Understanding programmed ribosomal frameshifting in coronaviruses

Project Summary Coronaviruses (CoV) are associated with severe diseases as demonstrated by the 2003 severe acute respiratory syndrome (SARS)-CoV1 epidemic and the SARS-CoV2 pandemic. One of the critical steps of infection involves viral mRNA mediated recoding of gene expression; a -1 frameshifting event that occurs during translation. It is this elegant mechanism that allows the ribosome to bypass a stop codon and synthesize viral enzymatic proteins. Furthermore, the frequency by which this event occurs is important for efficient viral infectivity and is regulated by domains in the translating mRNA (in the case of the SARS-CoV, this domain is a pseudoknot). Although structural studies of frameshifting have received considerable aOention and various structures have been proposed and solved, information on exactly which structure causes the frameshifting is lacking. Our preliminary studies indicate that CoV gene expression is regulated by a dynamic, proton-driven equilibrium between an active, and two inactive pseudoknot conformations that allows for strict control over the protein ratios. This proposal aims to gain a complete structural and mechanistic understanding of the frameshifting frequency in CoV by combining structural studies with biochemical and in vivo experiments. Our aims will be: (#1) to understand the basis for how the frameshifting frequency is maintained by engineering structure-guided mutants to test our equilibrium model, (#2) to determine the structures of the pseudoknot signal in both configurations: permissive and nonpermissive for frameshifting, and (#3) to determine the structure of ribosomes as they encounter the permissive conformation of the pseudoknot.