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

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has triggered a global pandemic with devastating consequences for health-care and social-economic systems. Thus, the understanding of fundamental aspects of SARS-CoV-2 is of extreme importance.In this work, we have focused our attention on the viral ribonuclease (RNase) nsp14, since this protein was considered one of the most interferon antagonists from SARS-CoV-2, and affects viral replication. This RNase is a multifunctional protein that harbors two distinct activities, an N-terminal 3’-to-5’ exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), both with critical roles in coronaviruses life cycle. Namely, SARS-CoV-2 nsp14 ExoN knockout mutants are non-viable, indicating nsp14 as a prominent target for the development of antiviral drugs.Nsp14 ExoN activity is stimulated through the interaction with the nsp10 protein, which has a pleiotropic function during viral replication. In this study, we have performed the first biochemical characterization of the complex nsp14-nsp10 from SARS-CoV-2. Here we confirm the 3’-5’ exoribonuclease and MTase activities of nsp14 in this new Coronavirus, and the critical role of nsp10 in upregulating the nsp14 ExoN activity in vitro. Furthermore, we demonstrate that SARS-CoV-2 nsp14 N7-MTase activity is functionally independent of the ExoN activity. The nsp14 MTase activity also seems to be independent of the presence of nsp10 cofactor, contrarily to nsp14 ExoN.Until now, there is no available structure for the SARS-CoV-2 nsp14-nsp10 complex. As such, we have modelled the SARS-CoV-2 nsp14-nsp10 complex based on the 3D structure of the complex from SARS-CoV (PDB ID 5C8S). We also have managed to map key nsp10 residues involved in its interaction with nsp14, all of which are also shown to be essential for stimulation of the nsp14 ExoN activity. This reinforces the idea that a stable interaction between nsp10 and nsp14 is strictly required for the nsp14-mediated ExoN activity of SARS-CoV-2, as observed for SARS-CoV.We have 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 conserved catalytic residues in SARS-CoV, and in the Middle East respiratory syndrome coronavírus (MERS). The differences here revealed can have important implications regarding the specific pathogenesis of SARS-CoV-2.The nsp10-nsp14 interface is a recognized attractive target for antivirals against SARS-CoV-2 and other coronaviruses. This work has unravelled a basis for discovering inhibitors targeting the specific amino acids here reported, in order to disrupt the assembly of this complex and interfere with coronaviruses replication.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp14/nsp10 exoribonuclease

“…Previously, nsp14/nsp10 exonuclease activity had only been studied by monitoring cleavage of RNA substrates in gel-based assays using an excess of enzyme over substrate concentration [ 26 , 36 , 54 , 55 ]. We have purified nsp14 and nsp10 proteins from E. coli and from S. frugiperda insect cells and shown that both expression systems can be used to produce active nsp14/nsp10 exonuclease (ExoN).…”

Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role inBetacoronavirusreplication

“…Additional support came from immunoprecipitation experiments using a cap-specific monoclonal antibody (recognizing both the rare nucleoside 2,2,7-trimethylguanosine and 7-methylguanosine (m7G) cap structures ) that brought down the mRNAs of equine torovirus (65), a distant CoV relative for whichperhaps strikinglyan N7-MTase domain still remains to be identified (45). The presence of enzymes required for capping in CoVs and many of their relatives (6,17,45,47,66) and the in vitro activity profile of recombinant CoV nsp14 (26,27,32,33,37,38) lend additional credibility to CoV capping and cap methylation, but do not exclude the possibility that the CoV N7-MTase may target other substrates as well.…”

Coronavirus genomic nsp14‐ExoN, structure, role, mechanism, and potential application as a drug target

“…Also, nsp14 is involved in many other virus life cycles and pathogenicity processes, including recombination of the viral genome and control of innate immune responses 31 . The exoribonucleolytic activity of nsp14 3ʹ−5ʹ relies on metal ions, preferably Mg 2+ over Mn 2+ , Co 2+ , and Zn 2+ promote residual activity, while catalysis was not supported by Ca 2+ , Ni 2+ , and Cu 2+ 32 …”