Significance SARS-CoV-2 nonstructural protein 14 (nsp14) exoribonuclease (ExoN) plays important roles in the proofreading during viral RNA synthesis and the evasion of host immune responses. We used X-ray crystallography, molecular dynamics simulations, and biochemical assays to investigate the structure, dynamics, and RNA-binding mechanisms of nsp14-ExoN and how its activity is regulated by another viral protein, nsp10. We also demonstrated that nsp14-ExoN can collaborate with the viral RNA polymerase to enable RNA synthesis in the presence of a chain-terminating drug, biochemically recapitulating the proofreading process. Our studies provide mechanistic insights into the functions of a key viral enzyme and a basis for future development of chemical inhibitors. 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.
【저자키워드】 SARS-CoV-2, molecular dynamics simulations, crystal structure, Exoribonuclease, Proofreading, 【초록키워드】 Innate immunity, X-ray crystallography, drug, inhibitors, antiviral drug, Molecular dynamics simulation, RNA, Replication, nsp14, modeling, RdRP, RNA-dependent RNA polymerase, CoV, Viral RNA, molecular, platform, SARS-CoV-2 replication, mechanism, function, Viral virulence, binding, exonuclease, nucleotide, double-stranded RNA, RNA recombination, nonstructural protein 14, cofactor, Support, acute respiratory syndrome, Viral protein, enzyme, RNA synthesis, complex, residue, CoVs, viral RNA synthesis, host immune responses, 3′ end, nsp14-ExoN, RNA genome, substrate, extension, polymerase, N-terminal, high-resolution structure, biochemical assay, bind, coronavirus, in viral, biochemically, demonstrated, regulated, analyses, implicated, attenuate, RNA primer, Significance, the SARS-CoV-2, 【제목키워드】 Structure,