Since the emergence of SARS-CoV-2, mutations in all subunits of the RNA-dependent RNA polymerase (RdRp) of the virus have been repeatedly reported. Although RdRp represents a primary target for antiviral drugs, experimental studies exploring the phenotypic effect of these mutations have been limited. This study focuses on the phenotypic effects of substitutions in the three RdRp subunits: nsp7, nsp8, and nsp12, selected based on their occurrence rate and potential impact. We employed nano-differential scanning fluorimetry and microscale thermophoresis to examine the impact of these mutations on protein stability and RdRp complex assembly. We observed diverse impacts; notably, a single mutation in nsp8 significantly increased its stability as evidenced by a 13°C increase in melting temperature, whereas certain mutations in nsp7 and nsp8 reduced their binding affinity to nsp12 during RdRp complex formation. Using a fluorometric enzymatic assay, we assessed the overall effect on RNA polymerase activity. We found that most of the examined mutations altered the polymerase activity, often as a direct result of changes in stability or affinity to the other components of the RdRp complex. Intriguingly, a combination of nsp8 A21V and nsp12 P323L mutations resulted in a 50% increase in polymerase activity. To our knowledge, this is the first biochemical study to demonstrate the impact of amino acid mutations across all components constituting the RdRp complex in emerging SARS-CoV-2 subvariants.

• Klíčová slova
• RNA‐dependent RNA polymerase (RdRp), SARS‐CoV‐2, mutations, phenotypic effect,
• MeSH
• COVID-19 virologie   MeSH
• koronavirová RNA-replikasa * genetika   metabolismus   chemie   MeSH
• lidé MeSH
• mutace * MeSH
• RNA-dependentní RNA-polymerasa genetika   chemie   metabolismus   MeSH
• SARS-CoV-2 * genetika   enzymologie   MeSH
• stabilita proteinů MeSH
• vazba proteinů MeSH
• virové nestrukturální proteiny * genetika   chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• koronavirová RNA-replikasa * MeSH
• NS8 protein, SARS-CoV-2 MeSH Prohlížeč
• NSP12 protein, SARS-CoV-2 MeSH Prohlížeč
• NSP7 protein, SARS-CoV-2 MeSH Prohlížeč
• RNA-dependentní RNA-polymerasa MeSH
• virové nestrukturální proteiny * MeSH