Biochemical characterization of SARS-CoV-2 nsp14/nsp10 complex: a promising target for drug design

ABSTRACT

The global pandemic prompted by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has already caused more than 6 million deaths worldwide, calling for urgent effective therapeutic measures. A deep understanding of the mechanisms involved in viral replication is required. Among the nonstructural proteins (nsps) encoded by SARS-CoV-2 genome, there is the nsp14 ribonuclease, the main object of study in this work. Ribonucleases are key factors in the control of all biological processes, ensuring maturation, degradation, and quality control of all types of RNAs. Nsp14 is a bifunctional protein, holding a 3'- 5' exoribonucleolytic activity (ExoN) in the N-terminal domain, stimulated through the interaction with nsp10, and a C-terminal N7-methyltransferase activity (MTase). Both are critical for the coronavirus life cycle. In this work, we provide a complete biochemical characterization of SARS-CoV-2 nsp14-nsp10, addressing several aspects of the complex for the first time. Moreover, using a homology model, we have identified residues involved in the nsp14-nsp10 interaction that were extensively studied. We have confirmed the SARS-CoV-2 nsp14 dual function and we have shown that both ExoN and MTase activities are functionally independent. We demonstrate that the nsp14 MTase activity is independent of nsp10, contrarily to nsp14 ExoN that is upregulated in the presence of the cofactor. Additionally, our results show that the ExoN motif I has a prominent role on the ribonucleolytic activity of SARS-CoV-2 nsp14, contrasting to what was previously observed in other coronaviruses, which can be related to the pathogenesis of SARS-CoV-2. The knowledge provided in this work can serve as a basis to design effective drugs that target the pinpointed residues in order to disturb the complex assembly and affect the viral replication, ultimately, treating COVID-19 and other CoV infections.