Activation of the SARS-CoV-2 NSP14 3′–5′ exoribonuclease by NSP10 and response to antiviral inhibitors

Abstract

Understanding the core replication complex of 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 NSP14, a bifunctional enzyme with an N-terminal 3'-to-5' exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and its accessory protein, NSP10. The difficulty in producing pure, 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 E. coli and for the in vitro assembly and purification of a stoichiometric NSP10/14 complex with high yields. Using these methods, we observe 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-adenosyl homocysteine (SAH). Our analysis highlights two important factors for drug development: first, unlike other exonucleases, the monophosphate nucleoside analogue intermediate of remdesivir does not inhibit NSP14 activity; and second, SAH 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 COVID-19.

Despite the odds: formation of the SARS-CoV-2 methylation complex

“…Alongside N7-methyltransferase activity, nsp14 harbors an ExoN domain characterized by exoribonuclease activity; an activity essential for proofreading during virus replication. However, in vitro , nsp14 is characterized by the high processivity and non-specifically degrades nucleic acids 17,24,25 . As the nuclease activity must be tightly controlled, we assessed whether the complex formation regulates this process.…”

“…Nsp14 also harbours exoribonuclease activity, essential for proofreading during virus replication. However, in vitro, the protein acts as a highly processive ribonuclease, unspecifically degrading nucleic acids 19 . As the nuclease activity must be tightly controlled, we assessed whether the complex formation regulates this process.…”

“…Both nsp14 and nsp16 rely on nsp10 as an activating co-factor, the degradation activity of nsp14 increasing 35-fold with the presence of nsp10 [19][20][21] .…”

Recent insights into the structure and function of coronavirus ribonucleases

“…In the absence of Nsp10, Nsp14 exonuclease activity is negligible; however, the mechanism of stimulation is still unknown. Possible explanations include structural support or increased RNA‐binding contacts [ 35 ]. Cryo‐Electron Microscopy (EM) structures of the core RTC with multiple accessory factors revealed that the ExoN domain also makes contacts with additional RTC members including Nsp12, Nsp8, and Nsp9 (Fig.…”

Unraveling the Enzymatic Mechanism of the SARS-CoV-2 RNA-Dependent-RNA-Polymerase. An Unusual Active Site Leading to High Replication Rates

“…24,25 It uses the original viral positive-sense RNA strand to produce an intermediate negative-sense strand which is further used as a template allowing the synthesis of a novel duplicated positive-sense RNA strand. Of note, the RNA replication takes place in the cytoplasm in double-membrane vesicles and differently from other RNA viruses, SARS-CoV-2 also disposes of a replication proof-reading system based on a viral exonuclease, 26,27 which limits replication errors and considerably slows down its mutation rate. Obviously SARS-CoV-2 RNAP constitutes an ideal target for possible pharmacological development., [28][29][30] Its inhibition would, indeed, result in the arrest of the viral replication cycle and of the infection.…”