期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：9
DOI：10.1073/pnas.2106379119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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.
