title: A Balance between Inhibitor Binding and Substrate Processing Confers Influenza Drug Resistance.
abstract: The therapeutic benefits of the neuraminidase (NA) inhibitor oseltamivir are dampened by the emergence of drug resistance mutations in influenza A virus (IAV). To investigate the mechanistic features that underlie resistance, we developed an approach to quantify the effects of all possible single-nucleotide substitutions introduced into important regions of NA. We determined the experimental fitness effects of 450 nucleotide mutations encoding positions both surrounding the active site and at more distant sites in an N1 strain of IAV in the presence and absence of oseltamivir. NA mutations previously known to confer oseltamivir resistance in N1 strains, including H275Y and N295S, were adaptive in the presence of drug, indicating that our experimental system captured salient features of real-world selection pressures acting on NA. We identified mutations, including several at position 223, that reduce the apparent affinity for oseltamivir in vitro. Position 223 of NA is located adjacent to a hydrophobic portion of oseltamivir that is chemically distinct from the substrate, making it a hotspot for substitutions that preferentially impact drug binding relative to substrate processing. Furthermore, two NA mutations, K221N and Y276F, each reduce susceptibility to oseltamivir by increasing NA activity without altering drug binding. These results indicate that competitive expansion of IAV in the face of drug pressure is mediated by a balance between inhibitor binding and substrate processing.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Ping
authors.initials: P
authors.lastname: Liu
authors.affiliation: School of Life Sciences, École Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.
authors.forename: Claudia
authors.initials: C
authors.lastname: Bank
authors.affiliation: Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Renzette
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Kristina
authors.initials: K
authors.lastname: Prachanronarong
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Lutfu S
authors.initials: LS
authors.lastname: Yilmaz
authors.affiliation: Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Caffrey
authors.affiliation: Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Konstantin B
authors.initials: KB
authors.lastname: Zeldovich
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Celia A
authors.initials: CA
authors.lastname: Schiffer
authors.affiliation: Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Timothy F
authors.initials: TF
authors.lastname: Kowalik
authors.affiliation: School of Life Sciences, École Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Jensen
authors.affiliation: Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Finberg
authors.affiliation: Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA. Electronic address: Jennifer.Wang@umassmed.edu.
authors.forename: Jennifer P
authors.initials: JP
authors.lastname: Wang
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA. Electronic address: Dan.Bolon@umassmed.edu.
authors.forename: Daniel N A
authors.initials: DNA
authors.lastname: Bolon
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D004285:Dogs; D024882:Drug Resistance, Viral; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D009980:Influenza A virus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D007251:Influenza, Human / Q000188:drug therapy* / Q000821:virology; D008958:Models, Molecular; D009439:Neuraminidase / Q000235:genetics* / Q000378:metabolism; D009976:Orthomyxoviridae Infections / Q000188:drug therapy / Q000821:virology; D053139:Oseltamivir / Q000494:pharmacology*; D017354:Point Mutation; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D014764:Viral Proteins; D053139:Oseltamivir; D009439:Neuraminidase
keywords: adaptive; experimental fitness; neuraminidase inhibitor; oseltamivir; systematic mutation