Dataset Information

Activation of the SARS-CoV-2 NSP14 3'-5' exoribonuclease by NSP10 and response to antiviral inhibitors.

ABSTRACT: Understanding the core replication complex of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to the development of novel coronavirus-specific antiviral therapeutics. Among the proteins required for faithful replication of the SARS-CoV-2 genome are nonstructural protein 14 (NSP14), a bifunctional enzyme with an N-terminal 3'-to-5' exoribonuclease (ExoN) and a C-terminal N7-methyltransferase, and its accessory protein, NSP10. The difficulty in producing pure and high quantities of the NSP10/14 complex has hampered the biochemical and structural study of these important proteins. We developed a straightforward protocol for the expression and purification of both NSP10 and NSP14 from Escherichia coli and for the in vitro assembly and purification of a stoichiometric NSP10/14 complex with high yields. Using these methods, we observe that NSP10 provides a 260-fold increase in k_cat/K_m in the exoribonucleolytic activity of NSP14 and enhances protein stability. We also probed the effect of two small molecules on NSP10/14 activity, remdesivir monophosphate and the methyltransferase inhibitor S-adenosylhomocysteine. Our analysis highlights two important factors for drug development: first, unlike other exonucleases, the monophosphate nucleoside analog intermediate of remdesivir does not inhibit NSP14 activity; and second, S-adenosylhomocysteine modestly activates NSP14 exonuclease activity. In total, our analysis provides insights for future structure-function studies of SARS-CoV-2 replication fidelity for the treatment of coronavirus disease 2019.

SUBMITTER: Riccio AA

PROVIDER: S-EPMC8685350 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Coronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains 3'-to-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) domains. While the latter presumably supports mRNA capping, ExoN is thought to mediate proofreading during genome replication. In line with such a role, ExoN knockout mutants of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) were previously reported to have crippled but viable hypermutation phenotypes. Remarkably, using reverse genetics, a large set of corresponding ExoN knockout mutations has now been found to be lethal for another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). For 13 mutants, viral progeny could not be recovered, unless-as happened occasionally-reversion had first occurred. Only a single mutant was viable, likely because its E191D substitution is highly conservative. Remarkably, a SARS-CoV-2 ExoN knockout mutant was found to be unable to replicate, resembling observations previously made for alpha- and gammacoronaviruses, but starkly contrasting with the documented phenotype of ExoN knockout mutants of the closely related SARS-CoV. Subsequently, we established in vitro assays with purified recombinant MERS-CoV nsp14 to monitor its ExoN and N7-MTase activities. All ExoN knockout mutations that proved lethal in reverse genetics were found to severely decrease ExoN activity while not affecting N7-MTase activity. Our study strongly suggests that CoV nsp14 ExoN has an additional function, which apparently is critical for primary viral RNA synthesis and thus differs from the proofreading function that, based on previous MHV and SARS-CoV studies, was proposed to boost longer-term replication fidelity.IMPORTANCE The bifunctional nsp14 subunit of the coronavirus replicase contains 3'-to-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase domains. For the betacoronaviruses MHV and SARS-CoV, ExoN was reported to promote the fidelity of genome replication, presumably by mediating a form of proofreading. For these viruses, ExoN knockout mutants are viable while displaying an increased mutation frequency. Strikingly, we have now established that the equivalent ExoN knockout mutants of two other betacoronaviruses, MERS-CoV and SARS-CoV-2, are nonviable, suggesting an additional and critical ExoN function in their replication. This is remarkable in light of the very limited genetic distance between SARS-CoV and SARS-CoV-2, which is highlighted, for example, by 95% amino acid sequence identity in their nsp14 sequences. For (recombinant) MERS-CoV nsp14, both its enzymatic activities were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development.

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

Activation of the SARS-CoV-2 NSP14 3'-5' exoribonuclease by NSP10 and response to antiviral inhibitors.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets