The nucleotide addition cycle of the SARS-CoV-2 polymerase.

Subhas Chandra Bera,Mona Seifert,Salina Quack,Flávia S Papini,Yibulayin Wubulikasimu,David Dulin,Martin Depken,Robert N Kirchdoerfer,Craig E Cameron,Jamie J Arnold,Bruno Canard,Pauline Van Nies

doi:10.1016/j.celrep.2021.109650

Abstract

Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We use a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide evidence that the RNA-dependent RNA polymerase (RdRp) uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enable the direct observation of coronavirus polymerase deep and long-lived backtracking that is strongly stimulated by secondary structures in the template. The framework we present here elucidates one of the most important structure-dynamics-function relationships in human health today and will form the grounds for understanding the regulation of this complex.

Highlights

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third zoonotic coronavirus outbreak in less than 20 years, after SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV)
We provide evidence that the RNA-dependent RNA polymerase (RdRp) uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state
Ultra-stable magnetic tweezers enable the direct observation of coronavirus polymerase deep and long-lived backtracking that is strongly stimulated by secondary structures in the template

Summary

Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third zoonotic coronavirus outbreak in less than 20 years, after SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). The $30 -kb-long positive single-stranded RNA ((+)ssRNA) genome of coronaviruses encodes many structural and nonstructural proteins (nsp) Among the latter are the viral proteins that constitute the multi-subunits RNA-dependent RNA polymerase (RdRp) responsible for replication and transcription of the viral genome (Snijder et al, 2016). Because of its central role in the virus life cycle, the coronavirus polymerase represents a major drug target (Robson et al, 2020). Nucleotide analogs, such as remdesivir, are the only therapeutic option to treat coronavirus infection, and a precise understanding of the nucleotide addition cycle would tremendously help the development of antiviral drugs

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