Molecular Mechanisms of Serum Resistance of Human Influenza H3N2 Virus and Their Involvement in Virus Adaptation in a New Host

  1. Yoshihiro Kawaoka2,3,4
  1. M. P. Chumakov Institute of Poliomyelitis and Viral Encephalitides, 142 782 Moscow, Russia1;
  2. Department of Virology and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee 381052;
  3. Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin—Madison, Madison, Wisconsin 537063; and
  4. Department of Pathology, University of Tennessee, Memphis, Memphis, Tennessee 381634
  1. Fig. 1.
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    Fig. 1.

    Binding of inhibitors from animal sera by the LA/87 strain and its serum-resistant variants. Horse (closed bars), pig (open bars), and rabbit (dashed bars) sera were heat inactivated and either treated with LA/87 NA or mock treated as described in Materials and Methods. Association constants of the virus complexes with inhibitors of mock-treated (A) or NA-treated (B) sera (K MTand K NA, respectively) were determined in a competitive solid-phase assay; 5 mM EDTA was present in the assay buffer to inactivate possible residual β-inhibitor activity in the sera (references 2 and 3 and this paper). The ratio K MT/K NA(C) reflects a decrease in the affinity of the serum inhibitors for the virus after NA treatment. The lower this ratio is, the more resistant is the inhibitor to inactivation by NA.

  2. Fig. 2.
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    Fig. 2.

    Binding of receptor analogs by LA/87 and its serum-resistant variants. The association constants of the virus complexes with free Neu5Ac, 3′SL, 6′SLN, and sialylglycopolymers of 3′SL and 6′SLN (3′SL-PAA and 6′SLN-PAA) were determined in a competitive solid-phase assay as described in Materials and Methods. The constants are expressed in mM−1 for the monovalent sialosides and in μM−1 sialic acid for the sialylglycopolymers. Our data on the binding of total heat-inactivated pig serum from Fig. 1 (K MT, mU−1) are also represented (Pig serum) to facilitate the comparison of binding patterns.

  3. Fig. 3.
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    Fig. 3.

    Key mutations (black) in the HA of serum-resistant variants of LA/87 shown on a model depicting the complex between X31 virus HA and the α-methyl glycoside of 4-O-acetyl-N-acetylneuraminic acid (1HGI structure; Brookhaven Protein Databank [36]). The solvent-accessible surface of the receptor-binding site of the HA monomer A (white) and the adjacent part of the second monomer C (gray) are represented. The molecule of Neu4,5Ac22Me is shown as a stick model (heavy atoms only); the carbon and oxygen atoms of the 4-O-acetyl group and the carbon atom of the 2-O-methyl group are shown as white, gray, and black balls, respectively. Black numbers indicate the positions of the amino acids discussed in the text. The figure was generated using the Preview version of WebLab ViewerPro 3.0, Molecular Simulations, Inc., San Diego, Calif.

  4. Fig. 4.
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    Fig. 4.

    Properties of the β inhibitor present in rabbit serum after heat inactivation for 30 min at 56°C and periodate treatment. (A) Effect of bivalent ions on the ability of the preparation to inhibit the binding of the fetuin (fet)-HRP conjugate to LA/87 virus (see Materials and Methods for assay details). Serum was diluted in 0.05 M TBS (pH 7.3) (closed circles), TBS supplemented with 5 mM EDTA (TBS-E) (open circles), or TBS-E containing either 25 mM CaCl2 (triangles) or 25 mM MgCl2 (solid squares). (B) Sensitivity of the HAI activity of the β inhibitor to competitive blocking by monosaccharides. The ordinates show the minimal concentrations of the sugars d-mannose (Man),l-fucose (Fuc),N-acetyl-d-glucosamine (GlcNAc),d-glucose (Glc), d-galactose (Gal), andN-acetyl-d-galactosamine (GalNAc) that are required to completely inhibit the HAI activity of 4 HAI units of the β inhibitor. The assay was performed in microplates on 4 HAI units of LA/87 virus and 0.5% chicken erythrocytes as described previously (17). No inhibition was observed at the highest concentration (200 mM) of Gal or GalNAc used. (C) HAI activity of the β inhibitor against LA/87 and its variants. Four HAI units of the viruses and 0.5% chicken erythrocytes were used for the assay.

  5. Fig. 5.
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    Fig. 5.

    Effect of V. cholerae NA treatment of plasma membranes of chicken embryo CAM cells on the binding of LA/87 and its variants.

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  1. J. Virol. vol. 72 no. 8 6373-6380
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