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. 2002 Dec;46(12):3809-16.
doi: 10.1128/AAC.46.12.3809-3816.2002.

Mechanism by which mutations at his274 alter sensitivity of influenza a virus n1 neuraminidase to oseltamivir carboxylate and zanamivir

Michael Z Wang  1 , Chun Y Tai, Dirk B Mendel
Affiliations

Mechanism by which mutations at his274 alter sensitivity of influenza a virus n1 neuraminidase to oseltamivir carboxylate and zanamivir

Michael Z Wang et al. Antimicrob Agents Chemother. 2002 Dec.

Abstract

Oseltamivir carboxylate is a potent and specific inhibitor of influenza neuraminidase (NA). An influenza A/H1N1 variant selected in vitro with reduced susceptibility to oseltamivir carboxylate contains a His274Tyr mutation. To understand the mechanism by which a His274Tyr mutation gives rise to drug resistance, we studied a series of NA variant proteins containing various substitutions at position 274. Replacement of His274 with larger side chain residues (Tyr or Phe) reduced the NA sensitivity to oseltamivir carboxylate. In contrast, replacement of His274 with smaller side chain residues (Gly, Asn, Ser, and Gln) resulted in enhanced or unchanged sensitivity to oseltamivir carboxylate. Previous studies have suggested that the slow-binding inhibition of NA by oseltamivir carboxylate is a result of the reorientation of Glu276. Loss of this slow-binding inhibition in the His274Tyr and His274Phe mutant NA but not in His274Asn, His274Gly, His274Ser, or His274Gln supports the conclusion that the conformational change of Glu276 is restricted in the His274Tyr and His274Phe mutant NA upon oseltamivir carboxylate binding. Interestingly, His274Asn, as well as His274Gly, His274Ser, and His274Gln, also displayed reduced sensitivity to zanamivir and its analogue, 4-amino-Neu5Ac2en. Substitution of His274 with Tyr in influenza A/Tokyo/3/67 (H3N2) recombinant NA did not affect the susceptibility to oseltamivir carboxylate. These data indicate that the volume occupied by the amino acid side chain at position 274 can influence the sensitivities of influenza N1 NA but not of N2 NA to both oseltamivir carboxylate and zanamivir.

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Figures

FIG. 1.
FIG. 1.
Chemical structures of sialic acid and influenza NAI.
FIG. 2.
FIG. 2.
Inhibition of wild-type (solid circle) and H274Y mutant (open circle) influenza NA by oseltamivir carboxylate. (A) Native NA from A/WS/33 (H1N1) virus. (B) Recombinant A/WS/33 (H1N1) NA expressed in HeLa cells. (C) Recombinant A/Tokyo/3/67 (H3N2) NA expressed in HeLa cells.
FIG. 3.
FIG. 3.
Influenza NA recognition of substrate (MUNANA) (A) and inhibitors oseltamivir carboxylate (B), zanamivir (C), and 4-amino-Neu5Ac2en (D) is affected by the side chain volume of amino acid at position 274 in A/WS/33 (H1N1).
FIG. 4.
FIG. 4.
Evaluation of the slow-binding inhibition kinetics of wild-type (inhibitor concentrations of 0, 1.9, 2.5, and 6.3 nM) (A), H274Y mutant (inhibitor concentrations of 0, 125, 250, and 1,250 nM) (B), and H274N mutant (inhibitor concentrations of 0, 5, 10, and 20 nM) (C) recombinant NA of A/WS/33 (H1N1) by oseltamivir carboxylate.
FIG. 5.
FIG. 5.
Interaction of oseltamivir carboxylate (brown) and zanamivir (green) with amino acid residues located in the active site of influenza A NA (N9).
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