HSV-1 Delta ribonucleotide reductase as a Model to Investigate Molnupiravir-Induced Mutagenesis

Abstract Molnupiravir, a nucleoside analog effective against RNA viruses such as Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2), exerts antiviral effects primarily through lethal mutagenesis by incorporation into viral RNA. However, its ambiguous base-pairing properties raise concerns about potential mutagenesis via conversion to deoxycytidine diphosphate (dCDP) by cellular ribonucleotide reductase (RNR) and subsequent incorporation into DNA. This study evaluates whether herpes simplex virus type 1 (HSV-1), in comparison to HSV-1 lacking RNR (HSV-1 ΔRR), can serve as a model to investigate molnupiravir’s mutagenic effects on DNA viruses. Using CRISPR-Cas9, the ICP6 RNRdomain has already been deleted to generate HSV-1 ΔRR. Human primary fibroblasts with low endogenous deoxyribonucleotide pools were infected at low multiplicity and treated with varying concentrations of molnupiravir. Viral titers were measured by TCID50 assays, and viral DNA from treated cultures was sequenced, targeting nonessential genes to detect mutations. Molnupiravir significantly reduced viral titers of wild-type HSV-1 in both fibroblasts and Vero cells, while HSV-1 ΔRR exhibited reduced sensitivity, with significant inhibition only at higher drug concentrations in fibroblasts. Importantly, no mutations were detected in viral DNA from either strain at any molnupiravir concentration. These findings indicate that molnupiravir’s mutagenic potential is limited in DNA viruses like HSV-1, likely due to restricted incorporation into DNA. This highlights the need for further research to optimize molnupiravir’s antiviral use beyond RNA viruses.