Double-stranded RNA dictates SARS-CoV-2 nucleocapsid condensation temperature

Project Summary/Abstract: INTRODUCTION: RNA viruses such as SARS-CoV-2 represent an ongoing public health threat. To replicate properly, RNA viruses must perform multiple functions in the same cytoplasm (i.e., genome replication, circularization, generation of sub-genomic RNA, packaging etc.) and for betacoronaviruses like SARS-CoV-2 the structural nucleocapsid protein (N) is required for all of these processes. How are RNA viruses able to perform multiple functions in the same cytoplasm with their limited proteome (for example ~24 proteins for SARS-CoV-2)? I hypothesize that one way RNA viruses achieve biochemical complexity is by condensation of RNA and protein components. Biochemical complexity can emerge by virtue of the co-condensing RNA as different viral RNA sequences confer different condensate properties. In this way, a single protein, N, can perform multiple functions in the same cytoplasm. I uncovered the mechanism by which SARS-CoV-2 N recognizes RNA to undergo condensation. Now, I am interested in understanding how the interaction of N with viral RNA controls condensation and impacts important viral functions. RESEARCH: In my K99/R00 research; (Aim 1) I will unbiasedly probe RNA-RNA interactions to understand how RNA is arranged and how interactions are modulated by condensation for genome circularization. (Aim 2) I will develop BSL2 assays to determine which viral processes require N condensation. (Aim 3) I will design synthetic RNA-binding proteins and small molecules to disrupt N condensation. TRAINING: I will complete my training period in Dr. Amy Gladfelter's lab. During the training period, I will further develop my skills in RNA structure probing SHAPE (RNA structure), RNP-map (RNA/protein crosslinking), and SHAPE-Jump (RNA-RNA crosslinking) in collaboration with Weeks lab. I will learn how to design synthetic IDPs for the creation of bespoke RNA-binding proteins (Chilkoti). I will screen an existing small molecule library to identify inhibitors of N condensation (Fiorti). I will learn how to culture and genetically manipulate a BSL2 model betacoronavirus, MHV (Sheahan and Cameron). ENVIRONMENT: Dr. Amy Gladfelter is one of the world's leading experts on the role of RNA in condensation. Additionally, UNC has some of the world's leading experts in coronavirus biology including labs with BSL3 facilities offering the possibility to test newly developed N condensation inhibitors with live virus. I plan to further my growth through participation in RNA and condensation conferences, and attendance of courses in virology. I also plan on using my K99/R00 to ease my transition into running my own lab as an independent investigator. I am particularly interested in joining a university with strong biochemistry, molecular biology, virology, and biomedical engineering departments who are looking for an RNA structure enthusiast to join their ranks! IMPACT ON PUBLIC HEALTH: This work will advance our understanding of how betacoronavirus N recognize viral RNA sequences and may one day lead to novel therapeutics.