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

Nicholas H. Moeller, Ke Shi, Özlem Demir, Christopher Belica, Surajit Banerjee, Lulu Yin, Cameron Durfee, Rommie E. Amaro, Hideki Aihara

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 30-to-50 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 30 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.

Original languageEnglish (US)
Article numbere2106379119
JournalProceedings of the National Academy of Sciences of the United States of America
Volume119
Issue number9
DOIs
StatePublished - Mar 1 2022

Bibliographical note

Funding Information:
ACKNOWLEDGMENTS. We thank Daniel Harki and Reuben Harris for thoughtful comments. This work was supported by grants from the US NIH [National Institute of General Medical Sciences (NIGMS) R35-GM118047 to H.A., R01-GM132826 to R.E.A., and National Cancer Institute P01-CA234228 to R.E.A. and H.A.], NSF Rapid Response Research MCB-2032054, an award from the RCSA Research Corp., and a University of California San Diego Moores Cancer Center 2020 SARS-COV-2 seed grant to R.E.A. This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the US NIH (NIGMS P30 GM124165). The Pilatus 6M detector on 24-ID-C beamline is funded by NIH Office of Research Infrastructure Programs High-End Instrumentation Grant S10 RR029205. This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357, and those of the Minnesota Supercomputing Institute. We are grateful for the efforts of the Texas Advanced Computing Center Frontera team and for the computing time made available through a Director’s Discretionary Allocation (made possible by NSF Award OAC-1818253).

Funding Information:
We thank Daniel Harki and Reuben Harris for thoughtful comments. This work was supported by grants from the US NIH [National Institute of General Medical Sciences (NIGMS) R35-GM118047 to H.A., R01-GM132826 to R.E.A., and National Cancer Institute P01-CA234228 to R.E.A. and H.A.], NSF Rapid Response Research MCB-2032054, an award from the RCSA Research Corp., and a University of California San Diego Moores Cancer Center 2020 SARS-COV-2 seed grant to R.E.A. This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the US NIH (NIGMS P30 GM124165). The Pilatus 6M detector on 24-ID-C beamline is funded by NIH Office of Research Infrastructure Programs High-End Instrumentation Grant S10 RR029205. This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357, and those of the Minnesota Supercomputing Institute. We are grateful for the efforts of the Texas Advanced Computing Center Frontera team and for the computing time made available through a Director?s Discretionary Allocation (made possible by NSF Award OAC-1818253).

Publisher Copyright:
© 2022 National Academy of Sciences. All rights reserved.

Keywords

  • Crystal structure
  • Exoribonuclease
  • Molecular dynamics simulations
  • Proofreading
  • SARS-CoV-2

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

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