UC Irvine

The RNA dependent RNA polymerase (RdRp) in SARS-CoV-2, the virus responsible forthe COVID-19 pandemic, is a highly conserved enzyme responsible for viral genome repli- cation/transcription. While there are many SARS-CoV-2 variants, the RdRp protein has remained relatively conserved, making it an attractive target for antiviral drugs. This dis- sertation investigates the nucleotide addition cycle (NAC) and nucleotide selectivity during the viral RdRp elongation, focusing on an early stage of the cycle from initial nucleotide sub- strate binding (enzyme active site open) to rate-limiting insertion states (active site closed). This is in contrast to common computational or modeling works which examine a generic one-step substrate binding process. The interactions of the RdRp with representative incom- ing nucleoside triphosphates (NTPs) are studied: cognate ATP, RDV-TP (a drug analogue to ATP), non-cognates dATP and GTP, according to RNA template uracil. Ensemble equi- librium all-atom molecular dynamics (MD) simulations have been employed to explore the configuration space of each NTP in two kinetic states (open and closed). Due to the expected millisecond conformational change (from the open to closed) accompanying nucleotide in- sertion and selection, enhanced sampling methods have been conducted to calculate the free energy profiles or potentials of mean force (PMFs) of the NTP’s. The analyses reveal a marked difference in the stabilization of cognate ATP and the RDV-TP analog versus non- cognate dATP and GTP. Upon initial binding and subsequent insertion, ATP and RDV-TP show marginal free energy barriers, whereas dATP and GTP show substantial stabilization upon initial binding followed by notably high barriers for insertion into the active site. This pattern suggests an intrinsic mechanism of nucleotide selectivity in RdRp that rejects non- cognate NTPs. Specifically, ATP and RDV-TP, which are selected for incorporation, are favored in the closed or insertion state, while non-cognate dATP and GTP appear trapped off-path in the open or initial binding state. These mechanisms are facilitated by conserved structural motifs in the RdRp’s palm and fingers subdomain. Interestingly, the RDV-TP analog exhibits base stacking with the template Uracil upon initial binding, contrasting with the Watson-Crick base pairing seen between cognate ATP and the template. Moreover, our study shows that while RDV-TP drug analog stabilization from initial binding to insertion is primarily energetically driven, the stabilization of natural cognate ATP is also contributed entropically. This dissertation offers physical insights into the nucleotide insertion and se- lection processes of SARS-CoV-2 RdRp prior to catalysis and can support the development of antiviral drugs targeting viral RdRps.