A multipronged investigation of SARS-CoV-2 genome packaging

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Project Summary The COVID-19 pandemic, caused by the virus SARS-CoV-2, represents an acute and ongoing threat to human life. A detailed molecular understanding of the viral life cycle is necessary to illuminate clinically accessible processes that can be targeted for therapeutic intervention. The Nucleocapsid (N) protein is a 420-residue multidomain protein with both folded and disordered regions that underlies genome packaging, an essential step in the virion lifecycle. N protein mediates cytosolic genome packaging by binding to and compacting genomic RNA in a process apparently conserved across the coronaviridae family. Our ability to disrupt genome packaging is limited by the absence of a molecular understanding of these processes. To address this knowledge gap, our proposal is focused on the molecular biophysics that underlies how N protein drives genome compaction. N protein is highly multivalent; it can simultaneously bind to both itself and RNA via a number of distinct interaction sites. Multivalency is encoded across both folded domains and intrinsically disordered regions. While there has been substantial work on the folded domains in other coronaviruses, the molecular biophysics of the disordered regions has been largely ignored. We hypothesize N protein multivalency underlies the molecular basis of RNA compaction, and that the three disordered regions play key roles in determining multivalency, binding affinity, and RNA binding specificity. Through the combination of single-molecule fluorescence and force spectroscopy, ensemble methods, and all-atom simulation, we will dissect the molecular details that underlie these interactions. We also present a novel approach to small-molecule screening that leverages the formation of phase separated protein:RNA liquid droplets as a readout for genome compaction. Our work will offer high-resolution structural insight into the physical basis for two critical steps in the viral life cycle, as well as reveal small molecules that can attenuate genome compaction. More generally, by focusing on fundamental biophysical phenomena that empirically explain behavior from other distant coronaviruses, we believe that our conclusions will be broadly transferable to existing coronaviruses that represent major public health threats (e.g., SARS, MERS) but also to future novel zoonotic coronaviruses.