Understanding the physiological mechanism of proofreading system of SARS-CoV-2 Replication Transcription Complex

Abstract

The fidelity of the unusually large viral RNA genome of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), consisting of ~30 kb, is ensured by a proofreading mechanism involving the nsp10/nsp14 exoribonuclease (ExoN). This counteracts the inherently error-prone RNA-dependent RNA polymerase (RdRP) activity of the viral replication-transcription complex (RTC, consisting of nsp12/nsp7/nsp8). The relationship between the RTC and ExoN was explored at a biochemical level, employing RNA extension, exonuclease activity, coupled proofreading, and RNA-exonuclease activities, together with electrophoretic mobility shift assay (EMSA) and Mass Photometry. The results of the Fluorophore-labelled RNA extension assay revealed that the RTC has the ability to carry out processive RNA synthesis, although it has the ability to misincorporate canonical and non canonical nucleotides. The fidelity of RNA polymerase was found to depend on pH, with the highest fidelity at pH 6 and misincorporation at pH 7, 8, and 9. The RTC's active site was found to be structurally flexible, allowing it to accommodate several nucleotide analogues. Exonuclease assay results revealed that the nsp10-nsp14 complex has strong 3' to 5' exonuclease activity, where the optimal activity was observed at a concentration of about 100nM. Coupled assay results revealed that RNA synthesis occurs normally even when ExoN is present, although RNA cleavage occurs after the RNA has left the RTC. The results of the EMSA assay revealed that there was no formation of a super-complex between the ExoN and the RTC, leading to the conclusion that the RNA surveillance model involves the RTC's RNA synthesis, where the ExoN surveys the RNA after it leaves the RTC, leading to genome stability during the SARS-CoV-2 viral replication process.