Skip to main content
OSTI.GOVU.S. Department of Energy Office of Scientific and Technical Information
U.S. Department of Energy
Office of Scientific and Technical Information
 

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

Journal Article · · Proceedings of the National Academy of Sciences of the United States of America
 [1];  [1];  [2];  [1];  [3];  [1];  [1];  [2];  [1]
  1. Univ. of Minnesota, Minneapolis, MN (United States)
  2. Univ. of California San Diego, La Jolla, CA (United States)
  3. Argonne National Lab. (ANL), Argonne, IL (United States). Northeastern Collaborative Access Team (NCAT); Cornell Univ., Lemont, IL (United States)
+ Show Author Affiliations
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.
Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Organization:
National Cancer Institute; National Institutes of Health (NIH); National Science Foundation (NSF); USDOE Office of Science (SC)
Grant/Contract Number:
AC02-06CH11357
OSTI ID:
1847131
Journal Information:
Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 9 Vol. 119; ISSN 0027-8424
Publisher:
National Academy of SciencesCopyright Statement
Country of Publication:
United States
Language:
ENGLISH

References (49)

Structural basis for the 3′-5′ exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. journal January 1991
Rates of Molecular Evolution in RNA Viruses: A Quantitative Phylogenetic Analysis journal February 2002
Novel Zinc Protein Molecular Dynamics Simulations: Steps Toward Antiangiogenesis for Cancer Treatment journal October 1999
MDTraj: A Modern Open Library for the Analysis of Molecular Dynamics Trajectories journal October 2015
Coupling of N7-methyltransferase and 3′-5′ exoribonuclease with SARS-CoV-2 polymerase reveals mechanisms for capping and proofreading journal June 2021
Coronavirus RNA Proofreading: Molecular Basis and Therapeutic Targeting journal September 2020
Nidovirales: Evolving the largest RNA virus genome journal April 2006
Nidovirus RNA polymerases: Complex enzymes handling exceptional RNA genomes journal April 2017
A Community Letter Regarding Sharing Biomolecular Simulation Data for COVID-19 journal April 2020
ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB journal July 2015
Cap binding and immune evasion revealed by Lassa nucleoprotein structure journal November 2010
A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease journal November 2012
Rapid incorporation of Favipiravir by the fast and permissive viral RNA polymerase complex results in SARS-CoV-2 lethal mutagenesis journal September 2020
Structure of replicating SARS-CoV-2 polymerase journal May 2020
Sofosbuvir terminated RNA is more resistant to SARS-CoV-2 proofreader than RNA terminated by Remdesivir journal October 2020
Scalable molecular dynamics on CPU and GPU architectures with NAMD journal July 2020
Discovery of an RNA virus 3'->5' exoribonuclease that is critically involved in coronavirus RNA synthesis journal March 2006
RNA 3'-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex journal May 2012
Structural basis and functional analysis of the SARS coronavirus nsp14–nsp10 complex journal July 2015
Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA journal December 2017
Electrostatics of nanosystems: Application to microtubules and the ribosome journal August 2001
Mutation rates among RNA viruses journal November 1999
Structures of Arenaviral Nucleoproteins with Triphosphate dsRNA Reveal a Unique Mechanism of Immune Suppression journal June 2013
Coronavirus Nsp10, a Critical Co-factor for Activation of Multiple Replicative Enzymes journal July 2014
In vitro antiviral activity of the anti-HCV drugs daclatasvir and sofosbuvir against SARS-CoV-2, the aetiological agent of COVID-19 journal April 2021
Characterization of the SARS-CoV-2 ExoN (nsp14ExoN–nsp10) complex: implications for its role in viral genome stability and inhibitor identification journal January 2022
PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations journal July 2004
Phaser crystallographic software journal July 2007
XDS journal January 2010
Features and development of Coot journal March 2010
Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix journal October 2019
New targets for drug design: importance of nsp14/nsp10 complex formation for the 3’‐5’ exoribonucleolytic activity on SARS‐CoV‐2 journal April 2021
Structure of DNA polymerase I Klenow fragment bound to duplex DNA journal April 1993
Structural basis of mismatch recognition by a SARS-CoV-2 proofreading enzyme journal September 2021
Structure-Function Analysis of Severe Acute Respiratory Syndrome Coronavirus RNA Cap Guanine-N7-Methyltransferase journal March 2013
Viral Mutation Rates journal July 2010
The Enzymatic Activity of the nsp14 Exoribonuclease Is Critical for Replication of MERS-CoV and SARS-CoV-2 journal November 2020
High Fidelity of Murine Hepatitis Virus Replication Is Decreased in nsp14 Exoribonuclease Mutants journal November 2007
Murine Hepatitis Virus nsp14 Exoribonuclease Activity Is Required for Resistance to Innate Immunity journal January 2018
Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease journal March 2018
Continuous and Discontinuous RNA Synthesis in Coronaviruses journal November 2015
Structural Basis for the dsRNA Specificity of the Lassa Virus NP Exonuclease journal August 2012
Infidelity of SARS-CoV Nsp14-Exonuclease Mutant Virus Replication Is Revealed by Complete Genome Sequencing journal May 2010
Coronaviruses Lacking Exoribonuclease Activity Are Susceptible to Lethal Mutagenesis: Evidence for Proofreading and Potential Therapeutics journal August 2013
Coronaviruses as DNA Wannabes: A New Model for the Regulation of RNA Virus Replication Fidelity journal December 2013
The coronavirus proofreading exoribonuclease mediates extensive viral recombination journal January 2021
Coronaviruses: An RNA proofreading machine regulates replication fidelity and diversity journal March 2011
Biochemical Characterization of Exoribonuclease Encoded by SARS Coronavirus journal September 2007
Mutations of SARS-CoV-2 nsp14 exhibit strong association with increased genome-wide mutation load journal January 2020

Cited By (1)

Additional file 1 of The RdRp genotyping of SARS-CoV-2 isolated from patients with different clinical spectrum of COVID-19 dataset January 2024

Similar Records

Structures of SARS-CoV-2 N7-methyltransferase with DOT1L and PRMT7 inhibitors provide a platform for new antivirals
Journal Article · Sun Jul 30 20:00:00 EDT 2023 · PLoS Pathogens · OSTI ID:1994568

Related Subjects

60 APPLIED LIFE SCIENCES
SARS-CoV-2
biological sciences
biophysics and computational biology
crystal structure
exoribonuclease
molecular dynamics simulations
proofreading