许多RNA病毒的衣壳在胞质中的病毒RNA周围赞助 感染的宿主细胞。绘制组件途径，即产生的分子重排集 最终的传染性结构是开发抗病毒药的第一步，以阻止该过程并停止 致病病毒的扩散。可用的实验技术，衡量大量发生的事情 capsids的溶液而不是单个衣壳中的溶液，掩盖了基本动力学。结果，我们没有答案 关于capsids如何形成的基本问题，例如限制步骤是成核的，还是生长，还是 在最初无序附着在RNA上的无序附着后，外套蛋白的重排。 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 在单相尺度上了解体外的衣壳组件：一种强大的纳米级成像技术称为 采用干涉散射（ISCAT）显微镜来监测单个病毒式衣壳在上的生长 RNA的单个链。这些测量的数据可用于构建组件的新模型 以前所未有的细节来描述过程的途径。特殊重点是在病毒RNA中的作用 指导这些途径。开发了新的基于DNA的技术来揭示复杂的折叠模式 病毒RNA并有选择地探究已提出发挥作用的特定RNA - 蛋白质相互作用 在衣壳组件中。快速纳米级成像和基于DNA的方法的合并方法 操纵特定的分子相互作用可能揭示了阻塞衣壳组件的意外途径，并且 打击病毒疾病。