Molecular Mechanism for Genomic RNA Delivery in ssRNA Phages

单链RNA噬菌体(或单链RNA噬菌体)是一种感染革兰氏阴性细菌的病毒，其基因组中含有RNA，而不是DNA。与许多双链DNA噬菌体不同，单链RNA噬菌体没有加压衣壳或“尾巴”来将基因组物质注入宿主。相反，他们通过单一的噬菌体衣壳蛋白(MP)识别特定的细菌，成熟蛋白(MP)结合了噬菌体基因组RNA(GRNA)和宿主细菌的一种特殊结构，称为可伸缩的菌毛，这是细菌分泌系统中的一种矛状成分。据认为，菌毛的收回使单链RNA噬菌体更接近宿主；当菌毛进一步收回时，细菌与回收的菌毛蛋白亚单位一起摄取MP/gRNA复合体，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”。虽然这一性质可以立即在生物技术上应用，有效地将大量外来RNA输送到宿主细菌中，但MP如何识别特定类型的菌毛以及MP/gRNA复合体如何从噬菌体衣壳转移到细胞的机制仍不清楚。本项目旨在研究这些过程，并比较两个相关的模型大肠杆菌单链RNA噬菌体MS2和Qbeta的结果，这为深入了解病毒与其宿主之间相互作用的结构和动力学提供了见解。该项目将伴随着一项丰富而广泛的影响计划，其中包括独特的跨学科培训活动，为未来的STEM教师举办的一系列研讨会，以及为公众和STEM教师制作在线可访问的动画和电影，供公众和STEM教师在课堂上使用。将通过单粒子冷冻电子显微镜(Cryo-EM)、荧光显微镜、冷冻电子断层扫描(Cryo-ET)、分子生物学和计算建模来研究单链RNA噬菌体MS2与大肠杆菌F型IV型分泌系统(F-T4SS)之间相互作用的结构和动力学。这个项目有两个目标。目的1：单链RNA噬菌体与宿主结合的分子机制。(A)MS2/PILUS复合体的体外原子结构将用单粒子冷冻EM解决。(B)体内单链RNA噬菌体与细胞包膜的结合方向用冷冻法测定。(C)将对单链RNA噬菌体/菌毛界面上的氨基酸残基进行基因特征分析。(D)与MS2相比，将确定单链RNA噬菌体Qbeta与F-菌毛之间的相互作用。目的2：gRNA宿主穿透细胞膜的分子机制。(A)将通过时间分辨广场荧光显微镜建立毛状体回缩和单链RNA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)将使用smFISH超分辨率显微镜和电子断层扫描来揭示gRNA进入细胞的路径。(D)MS2与F-T4SS之间的相互作用将通过低温等离子体原位观测来揭示。(E)将对gRNA从衣壳转移到细胞内的过程进行计算建模。这一裁决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智力优势和更广泛的影响审查标准进行评估，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.