The SARS-CoV ORF1b enzymes : Fast, furious, and unique

With the current Covid-19 ongoing outbreak originating inWuhan, human pathogenic coronaviruses demonstrate their ability to emerge abruptly and spread serious pulmonary disease The Orf1b enzymes promote replication and transcription within a complex with unique enzyme having outstanding properties The RdRp core sequence of the Covid-19 isolate published in january 2020 shows a high (>95% aa) sequence homology to the SARSCoV emerged in 2003 Analysis of polymorphisms show that aa changes are mostly located at the protein surface, unlikely to affect any basic function of the RdRp We have reconstituted a highly active SARS-CoV RdRp complex made of nsp7, nsp8, and nsp12, and studied its polymerization activity on a variety of RNA templates using steady-state and pre-steady state kinetics The RdRp is able to incorporate single NTPs at the astonishing rate of >500 s-1, about 10-fold faster than any known viral RdRp Fast synthesis occurs at the expense of fidelity, which is at least 10-fold lower than that of Dengue virus NS5 or Coxsackie virus RdRps Such low fidelity must be corrected by the nsp14 ExoN subdomain -able to remove 3'-terminal mismatches to match genome stability observed in infected cells, and account for the large size of the Coronavirus RNA genome The nsp14 enzyme is a bi-functional enzyme made of an Exonuclease domain, activated through binding to nsp10, and an RNA methyltransferase able to execute N7-guanine methylation of RNA caps It is the only example of RNA cap MTase which does not have a Rossmann fold, which poses interesting questions given the overwhelming success of the Rossmann fold through evolution Nsp13 is a type 1 helicase, whose role is unclear The nsp15 RNA endonuclease is a RNase A type endonuclease specific for Uracile, inhibited by 2'-O methylation of RNA, while nsp16 is a 2'-O methyltransferase also activated by nsp10 Our work provides a structural and functional view of this sophisticated replication complex, with highly active enzyme preparations suitable for robotized high-throughput inhibition assays aiming at the discovery of pan-coronavirus inhibitors Orf1b enzymes