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

ABSTRACT: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus that causes COVID-19, continues to evolve resistance to vaccines and existing antiviral therapies at an alarming rate, increasing the need for new direct-acting antiviral drugs. Despite significant advances in our fundamental understanding of the kinetics and mechanism of viral RNA replication, there are still open questions regarding how the proofreading exonuclease (NSP10/NSP14 complex) contributes to replication fidelity and resistance to nucleoside analogs. Through single turnover kinetic analysis, we show that the preferred substrate for the exonuclease is double-stranded RNA without any mismatches. Double-stranded RNA containing a 3'-terminal remdesivir was hydrolyzed at a rate similar to a correctly base-paired cognate nucleotide. Surprisingly, single-stranded RNA or duplex RNA containing a 3'-terminal mismatch was hydrolyzed at rates 125- and 45-fold slower, respectively, compared to the correctly base-paired double-stranded RNA. These results define the substrate specificity and rate of removal of remdesivir for the exonuclease and outline rigorous kinetic assays that could help in finding next-generation exonuclease inhibitors or nucleoside analogs that are able to evade excision. These results also raise important questions about the role of the polymerase/exonuclease complex in proofreading during viral replication. Addressing these questions through rigorous kinetic analysis will facilitate the search for desperately needed antiviral drugs to combat COVID-19.

SUBMITTER: Dangerfield TL

PROVIDER: S-EPMC9718090 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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

Dangerfield Tyler L TL Johnson Kenneth A KA

ACS bio & med chem Au 20221116 6

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus that causes COVID-19, continues to evolve resistance to vaccines and existing antiviral therapies at an alarming rate, increasing the need for new direct-acting antiviral drugs. Despite significant advances in our fundamental understanding of the kinetics and mechanism of viral RNA replication, there are still open questions regarding how the proofreading exonuclease (NSP10/NSP14 complex) contributes to replication fidelity ...[more]

PMID: 36570070

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Project description:The interaction of exoribonuclease (ExoN) nonstructural protein (NSP14) with NSP10 co-factors is crucial for high-fidelity proofreading activity of coronavirus replication and transcription. Proofreading function is critical for maintaining the large genomes to ensure replication proficiency; therefore, while maintaining the viral replication fitness, quick resistance has been reported to the nucleotide analogue (NA) drugs. Therefore, targeting the NSP14 and NSP10 interacting interface with small molecules or peptides could be a better strategy to obstruct replication processes of coronaviruses (CoVs). A comparative study on the binding mechanism of NSP10 with the NSP14 ExoN domain of SARS-CoV-2, SARS-CoV, MERS-CoV, and four SARS-CoV-2 NSP14mutant complexes has been carried out. Protein-protein interaction (PPI) dynamics, per-residue binding free energy (BFE) analyses, and the identification of interface hotspot residues have been studied using molecular dynamics simulations and various computational tools. The BFE of the SARS-CoV NSP14-NSP10 complex was higher when compared to novel SARS-CoV-2 and MERS. However, SARS-CoV-2 NSP14mutant systems display a higher BFE as compared to the wild type (WT) but lower than SARS-CoV and MERS. Despite the high BFE, the SARS-CoV NSP14-NSP10 complex appears to be structurally more flexible in many regions especially the catalytic site, which is not seen in SARS-CoV-2 and its mutant or MERS complexes. The significantly high residue energy contribution of key interface residues and hotspots reveals that the high binding energy between NSP14 and NSP10 may enhance the functional activity of the proofreading complex, as the NSP10-NSP14 interaction is essential in maintaining the stability of the ExoN domain for the replicative fitness of CoVs. The factors discussed for SARS-CoV-2 complexes may be responsible for NSP14 ExoN having a high replication proficiency, significantly leading to the evolution of new variants of SARS-CoV-2. The NSP14 residues V66, T69, D126, and I201and eight residues of NSP10 (L16, F19, V21, V42, M44, H80, K93, and F96) are identified as common hotspots. Overall, the interface area, hotspot locations, bonded/nonbonded contacts, and energies between NSP14 and NSP10 may pave a way in designing potential inhibitors to disrupt NSP14-NSP10 interactions of CoVs especially SARS-CoV-2.

Dataset Information

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

Publications

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

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