Determining key residues involved in essential coronavirus nsp9 interactions

Abstract

Coronaviruses such as MERS-CoV and SARS-CoV-2 have caused devastating losses in human life and the economy. The replication and transcription complex (RTC) is crucial for the survival and reproduction of coronaviruses; thus, it has been an area of interest in drug inhibition. Within this complex, non-structural protein 9 (nsp9) is covalently bound to RNA by the NiRAN domain of nsp12 — a process known as RNAylation. Nsp12 then transfers guanine diphosphate to the 5’-pRNA (NMPylation), resulting in the RNA cap core structure 5’-GpppRNA. This RNA cap has been shown to increase viral replication and evasion of the host’s immune response; however, the exact molecular mechanisms of these processes are poorly understood. This thesis proposes potential drug targets against nsp9 function by investigating vital amino acid residues involved in nsp12 and RNA interactions. Although recent studies have uncovered the structural properties of nsp9 and nsp12 interaction, the data presented here (paper 1) determines their exact biochemical and biophysical details for the first time. The amino acid residues crucial for nsp9/12 binding were revealed through a mutational analysis using surface plasmon resonance (SPR) and size exclusion chromatography (SEC) and for these mutated residues, RNAylation of nsp9 was prevented in the presence of nsp12. Additionally, we established a set of antiviral peptides that recognised nsp9 and prevented binding to nsp12. These peptides present a good starting point in the development of a treatment for COVID-19. The mechanism of RNA binding was investigated using nuclear magnetic resonance (NMR) spectroscopy and we developed a structural model of the MERS-CoV nsp9 – RNA interaction (paper 2). Binding between the nsp9 protein from MERS-CoV and RNA has been previously demonstrated, however, the purpose and exact binding mechanism have not been shown. Furthermore, studies have previously identified potential RNA-binding residues on nsp9 that were confirmed by a mutational analysis of biolayer interferometry (BLI); one residue demonstrated a significant decrease in its RNAylation ability. The dimerization propensity of nsp9 protein was found to be lower in MERS-CoV compared to SARS-CoV-2, likely due to a unique glutamine residue in the dimerization interface of MERS-CoV. This study proposes that nsp9 guides the newly synthesized RNA from the RdRp domain of nsp12 to the NiRAN domain to be RNAylated. Drugs that prevent the nsp9-RNA interaction may therefore be developed to stop the replication of SARS-CoV-2.

Date of Award	2024
Original language	English
Awarding Institution	Western Sydney University
Supervisor	Roland Gamsjaeger (Supervisor) & Liza Cubeddu (Supervisor)