Significance The COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome is replicated and transcribed by its RNA-dependent RNA polymerase (RdRp), which is the target for antivirals such as remdesivir. We use a combination of approaches to show that backtracking (backward motion of the RdRp on the template RNA) is a feature of SARS-CoV-2 replication/transcription. Backtracking may play a critical role in proofreading, a crucial process for SARS-CoV-2 resistance against many antivirals. Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA–protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3′ segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3′ end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.
【저자키워드】 coronavirus, Cryo-electron microscopy, molecular dynamics, RNA-dependent RNA polymerase, backtracking, 【초록키워드】 viruses, Structure, SARS-CoV-2, coronavirus, Antiviral, antivirals, COVID-19 pandemic, Transcription, Remdesivir, Cryo-electron microscopy, molecular dynamics, severe acute respiratory syndrome Coronavirus, Molecular dynamics simulation, RNA, Replication, Helicase, nsp13, Regulatory, nucleic acid, electron microscopy, Viral, SARS-CoV-2 genome, RdRP, RNA-dependent RNA polymerase, target, backtracking, RNA polymerase, respiratory, Critical, cellular, SARS-CoV-2 RdRp, Evidence, Combination, nucleotide, Proofreading, structures, (RdRp, nucleoside triphosphate, (RdRp), acute respiratory syndrome, Template, acute respiratory syndrome coronavirus, acute respiratory syndrome coronavirus 2, approaches, enzyme, product, 3′ end, organism, transcriptase, cryo, viral transcription, single-stranded, caused, approach, virus, facilitate, suggested, transcribed, replicated, stimulate, NTP, Significance, 【제목키워드】 complex, the SARS-CoV-2,