Molnupiravir promotes SARS-CoV-2 mutagenesis via the RNA template

J Biol Chem. 2021 Jul;297(1):100770. doi: 10.1016/j.jbc.2021.100770. Epub 2021 May 11.


The RNA-dependent RNA polymerase of the severe acute respiratory syndrome coronavirus 2 is an important target in current drug development efforts for the treatment of coronavirus disease 2019. Molnupiravir is a broad-spectrum antiviral that is an orally bioavailable prodrug of the nucleoside analogue β-D-N4-hydroxycytidine (NHC). Molnupiravir or NHC can increase G to A and C to U transition mutations in replicating coronaviruses. These increases in mutation frequencies can be linked to increases in antiviral effects; however, biochemical data of molnupiravir-induced mutagenesis have not been reported. Here we studied the effects of the active compound NHC 5'-triphosphate (NHC-TP) against the purified severe acute respiratory syndrome coronavirus 2 RNA-dependent RNA polymerase complex. The efficiency of incorporation of natural nucleotides over the efficiency of incorporation of NHC-TP into model RNA substrates followed the order GTP (12,841) > ATP (424) > UTP (171) > CTP (30), indicating that NHC-TP competes predominantly with CTP for incorporation. No significant inhibition of RNA synthesis was noted as a result of the incorporated monophosphate in the RNA primer strand. When embedded in the template strand, NHC-monophosphate supported the formation of both NHC:G and NHC:A base pairs with similar efficiencies. The extension of the NHC:G product was modestly inhibited, but higher nucleotide concentrations could overcome this blockage. In contrast, the NHC:A base pair led to the observed G to A (G:NHC:A) or C to U (C:G:NHC:A:U) mutations. Together, these biochemical data support a mechanism of action of molnupiravir that is primarily based on RNA mutagenesis mediated via the template strand.

Keywords: Covid-19; RNA-dependent RNA polymerase; SARS-CoV-2; antiviral agent; coronavirus; drug development; mutagen; nucleoside analogue.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antiviral Agents / pharmacology*
  • COVID-19 / virology*
  • Cytidine / analogs & derivatives*
  • Cytidine / pharmacology
  • Humans
  • Hydroxylamines / pharmacology*
  • Mutagenesis
  • Point Mutation / drug effects
  • RNA, Viral / genetics*
  • SARS-CoV-2 / drug effects*
  • SARS-CoV-2 / genetics*
  • SARS-CoV-2 / metabolism


  • Antiviral Agents
  • Hydroxylamines
  • RNA, Viral
  • Cytidine
  • N(4)-hydroxycytidine
  • molnupiravir