title: The I427T neuraminidase (NA) substitution, located outside the NA active site of an influenza A(H1N1)pdm09 variant with reduced susceptibility to NA inhibitors, alters NA properties and impairs viral fitness.
abstract: Emergence of pan neuraminidase inhibitor (NAI)-resistant variants constitutes a serious clinical concern. An influenza A(H1N1)pdm09 variant containing the I427T/Q313R neuraminidase (NA) substitutions was previously identified in a surveillance study. Although these changes are not part of the NA active site, the variant showed reduced susceptibility to many NAIs. In this study, we investigated the mechanism of resistance for the I427T/Q313R substitution and its impact on the NA enzyme and viral fitness. Recombinant wild-type (WT), I427T/Q313R and I427T A(H1N1)pdm09 viruses were generated by reverse genetics and tested for their drug susceptibilities, enzymatic properties and replication kinetics in vitro as well as their virulence in mice. Molecular dynamics (MD) simulations were performed for NA structural analysis. The I427T substitution, which was responsible for the resistance phenotype observed in the double (I427T/Q313R) mutant, induced 17-, 56-, 7-, and 14-fold increases in IC50values against oseltamivir, zanamivir, peramivir and laninamivir, respectively. The I427T substitution alone or combined to Q313R significantly reduced NA affinity. The I427T/Q313R and to a lesser extent I427T recombinant viruses displayed reduced viral titers vs WT in vitro. In experimentally-infected mice, the mortality rates were 62.5%, 0% and 14.3% for the WT, I417T/Q313R and I427T viruses, respectively. There were about 2.5- and 2-Log reductions in mean lung viral titers on day 5 post-infection for the I427T/Q313R and I427T mutants, respectively, compared to WT. Results from simulations revealed that the I427T change indirectly altered the stability of the catalytic R368 residue of the NA enzyme causing its reduced binding to the substrate/inhibitor. This study demonstrates that the I427T/Q313R mutant, not only alters NAI susceptibility but also compromises NA properties and viral fitness, which could explain its infrequent detection in clinic.
authors.affiliation: Research Center in Infectious Diseases of the CHUQ-CHUL and Laval University, Québec City, QC, Canada.
authors.forename: Véronique
authors.initials: V
authors.lastname: Tu
authors.affiliation: Research Center in Infectious Diseases of the CHUQ-CHUL and Laval University, Québec City, QC, Canada.
authors.forename: Yacine
authors.initials: Y
authors.lastname: Abed
authors.affiliation: Proteo and IBIS, Department of Chemistry, Faculty of Science and Engineering, Laval University, Québec City, QC, Canada.
authors.forename: Xavier
authors.initials: X
authors.lastname: Barbeau
authors.affiliation: Research Center in Infectious Diseases of the CHUQ-CHUL and Laval University, Québec City, QC, Canada.
authors.forename: Julie
authors.initials: J
authors.lastname: Carbonneau
authors.affiliation: Research Center in Infectious Diseases of the CHUQ-CHUL and Laval University, Québec City, QC, Canada.
authors.forename: Clément
authors.initials: C
authors.lastname: Fage
authors.affiliation: Proteo and IBIS, Department of Biochemistry, Microbiology and Bioinformatics, Faculty of Science and Engineering, Laval University, Québec City, QC, Canada.
authors.forename: Patrick
authors.initials: P
authors.lastname: Lagüe
authors.affiliation: Research Center in Infectious Diseases of the CHUQ-CHUL and Laval University, Québec City, QC, Canada. Electronic address: Guy.Boivin@crchul.ulaval.ca.
authors.forename: Guy
authors.initials: G
authors.lastname: Boivin
mesh_terms: D000146:Acids, Carbocyclic; D019943:Amino Acid Substitution; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D020134:Catalytic Domain; D003198:Computer Simulation; D003517:Cyclopentanes / Q000494:pharmacology; D024882:Drug Resistance, Viral; D004791:Enzyme Inhibitors / Q000494:pharmacology; D056084:Genetic Fitness; D006146:Guanidines / Q000494:pharmacology; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D020128:Inhibitory Concentration 50; D051379:Mice; D056004:Molecular Dynamics Simulation; D009439:Neuraminidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009976:Orthomyxoviridae Infections / Q000821:virology; D053139:Oseltamivir / Q000494:pharmacology; D059386:Reverse Genetics
chemical_list: D000146:Acids, Carbocyclic; D000998:Antiviral Agents; D003517:Cyclopentanes; D004791:Enzyme Inhibitors; D006146:Guanidines; D053139:Oseltamivir; D009439:Neuraminidase; C414210:peramivir