Molecular Mechanism for Genomic RNA Delivery in ssRNA Phages

ssRNA 噬菌体中基因组 RNA 传递的分子机制

• 批准号: 1902392
• 负责人: Junjie Zhang
• 金额: $ 107.84万
• 依托单位: Texas A&M AgriLife Research
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1902392&HistoricalAwards=false
• 关键词: Molecular; Mechanism; Genomic; RNA; Delivery

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.

单链RNA噬菌体（或ssRNA噬菌体）是一种感染革兰氏阴性细菌并具有RNA而不是DNA作为其基因组的病毒。与许多双链DNA噬菌体不同，ssRNA噬菌体没有加压的衣壳或“尾巴”来将基因组材料注入宿主。取而代之的是，他们通过单个噬菌体衣壳蛋白，成熟蛋白（MP）识别特异性细菌，该蛋白（MP）结合了噬菌体基因组RNA（GRNA）和宿主细菌的特殊结构，称为伸缩菌Pilus，这是细菌分泌系统的长矛状成分。据认为，pili的缩回使ssrna噬菌体更接近宿主。在进一步缩回pili后，细菌摄入了MP/GRNA复合物以及再生的PILIN亚基，这是一种类似于特洛伊战争期间希腊人使用的“特洛伊木马”的策略。尽管该特性可以立即具有生物技术应用，以有效地将大量的外源RNA传递到宿主细菌中，但MP如何识别特定类型的Pili的机制以及MP/GRNA复合物如何从噬菌体上转移到细胞的细胞中。该项目旨在研究这些过程，并比较两个相关模型的大肠杆菌噬菌体MS2和QBETA的结果，这些噬菌体MS2和QBETA提供了有关病毒与其宿主之间相互作用的结构和动力学的见解。该项目将伴随着更广泛的影响计划，其中包括独特的跨学科培训活动，未来的STEM老师的研讨会系列以及公众和STEM老师在其课程中使用的在线动画和电影的产生。SSRNAPHAGE MS2和E. COLI FLICK IIV SYTES的结构和动态（F-T-PRAINS）将（F-T4S）（F-T4）（F-T4）（F-T4）（F-T4）冷冻电子显微镜（冷冻EM），荧光显微镜，冷冻电子层析成像（冷冻-ET），分子生物学和计算建模。该项目有两个目标。目标1：用于宿主噬菌体宿主附着的分子机制。 （a）在体外MS2/pilus复合物的原子结构应通过单粒子冷冻EM解决。 （b）在体内的细胞包膜的ssRNA噬菌体结合取向将通过冷冻-ET确定。 （c）将在基因表征ssRNA噬菌体/pili界面的氨基酸残基。 （d）与MS2相比，将确定SSRNA噬菌体QBeta与F-Pilus之间的相互作用。目标2：GRNA宿主进入细胞包膜的分子机制。 （a）将通过时间分辨的宽视野荧光显微镜建立pili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阐明。 （c）将使用Smfish超分辨率显微镜和电子断层扫描揭示GRNA进入细胞的路径。 （d）Cryo-Et将揭示MS2和F-T4SS机器原位的相互作用。 （e）将GRNA从CAPSID转移到单元格的易位。该奖项反映了NSF的法定任务，并被认为是值得通过基金会的知识分子优点和更广泛的影响评估标准通过评估来支持的。

项目成果

ssRNA phage penetration triggers detachment of the F-pilus

• DOI: 10.1073/pnas.2011901117
• 发表时间: 2020-10-13
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Harb, Laith;Chamakura, Karthik;Zeng, Lanying
• 通讯作者: Zeng, Lanying

Structural bases for F plasmid conjugation and F pilus biogenesis in Escherichia coli

• DOI: 10.1073/pnas.1904428116
• 发表时间: 2019-07-09
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Hu, Bo;Khara, Pratick;Christie, Peter J.
• 通讯作者: Christie, Peter J.

A Multiscale Model for the Self-Assembly of Coat Proteins in Bacteriophage MS2

噬菌体 MS2 中外壳蛋白自组装的多尺度模型

• DOI: 10.1021/acs.jcim.9b00514
• 发表时间: 2019
• 期刊: Journal of Chemical Information and Modeling
• 影响因子: 5.6
• 作者: Wang, Bo;Zhang, Junjie;Wu, Yinghao
• 通讯作者: Wu, Yinghao