Importance of Neuraminidase Active-Site Residues to the Neuraminidase Inhibitor Resistance of Influenza Viruses

ABSTRACT Neuraminidase inhibitors (NAIs) are antivirals designed to target conserved residues at the neuraminidase (NA) enzyme active site in influenza A and B viruses. The conserved residues that interact with NAIs are under selective pressure, but only a few have been linked to resistance. In the A/Wuhan/359/95 (H3N2) recombinant virus background, we characterized seven charged, conserved NA residues (R118, R371, E227, R152, R224, E276, and D151) that directly interact with the NAIs but have not been reported to confer resistance to NAIs. These NA residues were replaced with amino acids that possess side chains having similar properties to maintain their original charge. The NA mutations we introduced significantly decreased NA activity compared to that of the A/Wuhan/359/95 recombinant wild-type and R292K (an NA mutation frequently reported to confer resistance) viruses, which were analyzed for comparison. However, the recombinant viruses differed in replication efficiency when we serially passaged them in vitro; the growth of the R118K and E227D viruses was most impaired. The R224K, E276D, and R371K mutations conferred resistance to both zanamivir and oseltamivir, while the D151E mutation reduced susceptibility to oseltamivir only (∼10-fold) and the R152K mutation did not alter susceptibility to either drug. Because the R224K mutation was genetically unstable and the emergence of the R371K mutation in the N2 subtype is statistically unlikely, our results suggest that only the E276D mutation is likely to emerge under selective pressure. The results of our study may help to optimize the design of NAIs.

[1]  Y. Guan,et al.  Universal primer set for the full-length amplification of all influenza A viruses , 2001, Archives of Virology.

[2]  S Cusack,et al.  The 2.2 A resolution crystal structure of influenza B neuraminidase and its complex with sialic acid. , 1992, The EMBO journal.

[3]  W. Barclay,et al.  Characterization of recombinant influenza B viruses with key neuraminidase inhibitor resistance mutations. , 2005, The Journal of antimicrobial chemotherapy.

[4]  R. Webster,et al.  Evidence for zanamivir resistance in an immunocompromised child infected with influenza B virus. , 1998, The Journal of infectious diseases.

[5]  G. Boivin,et al.  A Reverse Genetics Study of Resistance to Neuraminidase Inhibitors in An Influenza A/H1N1 Virus , 2003, Antiviral therapy.

[6]  R. Webster,et al.  Detection of influenza virus resistance to neuraminidase inhibitors by an enzyme inhibition assay. , 2002, Antiviral research.

[7]  H. Goto,et al.  Mutations affecting the sensitivity of the influenza virus neuraminidase to 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid. , 1997, Virology.

[8]  R. Webster,et al.  Neuraminidase Inhibitor-Resistant Influenza Viruses May Differ Substantially in Fitness and Transmissibility , 2005, Antimicrobial Agents and Chemotherapy.

[9]  G. Air,et al.  Effects of site-specific mutation on structure and activity of influenza virus B/Lee/40 neuraminidase. , 1987, Virology.

[10]  Hideo Goto,et al.  Avian flu: Isolation of drug-resistant H5N1 virus , 2005, Nature.

[11]  F. Hayden,et al.  Neuraminidase Sequence Analysis and Susceptibilities of Influenza Virus Clinical Isolates to Zanamivir and Oseltamivir , 2003, Antimicrobial Agents and Chemotherapy.

[12]  P. Colman,et al.  The structure of the complex between influenza virus neuraminidase and sialic acid, the viral receptor , 1992, Proteins.

[13]  J. McKimm-Breschkin,et al.  Resistance of influenza viruses to neuraminidase inhibitors--a review. , 2000, Antiviral research.

[14]  R. Webster,et al.  Characterization of mutants of influenza A virus selected with the neuraminidase inhibitor 4-guanidino-Neu5Ac2en , 1996, Journal of virology.

[15]  P. Colman.  Influenza virus neuraminidase: Structure, antibodies, and inhibitors , 1994, Protein science : a publication of the Protein Society.

