Slow PPi release enhances fidelity of the SARS-CoV-2 RNA dependent RNA polymerase.
Viral RNA-dependent RNA polymerases (RdRps) must balance replication speed with fidelity, preserving genome integrity while permitting enough variability for viral adaptation. The SARS-CoV-2 RdRp complex (non-structural protein 12/7/8) achieves this through the interplay of its intrinsic replication fidelity and a potential proofreading exonuclease complex (NSP10/14). Here, we comprehensively quantify the intrinsic fidelity of the SARS-CoV-2 RdRp through direct pre-steady-state kinetic analyses of nucleotide incorporation across all possible templating bases paired with incoming nucleotides. We discovered substantial variation in discrimination against mismatches ranging from one error in 103 to 108 (median of 105). Crucially, our data reveal a slow pyrophosphate release step that significantly enhances fidelity by effectively introducing a kinetic checkpoint after nucleotide incorporation. The error rates we measured for the RdRp align closely with observed in vivo mutation rates, suggesting that the exonuclease complex may play a less critical role than previously assumed in correcting mistakes during polymerization. These insights advance our understanding of SARS-CoV-2 replication fidelity, and the role of various subcomplexes in genome maintenance and adaptation.