CAREER: Role of RNA structure in the packaging and discovery of RNA bacteriophage genomes

Bacteriophages are viruses that infect bacteria. They are the most numerous life-forms on the planet. In general, bacteriophages consist of a genome made either of DNA or RNA that is packaged into a protective protein shell called a capsid. While much is known about bacteriophages with DNA genomes, far less is understood about those with RNA genomes. This project will investigate how these RNA genomes are packaged into their protein capsids. In particular, the project will describe the shapes into which the RNA genomes are folded, and how these shapes are recognized by the proteins and selected for packaging. By identifying these structures, the project will inform the design of synthetic capsids capable of packaging synthetic RNA molecules for use in RNA-based technologies. Furthermore, these RNA structures will facilitate the discovery of new RNA viruses by learning how to recognize viral RNA in the sequence libraries obtained directly from the environment. These discoveries will advance the understanding of RNA bacteriophage diversity and enable the production of novel nanoscale materials derived from their coat proteins. Throughout, the project will train graduate and undergraduate students in challenging fields of physical chemistry, biochemistry, and computational biology. Bacteriophage MS2 is the canonical model system for studying viral RNA packaging, yet the mechanisms by which MS2 coat proteins selectively package their RNA genomes while excluding abundant cellular RNA remain unclear. While RNA of a model phage MS2 is believed to contain structural signals that are recognized by its coat proteins during packaging, the precise nature of these signals and their role in selective packaging is not fully understood. This project will use quantitative packaging experiments inside the cells to identify key RNA structures involved in selectivity. Plasmid-encoded RNA molecules with defined structures will compete with the cellular transcriptome for packaging by MS2 coat proteins. Single-particle imaging and high-throughput sequencing will quantify the packaged RNA composition, providing a direct measure of selectivity. These measurements will be used to critically evaluate current hypotheses of selective packaging and identify the key RNA structures involved. The project will then use these structures to screen metagenomic datasets for previously undescribed RNA bacteriophage genomes. Once discovered, the coat proteins from newly identified bacteriophages will be expressed in cells to produce protein capsids. In addition to verifying the discovered bacteriophages, these capsids will provide the research community with new protein nanoparticles for applications in biotechnology and nanoscience. 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.