New targets for drug design: importance of nsp14/nsp10 complex formation for the 3’‐5’ exoribonucleolytic activity on SARS‐CoV‐2

Abstract

SARS‐CoV‐2 virus has triggered a global pandemic with devastating consequences. The understanding of fundamental aspects of this virus is of extreme importance. In this work, we studied the viral ribonuclease nsp14, one of the most interferon antagonists from SARS‐CoV‐2. Nsp14 is a multifunctional protein with two distinct activities, an N‐terminal 3’‐to‐5’ exoribonuclease (ExoN) and a C‐terminal N7‐methyltransferase (N7‐MTase), both critical for coronaviruses life cycle, indicating nsp14 as a prominent target for the development of antiviral drugs. In coronaviruses, nsp14 ExoN activity is stimulated through the interaction with the nsp10 protein. We have performed a biochemical characterization of nsp14‐nsp10 complex from SARS‐CoV‐2. We confirm the 3’‐5’ exoribonuclease and MTase activities of nsp14 and the critical role of nsp10 in upregulating the nsp14 ExoN activity. Furthermore, we demonstrate that SARS‐CoV‐2 nsp14 N7‐MTase activity is functionally independent of the ExoN activity and nsp10. A model from SARS‐CoV‐2 nsp14‐nsp10 complex allowed mapping key nsp10 residues involved in this interaction. Our results show that a stable interaction between nsp10 and nsp14 is required for the nsp14‐mediated ExoN activity of SARS‐CoV‐2. We studied the role of conserved DEDD catalytic residues of SARS‐CoV‐2 nsp14 ExoN. Our results show that motif I of ExoN domain is essential for the nsp14 function, contrasting to the functionality of these residues in other coronaviruses, which can have important implications regarding the specific pathogenesis of SARS‐CoV‐2. This work unraveled a basis for discovering inhibitors targeting specific amino acids in order to disrupt the assembly of this complex and interfere with coronaviruses replication.

Exploring the therapeutic nature of limonoids and triterpenoids against SARS-CoV-2 by targeting nsp13, nsp14, and nsp15 through molecular docking and dynamics simulations

“…The nsp14 exonuclease requires divalent cations like Mg 2+ , Mn 2+ , Ca 2+ , Ni 2+ , Cu 2+ , Co 2+ , or Zn 2+ for inducing structural changes and reaction activity. 16 , 17 The proofreading activity of nsp14 was associated with nsp10, and this activity could be hindered by sofosbuvir. 18 …”

Identification of fragments binding to SARS-CoV-2 nsp10 reveals ligand-binding sites in conserved interfaces between nsp10 and nsp14/nsp16

“…While we still lack structural data for the SARS-CoV-2 full-length nsp14-nsp10 complex, the structure for its SARS homologue has been previously determined 3,4 confirming this type of arrangement deduced by our MST data in which only the ExoN domain but not the N7-MTase domain is involved in binding to nsp10. Owing to the very high protein sequence identify between SARS and SARS-CoV-2 nsp14 18 , we hypothesise that the SARS-CoV-2 nsp14-nsp10 complex displays the same characteristics as observed for the SARS complex and that the affinity is relatively weak with K d values in the low micromolar range. To the best of our knowledge, the work shown here is the first time the quantitative interaction between the two proteins is reported for coronaviruses.…”

“…Finally, we also tested for a potential interaction between the nsp14 N7-MTase domain and nsp10, but did not observe any interacting between these two proteins under similar experimental conditions (Figure 4C) thereby also excluding any artefacts due to the presence of the N-terminal tag. Recently we became aware of work showing the interaction of nsp10 and nsp14 from SARS-CoV-2 using SPR 18 . While the authors show an interaction between these two proteins, steady-state analysis of interaction curves did not allow to unambiguously quantify K d values for the interaction from these experiments 18 .…”

“…Recently we became aware of work showing the interaction of nsp10 and nsp14 from SARS-CoV-2 using SPR 18 . While the authors show an interaction between these two proteins, steady-state analysis of interaction curves did not allow to unambiguously quantify K d values for the interaction from these experiments 18 . MST therefore shows excellent potential to study and quantify the interaction between nsp14 and nsp10.…”

Spotlight on COVID‐19: eighteen months on

“…In line with this, Saramago et al . [ 10 ] performed an elegant biochemical characterisation of the SARS‐CoV‐2 nsp14/nsp10 complex. They demonstrated that complex formation is a critical requirement for optimal nsp14 exoribonuclease activity, which is, in turn, essential for completion of the viral life cycle.…”