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

Result code in 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>

Alternative codes found

RIV/60461373:22330/24:43928477

Result on the web

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DOI - Digital Object Identifier

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Result language

angličtina

Original language name

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

Original language description

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.

Czech name

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Czech description

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Type

O - Miscellaneous

CEP classification

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OECD FORD branch

10610 - Biophysics

Project

<a href="/en/project/LX22NPO5103" target="_blank" >LX22NPO5103: National Institute of Virology and Bacteriology</a><br>

Continuities

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

Publication year

2024

Confidentiality

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