DNA-dependent RNA polymerase incorporates β-D-N4-hydroxycytidine (NHC) linking Molnupiravir to host transcription-dependent mutagenesis.

Molnupiravir, a prodrug of β-D-N4-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. Full-length 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 RNA-dependent 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 RT-mediated 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.