Abstract: 

The RNA dependent RNA polymerase (RdRp) in SARS-CoV-2, the virus responsible for the COVID-19 pandemic, is a highly conserved enzyme responsible for viral genome replication/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 dissertation 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 substrate binding (enzyme active site open) to rate-limiting insertion states (active site closed). This is in contrast to most studies which focus primarily on the apo or closed state as a drug binding target. The interactions of the RdRp with representative incoming 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 equilibrium 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 insertion and selection, enhanced sampling methods have been conducted to calculate the free energy profiles or potentials of mean force (PMFs) of the NTP’s. Overall, ATP and RDV-TP are favored in the closed state, while dATP and GTP are notably more stable in the open state. Additionally, ATP exhibits Watson-Crick base pairing in initial binding, whereas RDV-TP, dATP, and GTP form unique, more stable configurations. This dissertation offers physical insights into the nucleotide insertion and selection processes of SARS-CoV-2 RdRp prior to catalysis and can support the development of antiviral drugs targeting viral RdRps.