Exploring the interplay of viral genomic RNA structure and protein biochemistry in coronavirus replication

PROJECT SUMMARY/ABSTRACT Coronaviruses are important human and animal pathogens. In addition to common colds, coronaviruses have been responsible for several major outbreaks, including the SARS-CoV epidemic in 2002, the MERS-CoV epidemic in 2012, and the SARS-CoV-2 pandemic in 2020. Although accelerated research on coronavirus pathogenicity and biology during the 2020 pandemic has led to the design of several successful vaccines and antivirals, coronaviruses are constantly evolving. Continued research is therefore imperative for the identification of additional therapeutic targets. Coronaviruses contain positive-sense, single-stranded RNA genomes. Once a viral particle has infected a cell, the genome must be effectively copied and packaged prior to viral egress. Key molecular processes of the viral life cycle, including genome replication and genome encapsidation during viral egress, necessarily rely upon interactions between genomic RNA and viral proteins. However, the specific RNA- protein interactions driving these processes are poorly understood on a biochemical and structural level. Using a combined approach of virology, biochemistry, and structural biology, the current proposal seeks to understand the relevance of viral RNA-protein biomolecular interactions in the coronavirus cellular infection cycle. Replication of the coronavirus RNA genome is mediated by the viral polymerase and its essential cofactors. Previous work in the field has suggested that there are RNA structures within the coronavirus genomic termini that are critical for genome replication. However, the precise genomic RNA structures that are required for effective initiation of coronavirus genome replication remain unknown. Specific Aim 1 of this proposal seeks to elucidate which genomic RNA structures interact with the polymerase complex to initiate genome replication. Another common antiviral target is viral egress. For coronaviruses, viral egress occurs following encapsidation of the genome with a viral structural protein termed Nucleocapsid (N). In addition to it necessary structural function in viral egress, N has been proposed to play manifold roles throughout the viral life cycle. A major limitation of these studies is that the structure of full-length coronavirus N has yet to be solved. Additionally, how N is organized on genomic RNA within a virion remains unknown. Specific Aim 2 of this proposal seeks to examine the structure of N-bound genomic RNA in the context of the packaged genome. The research described in this proposal will take place under the mentorship of Dr. Robert Kirchdoerfer (fellowship sponsor), who studies coronavirus protein biochemistry and structure, and Dr. Samuel Butcher (co- sponsor), who studies the RNA-mediated regulation of gene expression. Supported by facilities including the Cryo-Electron Microscopy Research Center and the Biophysics Instrument Facility, the current proposal aims to explore critical coronavirus RNA-protein interactions on a structural and biochemical level. This work is anticipated to illuminate key viral biomolecular interactions that could serve as targets for antivirals.