ABSTRACT The world is currently in the midst of a global pandemic caused by the second severe acute respiratory syndrome coronavirus (SARS2). In spite of the foreshadowing of such a pandemic by the emergence of SARS1 in 2002 and Middle East respiratory syndrome coronavirus (MERS) in 2012, we were ill equipped to address this scourge. Each of these early outbreaks yielded a substantial body of knowledge on the structures of coronavirus proteins. As with previous outbreaks, we are witnessing a redoubled coronavirus research effort. Structural biology continues to lead the way; however, our laboratory is now pledging a sustained commitment to elucidation of the fundamental enzymology and corresponding mechanisms of coronavirus genome replication. The SARS2 replisome has emerged as a clinically tractable target for development antiviral therapeutics. Remdesivir is a nucleotide analog prodrug, metabolized to the triphosphate in cells, and incorporated by the SARS2 replisome without excision by its proofreading exonuclease, ExoN. The mechanism of action of remdesivir is unclear, and the mechanism of escape from ExoN is unknown. This circumstance reflects the absence of a quantitative, mechanistic perspective of the SARS2 replisome. Such a perspective will be essential to elucidation of the mechanism of drug action and the mechanism of drug resistance. We have demonstrated the feasibility of elucidating the principles governing the dynamics and function of the SARS2 core replicase using state-of-the-art ensemble and single-molecule approaches. We will exploit these advances to pursue the following specific aims: study assembly and function of the SARS2 core replicase and its sub-assemblies (Aim 1); study utilization of incorrect nucleotides and nucleotide analogues by the SARS2 core replicase (Aim 2); and study the mechanism of error correction by the SARS2 exoribonuclease (Aim 3). Completion of these studies will represent the first, deep dive into the mechanistic enzymology of the coronavirus replisome.

Project Narrative Severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is currently causing substantial morbidity and mortality on a global scale. The SARS CoV-2 replisome is an established target for antiviral ribonucleotide therapeutics. A quantitative, mechanistic framework to understand replisome function and mechanisms of inhibitor action and resistance is lacking. The proposed studies will fill this gap and therefore contribute to development of strategies to treat and/or prevent SARS CoV-2 infection and corresponding disease.