Elucidating the mechanisms of viral life cycles under near-native conditions

项目摘要 单负性病毒目是病毒的一个分类目， 链RNA基因组及其多形膜包膜病毒体。单负病毒属生活 循环涉及许多不同的事件，包括病毒体进入宿主细胞，病毒mRNA 转录、基因组复制和病毒组装以及来自宿主细胞的病毒出芽。这些 事件通过病毒和宿主细胞机制之间的分子相互作用进行; 阐明这些相互作用是理解病毒生命周期和确定潜在的 治疗目标 对病毒机制的研究通常仅限于孤立的颗粒或集合体;这就排除了 使它们脱离原生环境，并剥夺了重要的分子相互作用。到 为了保护生物学背景，必须原位研究病毒机制，即在近天然条件下， 条件下，如在完整的病毒体或细胞内。这些环境是一个复杂的，无序的 分子的混合物，使得特别难以获得分子分辨率信息。 在这里，我们打算研究三种单负病毒：麻疹、狂犬病和埃博拉病毒。每个 作为其分类学家族的原型病毒，每种都是重要的病原体， 全球健康。 为了进行我们提出的研究，我们将使用和开发原位结构生物学方法。 我们的主要方法将是冷冻电子断层扫描（cryo-ET），一种冷冻电子 显微镜（cryo-EM），其允许三维体积的可视化。这 克服了纯化颗粒的薄单层的典型冷冻EM要求， 在接近自然环境中获得分子分辨率的信息。我们将开发 低温ET的数据收集和计算方法，以实现快速、自动化的数据收集 收集、高分辨率结构测定和精确的分子鉴定。我们将 我还使用和开发补充冷冻ET的方法，包括聚焦离子束铣削 以及相关的光学和电子显微镜方法。 我们的研究将提供三种重要病毒的新生物学见解，但更广泛地说， 将展示研究病毒的变革性方法。而不是试图戏弄 除了通过间接生物化学手段的功能和相互作用外，我们的研究将提供一个 直接观察病毒和宿主细胞机制的基础设施， 在接近自然的条件下。

Project Summary Mononegavirales is a taxonomic order of viruses, so classified for their negative sense single stranded RNA genome and their pleomorphic membrane-enveloped virions. Mononegavirus life cycles involve a number of different events, including entry of virions into host cells, viral mRNA transcription, genome replication and virus assembly, and viral budding from host cells. These events are carried out through molecular interactions between virus and host cell machinery; elucidating these interactions is key to understanding viral life cycles and identifying potential therapeutic targets. Studies of viral machinery are typically limited to isolated particles or assemblies; this removes them from their native environments and strips away important molecular interactions. To preserve biological context, viral machinery must be studied in situ, i.e. under near-native conditions, such as within intact virions or cells. These environments are a complex, disordered mixture of molecules, making it particularly difficult to obtain molecular resolution information. Here, we propose to study three mononegaviruses: measles, rabies, and Ebola viruses. Each serve as prototypical viruses for their taxonomic families, and each are pathogens important to global health. To carry out our proposed research, we will use and develop in situ structural biology methods. Our primary method will be cryo-electron tomography (cryo-ET), a type of cryo-electron microscopy (cryo-EM) that allows for visualization of three-dimensional volumes. This overcomes the typical cryo-EM requirement of thin monolayers of purified particles, allowing for the acquisition of molecular-resolution information in near-native environments. We will develop data collection and computational methods for cryo-ET to enable rapid, automated data collection, high-resolution structure determination, and accurate molecular identification. We will also use and develop methods complementary to cryo-ET including focused ion-beam milling and correlative light and electron microscopy approaches. Our research will provide novel biological insights three important viruses, but more broadly, it will demonstrate a transformative approach for studying viruses. Rather than trying to tease apart function and interactions through indirect biochemical means, our research will provide an infrastructure to directly observe the virus and host cell machinery with complete biological contexts under near-native conditions.