title: Understanding the Mechanism of the Broad-Spectrum Antiviral Activity of Favipiravir (T-705): Key Role of the F1 Motif of the Viral Polymerase.
abstract: Favipiravir (T-705) is a broad-spectrum antiviral agent that has been approved in Japan for the treatment of influenza virus infections. T-705 also inhibits the replication of various RNA viruses, including chikungunya virus (CHIKV). We demonstrated earlier that the K291R mutation in the F1 motif of the RNA-dependent RNA polymerase (RdRp) of CHIKV is responsible for low-level resistance to T-705. Interestingly, this lysine is highly conserved in the RdRp of positive-sense single-stranded RNA (+ssRNA) viruses. To obtain insights into the unique broad-spectrum antiviral activity of T-705, we explored the role of this lysine using another +ssRNA virus, namely, coxsackievirus B3 (CVB3). Introduction of the corresponding K-to-R substitution in the CVB3 RdRp (K159R) resulted in a nonviable virus. Replication competence of the K159R variant was restored by spontaneous acquisition of an A239G substitution in the RdRp. A mutagenesis analysis at position K159 identified the K159M variant as the only other viable variant which had also acquired the A239G substitution. The K159 substitutions markedly decreased the processivity of the purified viral RdRp, which was restored by the introduction of the A239G mutation. The K159R A239G and K159M A239G variants proved, surprisingly, more susceptible than the wild-type virus to T-705 and exhibited lower fidelity in polymerase assays. Furthermore, the K159R A239G variant was found to be highly attenuated in mice. We thus demonstrate that the conserved lysine in the F1 motif of the RdRp of +ssRNA viruses is involved in the broad-spectrum antiviral activity of T-705 and that it is a key amino acid for the proper functioning of the enzyme.IMPORTANCEIn this study, we report the key role of a highly conserved lysine residue of the viral polymerase in the broad-spectrum antiviral activity of favipiravir (T-705) against positive-sense single-stranded RNA viruses. Substitutions of this conserved lysine have a major negative impact on the functionality of the RdRp. Furthermore, we show that this lysine is involved in the fidelity of the RdRp and that the RdRp fidelity influences the sensitivity of the virus for the antiviral efficacy of T-705. Consequently, these results provide insights into the mechanism of the antiviral activity of T-705 and may lay the basis for the design of novel chemical scaffolds that may be endowed with a more potent broad-spectrum antiviral activity than that of T-705.
authors.affiliation: KU Leuven-University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium.
authors.forename: Rana
authors.initials: R
authors.lastname: Abdelnabi
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257, Aix-Marseille Université, Marseille, France.
authors.forename: Ana Theresa Silveira de
authors.initials: ATS
authors.lastname: Morais
authors.affiliation: KU Leuven-University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium.
authors.forename: Pieter
authors.initials: P
authors.lastname: Leyssen
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257, Aix-Marseille Université, Marseille, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Imbert
authors.affiliation: Institut Pasteur, Centre National de la Recherche Scientifique UMR 3569, Paris, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Beaucourt
authors.affiliation: Institut Pasteur, Centre National de la Recherche Scientifique UMR 3569, Paris, France.
authors.forename: Hervé
authors.initials: H
authors.lastname: Blanc
authors.affiliation: KU Leuven-University of Leuven, Department of Pharmaceutical and Pharmacological Sciences, Rega Institute for Medical Research, Laboratory of Medicinal Chemistry, Leuven, Belgium.
authors.forename: Mathy
authors.initials: M
authors.lastname: Froeyen
authors.affiliation: Institut Pasteur, Centre National de la Recherche Scientifique UMR 3569, Paris, France.
authors.forename: Marco
authors.identifier: http://orcid.org/0000-0002-4400-771X
authors.initials: M
authors.lastname: Vignuzzi
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257, Aix-Marseille Université, Marseille, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Canard
authors.affiliation: KU Leuven-University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium Johan.Neyts@kuleuven.be.
authors.forename: Johan
authors.initials: J
authors.lastname: Neyts
authors.affiliation: KU Leuven-University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium.
authors.forename: Leen
authors.initials: L
authors.lastname: Delang
mesh_terms: D000577:Amides / Q000008:administration & dosage / Q000494:pharmacology*; D020816:Amino Acid Motifs; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002646:Chikungunya virus / Q000187:drug effects / Q000235:genetics; D002522:Chlorocebus aethiops; D024882:Drug Resistance, Viral / Q000235:genetics; D029822:Enterovirus B, Human / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D007564:Japan; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D050296:Microbial Viability / Q000187:drug effects; D016296:Mutagenesis; D009154:Mutation; D011719:Pyrazines / Q000008:administration & dosage / Q000494:pharmacology*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics; D014709:Vero Cells; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000577:Amides; D000998:Antiviral Agents; D011719:Pyrazines; D012324:RNA-Dependent RNA Polymerase; C462182:favipiravir; D008239:Lysine
keywords: CVB3; RdRp; favipiravir; fidelity; mutagenesis