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Substrate Specificity and Kinetics of RNA Hydrolysis by SARS-CoV-2 NSP10/14 Exonuclease

Tyler L Dangerfield1, Kenneth A Johnson1

  • 1Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas, 2500 Speedway, Austin, Texas 78712, United States.

ACS Bio & Med Chem Au
|December 26, 2022
PubMed

Insights

The SARS-CoV-2 exonuclease preferentially removes remdesivir from double-stranded RNA. This finding helps in developing new antiviral drugs against COVID-19 by understanding viral RNA replication and resistance mechanisms.

Area of Science:

  • Virology
  • Molecular Biology
  • Drug Discovery

Background:

  • Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is evolving resistance to current therapies.
  • New direct-acting antiviral drugs are urgently needed to combat COVID-19.
  • The SARS-CoV-2 exonuclease complex (NSP10/NSP14) plays a role in viral RNA replication fidelity and antiviral resistance, but its precise function remains unclear.

Purpose of the Study:

  • To investigate the substrate specificity and kinetic properties of the SARS-CoV-2 exonuclease complex.
  • To determine the efficiency of remdesivir removal by the exonuclease.
  • To inform the development of next-generation antiviral drugs and inhibitors.

Main Methods:

  • Single turnover kinetic analysis was employed to study the exonuclease activity.
  • The hydrolysis rates of various RNA substrates, including those with mismatches or remdesivir incorporation, were measured.
  • Enzyme kinetics were rigorously analyzed to define substrate preferences.

Main Results:

  • The SARS-CoV-2 exonuclease preferentially hydrolyzes double-stranded RNA lacking mismatches.
  • Remdesivir at the 3'-terminus of double-stranded RNA was hydrolyzed at a rate comparable to a correctly base-paired nucleotide.
  • Single-stranded RNA or mismatched duplex RNA were hydrolyzed significantly slower (125- and 45-fold, respectively) than correctly paired double-stranded RNA.

Conclusions:

  • The study defines the substrate specificity and remdesivir removal rate for the SARS-CoV-2 exonuclease.
  • These findings provide a basis for designing improved exonuclease inhibitors and nucleoside analogs that evade excision.
  • Further kinetic analysis is crucial for understanding the polymerase/exonuclease complex's role in viral replication and for developing effective COVID-19 therapeutics.

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