biostudies-literatureSong MSNIAID NIH HHSCEIRSNCI NIH HHSHHS | NIH | National Institute of Allergy and Infectious DiseasesSt. Jude Cancer CenterPHS HHS3669-74https://www.ebi.ac.uk/biostudies/studies/S-EPMC4822642biostudies-literatureUnknown113(13)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.Proceedings of the National Academy of Sciences of the United States of AmericaIdentification and characterization of influenza variants resistant to a viral endonuclease inhibitor.PMC4822642R01 AI098757P30 CA021765HHSN266200700005CAI098757CA21765HHSN272201400006CShadrick WRKumar GLi ZFabrizio TPWhite SWWebb TRZhou WSlavish PJYoon SWSong MSWebby RJJeevan TfalseIdentification and characterization of influenza variants resistant to a viral endonuclease inhibitor.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.2016-01-01T00:00:00Z2016 Mar2024-11-21T08:50:42.377Z2019-03-27T03:11:20ZS-EPMC48226422697657510.1073/pnas.1519772113