Mismatch Recognition and Proofreading Dynamics Within the SARS-CoV-2 Replication-Transcription Complex

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60461373%3A22810%2F25%3A43932655" target="_blank" >RIV/60461373:22810/25:43932655 - isvavai.cz</a>

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RIV/60461373:22330/25:43932655

Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Mismatch Recognition and Proofreading Dynamics Within the SARS-CoV-2 Replication-Transcription Complex

Popis výsledku v původním jazyce

The replication–transcription complex (RTC), responsible for RNA synthesis, is a prime target for antiviral drug development against SARS‑CoV‑2 and other coronaviruses. The RTC comprises the RNA‑dependent RNA polymerase (RdRp) complex, composed of the catalytic subunit nonstructural protein (nsp) 12 and the cofactors nsp7 and nsp8, along with other accessory proteins. Crucially, it also incorporates a 3’ – 5’ exonuclease (ExoN) that performs proofreading and can remove antiviral nucleotide analogs. Despite the importance of RdRp‑ExoN regulation for designing novel antivirals, the precise molecular mechanism of mismatch recognition and removal remains unknown. In this study, we investigated the effect of misincorporated ribonucleotides at the 3’ end within the context of the dsRNA–RdRp complex, using a combination of enzymatic and biophysical methods. This revealed that dsRNA length and mismatch identity significantly affect the dsRNA–RdRp complex stability. Subsequent catalytic activity evaluation showed that mismatches can either slow polymerase kinetics, inhibit RdRp activity, or be ignored, correlating with prior stability assessment. We then assessed whether ExoN can cleave the RNA bound to RdRp and restore the polymerase activity. Consistentwith previous reports, cleavage was significantly reduced, while the activity was restored only to a very limited extent, suggesting the involvement of additional regulatory proteins. Finally, we explored the impact of additional RTC components, specifically the nsp13 helicase and RNA‑binding protein nsp9. Notably, nsp13 further stabilized the RNA, preventing cleavage while also suppressing the RdRp activity.Altogether, this study highlights the intricate regulatory interplay within the RTC and provides insights for future mechanistic investigations.This study was supported by the project National Institute of Virology and Bacteriology (Programme EXCELES, ID Project No. LX22NPO5103) – Funded by the European Union – Next Generation EU.

Název v anglickém jazyce

Mismatch Recognition and Proofreading Dynamics Within the SARS-CoV-2 Replication-Transcription Complex

Popis výsledku anglicky

The replication–transcription complex (RTC), responsible for RNA synthesis, is a prime target for antiviral drug development against SARS‑CoV‑2 and other coronaviruses. The RTC comprises the RNA‑dependent RNA polymerase (RdRp) complex, composed of the catalytic subunit nonstructural protein (nsp) 12 and the cofactors nsp7 and nsp8, along with other accessory proteins. Crucially, it also incorporates a 3’ – 5’ exonuclease (ExoN) that performs proofreading and can remove antiviral nucleotide analogs. Despite the importance of RdRp‑ExoN regulation for designing novel antivirals, the precise molecular mechanism of mismatch recognition and removal remains unknown. In this study, we investigated the effect of misincorporated ribonucleotides at the 3’ end within the context of the dsRNA–RdRp complex, using a combination of enzymatic and biophysical methods. This revealed that dsRNA length and mismatch identity significantly affect the dsRNA–RdRp complex stability. Subsequent catalytic activity evaluation showed that mismatches can either slow polymerase kinetics, inhibit RdRp activity, or be ignored, correlating with prior stability assessment. We then assessed whether ExoN can cleave the RNA bound to RdRp and restore the polymerase activity. Consistentwith previous reports, cleavage was significantly reduced, while the activity was restored only to a very limited extent, suggesting the involvement of additional regulatory proteins. Finally, we explored the impact of additional RTC components, specifically the nsp13 helicase and RNA‑binding protein nsp9. Notably, nsp13 further stabilized the RNA, preventing cleavage while also suppressing the RdRp activity.Altogether, this study highlights the intricate regulatory interplay within the RTC and provides insights for future mechanistic investigations.This study was supported by the project National Institute of Virology and Bacteriology (Programme EXCELES, ID Project No. LX22NPO5103) – Funded by the European Union – Next Generation EU.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

10607 - Virology

Projekt

<a href="/cs/project/LX22NPO5103" target="_blank" >LX22NPO5103: Národní institut virologie a bakteriologie</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2025

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů