Mechanistic insights into multifaceted roles of coronavirus exoribonuclease complex

As SARS-CoV-2 continues to wreak havoc across the globe, it is imperative to understand the mechanism and regulation of the viral genome replication and transcription, which are essential processes in coronavirus life cycle and represent important targets for therapeutic interventions. Coronavirus genome replication and transcription are carried out by a dynamic replication-transcription complex (RTC), assembled from an array of viral non-structural proteins (nsps). Within the RTC, a unique proofreading exoribonuclease (ExoN) complex, nsp14-nsp10, boosts replication fidelity by excising mis-incorporated nucleotides and many antiviral nucleotide analogs. In addition to its role in proofreading viral RNA synthesis, the ExoN complex is also involved in viral RNA 5′ capping, which is critical for immune evasion by coronaviruses. Despite extraordinary efforts in studying coronavirus biology and replication, major gaps remain in our understanding of the key roles ExoN complex plays in various fundamental aspects of coronavirus life cycle. First, it is unclear how ExoN complex coordinates with the low-fidelity viral polymerase to proofread RNA synthesis. Second, it is poorly understood how ExoN complex is modulated by viral cofactors. Third, it is unknown how the two different enzymatic functions, RNA cleavage and capping activities, of ExoN complex are coupled in the virus life cycle. The central objective of our proposed experiments is to fill these gaps in understanding through a systematic dissection of the structural basis and functional roles of ExoN complex and its dynamic interlay with viral cofactors in viral RNA synthesis and processing. We will use SARS-CoV-2 as a model system and employ a combination of cryo-electron microscopy, single-molecule biophysics, protein-RNA biochemistry, and cell virology to achieve this central goal through the following aspects: Project 1, coordination of polymerase and exoribonuclease during mismatch correction. We will define the mechanism by which RNA mismatches are transferred from polymerase to ExoN and identify the molecular determinants for their functional interplay. Project 2, modulation of ExoN complex by viral cofactors. We will elucidate the molecular details of the interaction between ExoN complex and a key RTC subunit, nsp8, and determine how this interaction modulates the proofreading activity of ExoN complex during mismatch correction. Project 3, coupling of RNA exonucleolytic digestion and 5′ capping activities of ExoN complex. Building on our newfound cryo-EM structure of a dimeric form of the ExoN complex in which its RNA digestion and capping activities are coupled, we will determine the molecular and biochemical underpinnings and establish the physiological significance of the functional link between the two enzymatic activities of ExoN complex. Through this research program, we will reveal the principles and molecular details governing the multifaceted roles of this unique viral RNA proofreader and provide new insights into the mechanisms and regulation of coronavirus genome replication and transcription. More broadly, the established tools and experimental platforms are readily applicable to studying other RNA viruses.