EFFECT OF NATURALLY OCCURRING SUBSTITUTION I42V IN SARS-COV-2 NSP14 ON EXORIBONUCLEASE ACTIVITY AND STABILITY

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60461373%3A22330%2F25%3A43932630" target="_blank" >RIV/60461373:22330/25:43932630 - isvavai.cz</a>

Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

EFFECT OF NATURALLY OCCURRING SUBSTITUTION I42V IN SARS-COV-2 NSP14 ON EXORIBONUCLEASE ACTIVITY AND STABILITY

Popis výsledku v původním jazyce

SARS-CoV-2 replication-transcription complex (RTC), a complex of non-structural proteins and RNA, is crucial for the replication of SARS-CoV-2, the causative agent of the global pandemic COVID-19. The RNA-dependent RNA polymerase (RdRp), which is responsible for the synthesis of 29.9kb single-stranded genomic RNA, plays a crucial role in this complex. Unlike cellular DNA polymerases, viral RdRp does not contain a high-fidelity exonuclease proofreading domain. High-fidelity replication of the viral genome is mediated by the 3′-to-5′ exoribonuclease (ExoN), which is comprised by the N-terminal part of non-structural protein 14 (nsp14). The formation of the nsp14 complex with another viral protein, nsp10, is essential for ExoN activity. ExoN excises nucleotides including nucleotide-based antivirotics misincorporated by low-fidelity viral RdRp. In the Omicron SARS-CoV-2 variant and its subvariants, including currently circulating variants, an amino acid substitution I42V in nsp14 is conserved. The mutation is situated outside of the active site of nsp14 and also it does not participate in interaction with nsp10 or with RNA substrate. We hypothesize that the mutation does not directly affect the ExoN activity of nsp14, but that it could play a role during the formation of the nsp14/nsp10 complex.We expressed, isolated and purified the nsp14 WT protein and its mutant variant nsp14 I42V to compare their properties. Protein production was carried out in the bacterial expression system. This was followed by multi-step isolation and purification by fast protein liquid chromatography according to the protocol available in the laboratory. In order to study the exoribonuclease activity of nsp14, it was also necessary to express, isolate and purify nsp10. The ExoN activity of prepared proteins was verified by activity assay.Preliminary data of thermal stability show that nsp14 I42V mutant exhibits lower stability than the wild-type protein. The effect was comparable to that obtained through chemical denaturation with urea.

Název v anglickém jazyce

EFFECT OF NATURALLY OCCURRING SUBSTITUTION I42V IN SARS-COV-2 NSP14 ON EXORIBONUCLEASE ACTIVITY AND STABILITY

Popis výsledku anglicky

SARS-CoV-2 replication-transcription complex (RTC), a complex of non-structural proteins and RNA, is crucial for the replication of SARS-CoV-2, the causative agent of the global pandemic COVID-19. The RNA-dependent RNA polymerase (RdRp), which is responsible for the synthesis of 29.9kb single-stranded genomic RNA, plays a crucial role in this complex. Unlike cellular DNA polymerases, viral RdRp does not contain a high-fidelity exonuclease proofreading domain. High-fidelity replication of the viral genome is mediated by the 3′-to-5′ exoribonuclease (ExoN), which is comprised by the N-terminal part of non-structural protein 14 (nsp14). The formation of the nsp14 complex with another viral protein, nsp10, is essential for ExoN activity. ExoN excises nucleotides including nucleotide-based antivirotics misincorporated by low-fidelity viral RdRp. In the Omicron SARS-CoV-2 variant and its subvariants, including currently circulating variants, an amino acid substitution I42V in nsp14 is conserved. The mutation is situated outside of the active site of nsp14 and also it does not participate in interaction with nsp10 or with RNA substrate. We hypothesize that the mutation does not directly affect the ExoN activity of nsp14, but that it could play a role during the formation of the nsp14/nsp10 complex.We expressed, isolated and purified the nsp14 WT protein and its mutant variant nsp14 I42V to compare their properties. Protein production was carried out in the bacterial expression system. This was followed by multi-step isolation and purification by fast protein liquid chromatography according to the protocol available in the laboratory. In order to study the exoribonuclease activity of nsp14, it was also necessary to express, isolate and purify nsp10. The ExoN activity of prepared proteins was verified by activity assay.Preliminary data of thermal stability show that nsp14 I42V mutant exhibits lower stability than the wild-type protein. The effect was comparable to that obtained through chemical denaturation with urea.

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