Molecular Mechanism for Genomic RNA Delivery in ssRNA Phages
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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.