Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis

Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, beta-d-N-4-hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp-RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir. Quantitative biochemical assays and high-resolution cryo-EM analysis reveal how the COVID-19 antiviral drug candidate molnupiravir causes lethal viral mutagenesis by the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2.

Automated summary

Molnupiravir is an oral antiviral drug candidate that increases viral RNA mutations frequency and inhibits replication of SARS-CoV-2 by altering the substrate preference of RdRp. This two-step mutagenesis mechanism can explain the broad-spectrum antiviral activity of molnupiravir, making it a promising COVID-19 treatment option.