Characterization of the SARS-CoV-2 ExoN (nsp14ExoN–nsp10) complex: implications for its role in viral genome stability and inhibitor identification

Abstract

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14–nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14–nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14–nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3′-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14–nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12–nsp7–nsp8 (nsp12–7–8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14–nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14–nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.

Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN

“…Previous studies showed that the ExoN activity of nsp14 is strongly stimulated by nsp10 for both SARS-CoV and SARS-CoV-2 ( 32 , 34 – 36 ). In our crystal structure, the N-terminal residues of ExoN and those of nsp10 are wrapped around each other in a “criss-cross” arrangement and forming several hydrogen bond contacts, including one between nsp14 Lys9 and nsp10 Ala1 ( SI Appendix , Fig.…”

Recent insights into the structure and function of coronavirus ribonucleases

“…Excision proceeds in single base increments as confirmed by sizing analysis performed by Baddock et al. [ 41 ], which indicates there is a nucleophilic attack of the scissile phosphodiester, and protonation of the leaving group, catalyzing a 3′–5′ nucleoside monophosphate exit. Likewise, a self‐assembled monolayer for matrix‐assisted laser desorption ionization (SAMDI) mass spectrometry assay indicates that Nsp14/Nsp10 requires a 3′OH, confirming that the enzyme acts in a 3′–5′ direction and that the first nucleotide likely is digested before proceeding with subsequent activities [ 42 ].…”

“…[ 46 ] have shown biochemically that Nsp10/14 hydrolyzes RNAs in dsRNA, ssRNA, and RNA DNA/hybrid. Likewise, cleavage is observed on a mismatch at the 3′ end of RNA, while longer flaps or gaps showed no strong preference in exonuclease activity [ 41 ]. At high concentrations Nsp14, in the absence of Nsp10, has also been shown to minimally act on dsRNA substrates.…”

“…Interestingly, in vitro and structural studies seem to support the observation that a 3′ OH is critical for the cleavage of misincorporated RNA bases [ 36 ]. Additionally, Nsp14 in both SARS‐CoV‐1 and ‐2 show no sequence specificity [ 41 ]. These same analyses show that activity is substantially reduced on poly‐U or poly‐A RNA perhaps supported by the hypothesis that Nsp14 would have specificity for certain types of ternary structures in SARS‐COV‐2 [ 26 ].…”

Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity

“…The structural basis of nsp14 interaction with nsp10 has been elucidated 11 . We have recently demonstrated that nsp14, nsp10 and nsp16 form a ternary complex, further modulating nsp14 catalytic activity 12 .…”