Abstract
RNA-dependent RNA polymerase (RdRp) is the central molecular machine for viral replication and transcription. It catalyzes the nucleotide addition reaction, in which the release of the pyrophosphate ion (PPi) is a critical step in the resumption of the catalysis cycle. However, the precise mechanism by which PPi is released in the viral RdRp remains elusive, thereby impeding a comprehensive comprehension of the transcription and replication mechanisms of RdRp. In this study, we elucidated the molecular mechanism of PPi release in SARS-CoV-2 through the construction of a tailor-made Markov state model informed by extensive molecular dynamics simulations. We identified three metastable states, which demonstrate the stepwise release of PPi over a timescale of 3.6 μs. Additionally, we discovered that four highly conserved and positively charged residues, K551, R553, R555, and K798, can collectively mediate the release of PPi, suggesting their typical roles in the transcription and replication of coronaviruses. A two-step NTP incorporation assay and a PPi-mediated cleavage assay (pyrophosphorolysis) were developed and performed, validating their impact on PPi release by assessing both forward nucleotide incorporation efficiency and reverse RNA cleavage efficiency under exogenous PPi treatment. Further comparisons with the mechanisms utilized by other polymerases indicate that our study reveals a distinctive model of PPi release. In summary, our investigation contributes to advancing our understanding of the transcription and replication mechanism of viral RdRps by offering a glimpse into the dynamics of PPi release in SARS-CoV-2 RdRp.