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

Saramago, Margarida; Bárria, Cátia; Costa, Vanessa G.; Souza, Caio S.; Viegas, Sandra C.; Domingues, Susana; Lousa, Diana; Soares, Cláudio M.; Arraiano, Cecília M.; Matos, Rute G.

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.