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, β-d-N⁴-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.

Molnupiravir 是一种口服抗病毒候选药物，目前正在进行治疗 COVID-19 患者的 III 期试验。 Molnupiravir 会增加动物模型和人类中病毒 RNA 突变的频率并损害 SARS-CoV-2 复制。在这里，我们建立了病毒 RNA 依赖性 RNA 聚合酶 (RdRp) 莫鲁匹拉韦诱导 RNA 突变的分子机制。生化测定表明，RdRp 使用莫努匹拉韦的活性形式、β- d - N ^{4 -羟基}胞苷 (NHC) 三磷酸作为底物，而不是三磷酸胞苷或三磷酸尿苷。当 RdRp 使用生成的 RNA 作为模板时，NHC 会指导 G 或 A 的掺入，从而产生突变的 RNA 产物。对含有诱变产物的 RdRp-RNA 复合物的结构分析表明，NHC 可以与 RdRp 活性中心的 G 或 A 形成稳定的碱基对，这解释了聚合酶如何逃脱校对并合成突变的 RNA。这种两步诱变机制可能适用于各种病毒聚合酶，并且可以解释莫努匹拉韦的广谱抗病毒活性。