ABSTRACT
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, ß-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.
Subject(s)
COVID-19/prevention & control , Cytidine/analogs & derivatives , Hydroxylamines/metabolism , Mutagenesis/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Base Sequence , COVID-19/virology , Cytidine/chemistry , Cytidine/metabolism , Cytidine/pharmacology , Humans , Hydroxylamines/chemistry , Hydroxylamines/pharmacology , Models, Molecular , Molecular Structure , Mutagenesis/drug effects , Mutation/drug effects , Mutation/genetics , Nucleic Acid Conformation , Protein Binding/drug effects , Protein Conformation , RNA, Viral/chemistry , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Virus Replication/drug effects , Virus Replication/genetics , COVID-19 Drug TreatmentABSTRACT
Coronaviruses use an RNA-dependent RNA polymerase to replicate and transcribe their RNA genome. The structure of the SARS-CoV-2 polymerase was determined by cryo-electron microscopy within a short time in spring 2020. The structure explains how the viral enzyme synthesizes RNA and how it replicates the exceptionally large genome in a processive manner. The most recent structure-function studies further reveal the mechanism of polymerase inhibition by remdesivir, an approved drug for the treatment of COVID-19.
ABSTRACT
Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3'-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3'-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3'-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.