Multiscale characterization of natural amino acid substitutions in RNA-dependent RNA polymerase of SARS-CoV-2

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60461373%3A22810%2F24%3A43928477" target="_blank" >RIV/60461373:22810/24:43928477 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/60461373:22330/24:43928477

Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Multiscale characterization of natural amino acid substitutions in RNA-dependent RNA polymerase of SARS-CoV-2

Popis výsledku v původním jazyce

Since the emergence of SARSCoV 2, mutations have been repeatedly reported in the sequencesencoding nonstructural proteins (nsps), including the components of its minimal RNA dependent RNApolymerase (RdRp) complex: nsp7, nsp8 and nsp12. Despite the RdRp is a prime target for antiviraldrugs, experimental studies focusing on these mutations and their resulting amino acid substitutionshave been limited. To address this, we carried out a comprehensive analysis of representative aminoacid substitutions with in the RdRp subunits We selected mutations based on their frequency,localization of the encoded residue within the RdRp complex, and the nature of the resulting aminoacid. Subsequently, we conducted mutagenesis and purification of all the respective prot eins.Combining Nano Differential Scanning Fluorimetry (nanoDSF) and Microscale Thermophoresis (MST)assay, we analyzed the impact of these substitutions on protein stability, protein protein interactionsand complex assembly. Our results indicated that th e selected substitutions led to an increase inthermal stability of nsp8 by 4 °C to 13 °C, while nsp7 variants exhibited thermal destabilization. Specificsubstitutions, namely T141M in nsp8 and S25L in nsp7, negatively affected the RdRp complexformation. To assess the impact on RdRp activity, we developed a fluorometric enzymatic assayrevealing that even a single amino acid substitution in either protein could alter the RdRp activity,correlating with biophysical properties. Interestingly, a combination of two co occurring proteinvariants nsp12 P323L and nsp8 A21V increased the overall activity by more than 50 %. Furthermore,we evaluated the response of various RdRp variants to a commonly used anti RdRp drug, Remdesivirand discovered that certain m utations significantly reduced the drug’s efficacy. In conclusion, ourresults provide a basis for understanding new RdRp variants and their potential importance for drugdevelopment.

Název v anglickém jazyce

Multiscale characterization of natural amino acid substitutions in RNA-dependent RNA polymerase of SARS-CoV-2

Popis výsledku anglicky

Since the emergence of SARSCoV 2, mutations have been repeatedly reported in the sequencesencoding nonstructural proteins (nsps), including the components of its minimal RNA dependent RNApolymerase (RdRp) complex: nsp7, nsp8 and nsp12. Despite the RdRp is a prime target for antiviraldrugs, experimental studies focusing on these mutations and their resulting amino acid substitutionshave been limited. To address this, we carried out a comprehensive analysis of representative aminoacid substitutions with in the RdRp subunits We selected mutations based on their frequency,localization of the encoded residue within the RdRp complex, and the nature of the resulting aminoacid. Subsequently, we conducted mutagenesis and purification of all the respective prot eins.Combining Nano Differential Scanning Fluorimetry (nanoDSF) and Microscale Thermophoresis (MST)assay, we analyzed the impact of these substitutions on protein stability, protein protein interactionsand complex assembly. Our results indicated that th e selected substitutions led to an increase inthermal stability of nsp8 by 4 °C to 13 °C, while nsp7 variants exhibited thermal destabilization. Specificsubstitutions, namely T141M in nsp8 and S25L in nsp7, negatively affected the RdRp complexformation. To assess the impact on RdRp activity, we developed a fluorometric enzymatic assayrevealing that even a single amino acid substitution in either protein could alter the RdRp activity,correlating with biophysical properties. Interestingly, a combination of two co occurring proteinvariants nsp12 P323L and nsp8 A21V increased the overall activity by more than 50 %. Furthermore,we evaluated the response of various RdRp variants to a commonly used anti RdRp drug, Remdesivirand discovered that certain m utations significantly reduced the drug’s efficacy. In conclusion, ourresults provide a basis for understanding new RdRp variants and their potential importance for drugdevelopment.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

10610 - Biophysics

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

2024

Kód důvěrnosti údajů

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