title: Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor.
abstract: The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. These mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containing the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. Using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN 38105;
authors.forename: Min-Suk
authors.initials: MS
authors.lastname: Song
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105;
authors.forename: Gyanendra
authors.initials: G
authors.lastname: Kumar
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN 38105;
authors.forename: William R
authors.initials: WR
authors.lastname: Shadrick
authors.affiliation: Bioscience Division, SRI International, Menlo Park, CA 94025.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhou
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN 38105;
authors.forename: Trushar
authors.initials: T
authors.lastname: Jeevan
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105;
authors.forename: Zhenmei
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN 38105;
authors.forename: P Jake
authors.initials: PJ
authors.lastname: Slavish
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN 38105;
authors.forename: Thomas P
authors.identifier: http://orcid.org/0000-0002-8960-0728
authors.initials: TP
authors.lastname: Fabrizio
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN 38105;
authors.forename: Sun-Woo
authors.initials: SW
authors.lastname: Yoon
authors.affiliation: Bioscience Division, SRI International, Menlo Park, CA 94025 Stephen.white@stjude.org richard.webby@stjude.org thomas.webb@sri.com.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Webb
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN 38105; Stephen.white@stjude.org richard.webby@stjude.org thomas.webb@sri.com.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Webby
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105; Stephen.white@stjude.org richard.webby@stjude.org thomas.webb@sri.com.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: White
mesh_terms: D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D004285:Dogs; D024882:Drug Resistance, Viral / Q000235:genetics; D004720:Endonucleases / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D014644:Genetic Variation; D006885:Hydroxybutyrates / Q000494:pharmacology*; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D009980:Influenza A virus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D061985:Madin Darby Canine Kidney Cells; D008958:Models, Molecular; D016296:Mutagenesis; D010880:Piperidines / Q000494:pharmacology*; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D006885:Hydroxybutyrates; C585288:L-742,001; D010880:Piperidines; D012324:RNA-Dependent RNA Polymerase; D004720:Endonucleases
keywords: diketo acid; infection; mutation; replication; virus