Biochemical Studies of SARS-CoV-2 Non-structural Protein NSP14 and NiRAN Domain of NSP12

Abstract

Proofreading enzymes are essential for the proper functioning of many organisms, including viruses. In coronaviruses such as SARS-CoV-2, NSP14 carries out this function with the help of the N-terminal ExoN domain using its 3’ to 5’ exoribonuclease activity. CoVs cap their genomic RNA and mRNA to evade the host immune system. The NiRAN domain of NSP12 is proposed to play a role in this capping process. Exonuclease assay results in this project show that NSP14 prefers single-stranded RNA substrates to double-stranded RNA substrates, contradictory to the recently published papers. The NiRAN domain is inactive in the absence of the rest of the NSP12 domains. Mass spectrometry confirmed that NSP12, in its intact form, is able to transfer an AMP from an ATP molecule to NSP9. Attempts to crystallize the NiRAN domain and NSP14 were not successful. The nature of different substrates that can bind to the active site of the NiRAN domain is yet to be found out. Further experiments are required to be carried out in order to unravel the influence of NSP14 on the activities of NSP12 and NSP13.