期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 298, 期 1, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.jbc.2021.101518
关键词
-
资金
- Division of Intramural Research of the National Institutes of Health, National Institute of Environmental Health Sciences [Z01 ES065078, Z01 ES065080]
Understanding the core replication complex of SARS-CoV-2 is crucial for the development of antiviral therapeutics. The researchers developed a simple protocol for the expression and purification of NSP10 and NSP14, and successfully assembled a purified NSP10/14 complex. They found that NSP10 enhances the activity and stability of NSP14. The study also investigated the effects of two small molecules on NSP10/14 activity.
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.
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