[16]  Brian J Smith,et al.  Structural studies of the resistance of influenza virus neuramindase to inhibitors. , 2002, Journal of medicinal chemistry.

[17]  G. Air,et al.  Site-directed mutagenesis of catalytic residues of influenza virus neuraminidase as an aid to drug design. , 1998, European journal of biochemistry.

[18]  F. Hayden,et al.  Position statement: global neuraminidase inhibitor susceptibility network. , 2001, Antiviral research.

[19]  M. Potier,et al.  Fluorometric assay of neuraminidase with a sodium (4-methylumbelliferyl-alpha-D-N-acetylneuraminate) substrate. , 1979, Analytical biochemistry.

[20]  Yoshihiro Kawaoka,et al.  oseltamivir: descriptive study , 2022 .

[21]  D. M. Ryan,et al.  Rational design of potent sialidase-based inhibitors of influenza virus replication , 1993, Nature.

[22]  Hideo Goto,et al.  Selective incorporation of influenza virus RNA segments into virions , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[23]  G. Air,et al.  Loss of enzyme activity in a site-directed mutant of influenza neuraminidase compared to expressed wild-type protein. , 1986, Virology.

[24]  J. N. Varghese,et al.  Structure of the catalytic and antigenic sites in influenza virus neuraminidase , 1983, Nature.

[25]  Larisa V. Gubareva,et al.  Comparison of the Activities of Zanamivir, Oseltamivir, and RWJ-270201 against Clinical Isolates of Influenza Virus and Neuraminidase Inhibitor-Resistant Variants , 2001, Antimicrobial Agents and Chemotherapy.

[26]  Paul W Smith,et al.  Drug design against a shifting target: a structural basis for resistance to inhibitors in a variant of influenza virus neuraminidase. , 1998, Structure.

[27]  J. McKimm-Breschkin.  Neuraminidase inhibitors for the treatment and prevention of influenza , 2002, Expert opinion on pharmacotherapy.

[28]  H. Klenk,et al.  Neuraminidase Is Important for the Initiation of Influenza Virus Infection in Human Airway Epithelium , 2004, Journal of Virology.

[29]  P. Colman,et al.  Mutations in a Conserved Residue in the Influenza Virus Neuraminidase Active Site Decreases Sensitivity to Neu5Ac2en-Derived Inhibitors , 1998, Journal of Virology.

[30]  P. Colman,et al.  Generation and characterization of an influenza virus neuraminidase variant with decreased sensitivity to the neuraminidase-specific inhibitor 4-guanidino-Neu5Ac2en. , 1995, Virology.

[31]  P. Colman,et al.  Mutation in the influenza virus neuraminidase gene resulting in decreased sensitivity to the neuraminidase inhibitor 4-guanidino-Neu5Ac2en leads to instability of the enzyme. , 1996, Virology.

[32]  M. von Itzstein,et al.  Evidence for a sialosyl cation transition-state complex in the reaction of sialidase from influenza virus. , 1992, European journal of biochemistry.

[33]  Jonathan Greer,et al.  Influenza neuraminidase inhibitors: structure-based design of a novel inhibitor series. , 2003, Biochemistry.

[34]  Brian J. Smith,et al.  Analysis of inhibitor binding in influenza virus neuraminidase , 2001, Protein science : a publication of the Protein Society.

[35]  L. Gubareva,et al.  Molecular mechanisms of influenza virus resistance to neuraminidase inhibitors. , 2004, Virus research.

[36]  R. Webster,et al.  A DNA transfection system for generation of influenza A virus from eight plasmids. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[37]  M. Lawrence,et al.  Sequence and structure alignment of paramyxovirus hemagglutinin-neuraminidase with influenza virus neuraminidase , 1993, Journal of virology.

[38]  G. Air,et al.  Site-directed mutation of the active site of influenza neuraminidase and implications for the catalytic mechanism. , 1987, Biochemistry.