Biochemical Studies of SARS-CoV-2 RTC

Abstract

COVID-19, caused by SARS-CoV-2, has been a catastrophic pandemic. While vaccines have helped restore normalcy, reinfections persist, highlighting the urgent need for antiviral drugs. The replication transcription complex (RTC) offers a promising target due to its conservation across strains. The NiRAN (Nidoviruses RdRp associated nucleotidyltransferase) domain of Nsp 12 (RNA polymerase), which carries out the initial steps of RNA capping, has three different nucleotide binding poses. Each pose has its own significance for capping viral mRNA and NMPylation reactions (Nucleotide monophosphate addition to Nsp 9). In this investigation, using mutagenesis, I tried to delineate the molecular significance of each pose. Additionally, specific NiRAN residues influence RdRp's extension activity, highlighting their role in RTC stability and supporting my observations of feeble polymerase activity upon deletion of the NiRAN domain. These findings contribute to understanding CoV-2 replication and capping reaction. In addition, my project also provides an RTC mutant for virologists to hunt for the physiological significance of the NMPylation reaction.