Understanding the core replication complex of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to the development of novel coronavirus-specific antiviral therapeutics. Among the proteins required for faithful replication of the SARS-CoV-2 genome are nonstructural protein 14 (NSP14), a bifunctional enzyme with an N-terminal 3′-to-5′ exoribonuclease (ExoN) and a C-terminal N7-methyltransferase, and its accessory protein, NSP10. The difficulty in producing pure and high quantities of the NSP10/14 complex has hampered the biochemical and structural study of these important proteins. We developed a straightforward protocol for the expression and purification of both NSP10 and NSP14 from Escherichia coli and for the in vitro assembly and purification of a stoichiometric NSP10/14 complex with high yields. Using these methods, we observe that NSP10 provides a 260-fold increase in k cat / K m in the exoribonucleolytic activity of NSP14 and enhances protein stability. We also probed the effect of two small molecules on NSP10/14 activity, remdesivir monophosphate and the methyltransferase inhibitor S-adenosylhomocysteine. Our analysis highlights two important factors for drug development: first, unlike other exonucleases, the monophosphate nucleoside analog intermediate of remdesivir does not inhibit NSP14 activity; and second, S-adenosylhomocysteine modestly activates NSP14 exonuclease activity. In total, our analysis provides insights for future structure–function studies of SARS-CoV-2 replication fidelity for the treatment of coronavirus disease 2019.
【저자키워드】 COVID-19, SARS-CoV-2, coronavirus, COVID-19, Coronavirus disease 2019, SARS-CoV-2, Severe acute respiratory syndrome coronavirus 2, nsp10, nsp14, protein–protein interaction, protein stability, DMSO, dimethyl sulfoxide, Exoribonuclease, enzyme kinetics, SEC, size-exclusion chromatography, Protein purification, Viral protein, protein complex, DSF, differential scanning fluorimetry, protein drug interaction, viral proofreading, CV, column volume, DDEE, residues forming the active site aspartic acid (D) and glutamic acid (E), FL, full-length, MP, monophosphate, MTase, methyltransferase, Ni, nickel, NP-40, Nonidet-P40, NSP14, nonstructural protein 14, NTA, nitrilotriacetic acid, RMP, remdesivir monophosphate, SAH, S-adenosylhomocysteine, TCEP, Tris(2-carboxyethyl)phosphine, 【초록키워드】 Treatment, protocol, Remdesivir, Proteins, Replication, Protein, methyltransferase, understanding, antiviral therapeutics, small molecule, inhibitor, expression, SARS-CoV-2 replication, exonuclease, Analysis, nonstructural protein 14, Escherichia coli, acute respiratory syndrome, Factor, biochemical, enzyme, complex, purification, monophosphate, N-terminal, highlight, ENhance, observé, inhibit, required, provide, increase in, activate, producing, C-terminal, exonucleases, the SARS-CoV-2 genome, 【제목키워드】 antiviral inhibitor, the SARS-CoV-2,