The RNA-dependent RNA polymerase of the severe acute respiratory syndrome coronavirus 2 remains a crucial target for developing drugs for treating coronavirus disease 2019. Molnupiravir, a broad-spectrum antiviral, is an orally bioavailable prodrug of the nucleoside analogue β-D-N4-hydroxycytidine (NHC). It enhances G to A and C to U transition mutations in replicating coronaviruses, which ultimately increases its antiviral effects; however, biochemical data of molnupiravir-induced mutagenesis is not documented. 

The effect of the active compound NHC 5’-triphosphate (NHC-TP) against the purified severe acute respiratory syndrome coronavirus 2 RNA-dependent RNA polymerase complex has been explained. The efficiency of incorporation of natural nucleotides over the efficiency of incorporation of NHC-TP into model RNA substrates follows the following order-

GTP > ATP > UTP > CTP, suggesting NHC-TP to compete primarily with CTP for incorporation. 

No marked inhibition of RNA synthesis occurs as a result of the incorporated monophosphate in the RNA primer strand. On infiltrating the template strand, NHC-monophosphate generates both NHC:G and NHC:A base pairs with similar efficiencies. The elongation of the NHC:G product is modestly inhibited, but higher nucleotide concentrations overcome this stoppage. Whereas, the NHC:A base pair caused G to A (G:NHC:A) or C to U (C:G:NHC:A:U) mutations. These biochemical data collectively support a mechanism of action of molnupiravir that depends on RNA mutagenesis mediated through the template strand.

Source- Journal of Biological Chemistry, 2021;297(1). DOI:https://doi.org/10.1016/j.jbc.2021.100770