Molnupiravir, a prodrug of -D-N-hydroxycytidine (NHC), is an antiviral RNA mutagen that is incorporated by viral RNA-dependent RNA polymerases (RdRp) during replication of viral RNA genomes, ultimately driving target viruses such as SARS-CoV-2 toward lethal mutagenesis.In this study, first, we biochemically tested whether DNA-dependent RNA polymerases (DdRps) including T7 RNA polymerase and host RNA polymerase II, can also incorporate NHC-triphosphate (NHC-TP) during RNA synthesis from double-stranded DNA (dsDNA) templates.In vitro transcription (IVT) by T7 RNA polymerase was evaluated under two conditions: (1) all four natural ribonucleoside triphosphates (rNTPs) and (2) three natural rNTPs (ATP, GTP, and UTP) supplemented with NHC-TP.Fulllength RNA products were generated in both conditions, indicating that T7 RNA polymerase incorporates NHC-TP during DNA-dependent RNA synthesis.Second, these IVT-derived RNA products were subsequently reverse-transcribed into single-stranded DNA (ssDNA) using HIV-1 reverse transcriptase (RT).Comparable ssDNA yields were also obtained from both RNA templates, suggesting that NHC-monophosphates embedded in RNA template do not affect the RNAdependent DNA polymerase (RdDp) activity of HIV-1 RT under the experimental conditions tested.Third, next-generation sequencing (NGS) analysis of the reverse-transcribed products revealed the expected NHC-mediated C to T transition mutations, confirming the mutagenic impact of NHC during the HIV-1 RTmediated RdDp reactions.Finally, incorporation of NHC-TP by human RNA polymerase II was further confirmed using IVT reactions performed with HeLa cell nuclear extracts.Overall, these biochemical investigations establish both the capacity of DdRps to incorporate NHC-TP and the characteristic mutagenic signature induced by NHC during HIV-1 RT-mediated RNA-dependent DNA synthesis.
Garnier et al. (Sun,) studied this question.