Molecular Mechanism for Genomic RNA Delivery in ssRNA Phages

单链RNA噬菌体（或ssRNA噬菌体）是一种感染革兰氏阴性细菌的病毒，它们的基因组是RNA，而不是DNA。与许多双链DNA噬菌体不同，ssRNA噬菌体没有加压衣壳或将基因组物质注入宿主的“尾巴”。相反，它们通过单一的噬菌体衣壳蛋白，成熟蛋白（MP）来识别特定的细菌，成熟蛋白结合噬菌体基因组RNA （gRNA）和宿主细菌的特殊结构，称为缩回菌毛，这是细菌分泌系统的矛状成分。据认为，毛的收缩使ssRNA噬菌体更接近宿主；当菌毛进一步收缩时，细菌会摄入MP/gRNA复合体以及回收的菌毛亚基，这一策略类似于希腊人在特洛伊战争期间使用的“特洛伊木马”。虽然这种特性可以立即应用于生物技术，以有效地将大量外源RNA递送到宿主细菌中，但MP如何识别特定类型的菌毛以及MP/gRNA复合物如何从噬菌体衣壳转运到细胞的机制仍不清楚。该项目旨在研究这些过程，并比较两种相关模型大肠杆菌ssRNA噬菌体MS2和Qbeta的结果，这些结果为病毒与其宿主之间相互作用的结构和动力学提供了见解。该项目将伴随一个丰富的更广泛的影响计划，包括独特的跨学科培训活动，面向未来STEM教师的系列研讨会，以及生成可供公众和STEM教师在课堂上使用的在线动画和电影。ssRNA噬菌体MS2与大肠杆菌f样IV型分泌系统（F-T4SS）相互作用的结构和动力学将通过单粒子冷冻电子显微镜（cro - em）、荧光显微镜、冷冻电子断层扫描（cro - et）、分子生物学和计算模型进行研究。这个项目有两个目标。目的1:ssRNA噬菌体附着宿主的分子机制。(a)体外MS2/菌毛络合物的原子结构将用单粒子低温电镜分析。(b) ssRNA噬菌体在体内与细胞包膜的结合方向将通过cryo-ET测定。(c) ssRNA噬菌体/菌毛界面处的氨基酸残基将被遗传表征。(d) ssRNA噬菌体Qbeta与F-pilus的相互作用将与MS2进行比较。目的2:gRNA宿主穿过细胞包膜进入细胞的分子机制。(a)通过时间分辨宽视场荧光显微镜建立毛缩回与ssRNA进入之间的关系。(b) MS2 gRNA进入动力学将通过活细胞超分辨率显微镜来阐明。(c)使用smFISH超分辨率显微镜和电子断层扫描将显示gRNA进入细胞的路径。(d) MS2与F-T4SS机器在原位的相互作用将被cryo-ET揭示。(e)将计算模拟gRNA从衣壳转移到细胞中的过程。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Single-stranded RNA bacteriophages (or ssRNA phages) are a type of viruses that infect Gram-negative bacteria and have RNA, not DNA as their genomes. Unlike many double-stranded DNA phages, ssRNA phages do not have a pressurized capsid or a "tail" to inject the genomic material into the host. Instead, they recognize specific bacteria through a single phage capsid protein, the maturation protein (MP), which binds both the phage genomic RNA (gRNA) and a special structure of the host bacterium termed the retractile pilus which is a spear-like component of the bacterial secretion system. It is thought that the retraction of the pili brings ssRNA phages closer to the host; and upon further retraction of the pili, the bacteria intake the MP/gRNA complex along with the recycled pilin subunits, a strategy similar to the "Trojan horse" used by the Greeks during the Trojan War. While this property can have immediate biotechnological application to efficiently deliver large quantities of foreign RNA into a host bacterium, the mechanisms of how the MP recognizes a specific type of pili and how the MP/gRNA complex translocates from the phage capsid to the cell are still not clear. This project is directed at investigating these processes and compares the results of two related model E. coli ssRNA phages, MS2 and Qbeta, which provide insights into the structure and dynamics of the interaction between the virus and its host. The project will be accompanied by a rich broader impact program that includes unique interdisciplinary training activities, a seminar series to future STEM teachers, and the generation of online-accessible animations and movies to be used by the public and STEM teachers in their classes.The structure and dynamics of the interaction between the ssRNA phage MS2 and the E. coli F-like type IV secretion system (F-T4SS) will be studied by single-particle cryo-electron microscopy (cryo-EM), fluorescence microscopy, cryo-electron tomography (cryo-ET), molecular biology and computational modeling. This project has two objectives. Objective 1: Molecular mechanism for the host attachment of ssRNA phage. (a) An atomic structure of the MS2/pilus complex in vitro is to be solved by single-particle cryo-EM. (b) The ssRNA phage-binding orientation to the cell envelope in vivo is to be determined by cryo-ET. (c) The amino acid residues at the ssRNA phage/pili interface will be genetically characterized. (d) The interaction between ssRNA phage Qbeta and the F-pilus will be determined in comparison to MS2. Objective 2: Molecular mechanism for the gRNA host entry across the cell envelope. (a) The relationship between pili retraction and ssRNA entry will be established by time-resolved wide-field fluorescence microscopy. (b) The MS2 gRNA entry dynamics will be elucidated by live-cell super-resolution microscopy. (c) The path of the gRNA into the cell will be revealed using smFISH super-resolution microscopy and electron tomography. (d) The interaction between MS2 and the F-T4SS machine in situ will be revealed by cryo-ET. (e) The translocation of the gRNA from the capsid into the cell will be computationally modeled.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.