Biochemical characterization of naturally occurring mutations in SARS-CoV-2 RNA-dependent RNA polymerase

Abstract 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. Additionally, some of the examined substitutions in the RdRp subunits notably influenced the sensitivity of RdRp to Remdesivir®, highlighting their potential implications for therapeutic strategies. 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. Significance statement While the impact of SARS-CoV-2 spike protein mutations has been extensively explored, our understanding of mutations within the RNA-dependent RNA polymerase (RdRp), crucial for viral replication and a key target for antivirals like Remdesivir, remains limited with studies conducted solely in silico . We focused on selected RdRp mutations identified from December 2019 to June 2022, assessing their effects on enzyme stability, complex assembly, and activity. Advanced biochemical analyses reveal how these mutations can alter RdRp functionality, providing insights into viral evolution and resistance mechanisms. This study, pioneering in assessing the biochemical implications of RdRp mutations, provides invaluable insights into their roles in viral replication and antiviral resistance, hereby opening new pathways for developing therapies against the continuously evolving SARS-CoV-2 variants.