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

Abstract

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3′-to-5′ exoribonuclease (ExoN) in nonstructural protein 14 (nsp14), which excises nucleotides including antiviral drugs misincorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here, we determined a 1.6-Å resolution crystal structure of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) ExoN in complex with its essential cofactor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3′ end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. We also show that the ExoN activity can rescue a stalled RNA primer poisoned with sofosbuvir and allow RdRp to continue its extension in the presence of the chain-terminating drug, biochemically recapitulating proofreading in SARS-CoV-2 replication. Molecular dynamics simulations further show remarkable flexibility of multidomain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA binding to support its exonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN–nsp10 complex serves as a platform for future development of anticoronaviral drugs or strategies to attenuate the viral virulence.

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

“…Further, the RNA-binding site is partially occluded by the lid fragment (Thr35-Lys47), which additionally buries the nsp10-binding site. It has been shown that Lys9 of the nsp14 is critical for exonuclease activity ( Moeller et al., 2022 ). This residue is not visible in our electron density.…”

Infectious Bronchitis Virus Nsp14 Degrades JAK1 to Inhibit the JAK-STAT Signaling Pathway in HD11 Cells

“…We also found that IBV Nsp14 inhibited the chIFN-γ-activated JAK-STAT signaling pathway in a dose-dependent manner. The CoV Nsp14 protein acts as a bifunctional protein, with the ExoN domain being crucial for CoV replication proofreading activity and the N7-MTase domain being involved in viral mRNA capping [ 38 , 39 ]. Recombinant MHV using reverse genetic techniques demonstrated that guanine N7-methylation of CoV Nsp14 helps the virus evade the type-I-IFN-mediated immune response and pathogenesis in mice [ 40 ].…”

Recent insights into the structure and function of coronavirus ribonucleases

“…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 ]. Comparing the structural coordinates of Nsp14 in metal‐free ( 7MC5 , [ 43 ]), metal B site bound ( 7DIY , [ 44 ]), and substrate and metal A and B site bound ( 7N0B , [ 36 ]) states, reveals that the engagement of substrate and metals results in a conformational change surrounding the active site and the substrate‐binding site (Fig 3B , right). While the structural studies do not fully detail the mechanism of Nsp14 exonuclease activity, these recent structures provide the framework for future structural studies.…”