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Materials and Methods are available as supporting material on Science Online.
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13
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33645980119
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note
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Viet04 HA at a concentration of 9 mg/ml was used to grow crystals in sitting drops with a precipitant solution of 22% polyethylene glycol 2000 and 0.1 M Hepes, pH 6.55 (see also supporting online material).
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14
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33646013690
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19A).
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33645998225
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note
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Scores from the molecular replacement program PHASER revealed superior scores for the 1918 H1 structure (Z score: 37.2; and log-likelihood gain, 3412), as compared with the Sing97 structure (Z scores, 33.8; and log-likelihood gain, 768).
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17
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33645977279
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note
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57, in a symmetry-related monomer.
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20
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33646016088
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note
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Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; O, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; X, any amino acid; and Y, Tyr.
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33645985263
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note
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Regions of antigenic variation have been identified in H1 and H3 serotypes. For H1, these sites were designated Sa, Sb, Ca, and Cb; for H3, sites were designated A, B, C, and D.
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28
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33646000688
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note
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HA binding can be analyzed not only for sialic acid-linkage preference, but also for additional features, such as charge; glycan length; or additional sulfation, fucosylation, and sialylation. Of the 265 glycans currently imprinted on the array, 6 are glycoproteins; 38 have sialic acids with α2-3 linkages; 16 have α2-6 linkages; 7 have α2-8 linkages; and a further 16 are β-linkages, modified sialic acid analogs, or glycolylsialic acid glycans. (See table 54 for the glycans analyzed in this study. Of the α2-6 sialosides, only natural full-sized N-linked glycans represented on the array are the biantennary structures (nos. 56 and 57). The remaining sialosides are fragments or terminal sequences found on glycoproteins. For full information on the array, contact the Consortium for Functional Glycomics (62). Previous binding data using this technology and cell-based assays with whole viruses show that N-linked glycans close to the receptor-binding site can affect receptor binding through steric hindrance (35, 63). Insect cells do not produce complex glycans containing terminal galactose and/or sialic acids, as seen in mammalian cells, although high-mannose glycans are produced (64). However, because of the presence of the influenza sialidase, complex glycans of influenza HAs usually terminate only in galactose, and thus the size of the N-glycans elaborated by insect cells approximate to the size of the complex N-glycans in mammalian host cells. Thus, any importance of complex glycans for HA function is still unknown. Indeed, results for the avian H3 HA (A/Duck/Ukraine/1/1963), published recently (35), are in agreement with previous whole viral studies (65). However, independent studies are ongoing to develop the array for whole-virus analyses so that a direct comparison can be made. Such initial experiments are promising, because the strict α2-3 specificity observed here for Dk76 is also seen with whole-virus studies (37) and preliminary experiments with A/Puerto Rico/8/1934 virus that reveal both α2-3 and α2-6 specificity (34), in agreement with experiments from cell-based assays (37).
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38
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33645990102
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note
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Whole-virus studies, including those for Viet04 virus, also revealed α2-3 specificity with a preference for sulfation in current H5N1 strains (39). However, this assay used only seven ligands (one α2-6 and six α2-3), which is considerably fewer than the 84 sialosides, sialoside analogs, and glycoproteins analyzed here. In our glycan array, sulfation on the second galactose was not tolerated (no. 37) for Viet04, although binding was apparent for sialosides with Gal in either β1-3 or β1-4 linkage to a GlcNAc or GalNAc (nos. 21 to 23, 32, 33), as well as to fucosylated glycans (nos. 26 to 29).
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40
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0019572737
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H. Debray, D. Decout, G. Strecker, G. Spik, J. Montreuil, Eur. J. Biochem. 117, 41 (1981).
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33645975930
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note
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Avian H1 bound only to nonbranched glycans and to sulfated and/or negatively charged glycans (Figs. 4C and 5, A to C). The single E190D mutation reduced binding to most α2-3 glycans, except to sulfated sialosides (Fig. 5A). These results suggest mutation at both 190 and 225 positions is always a requirement for H1 serotypes to adapt to a human host.
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48
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33646007193
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note
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The E190D mutation (Fig. 5D) reduced overall binding of α2-3 ligands and glycoproteins, except for the sulfated and/or negatively charged glycans (nos. 18, 20, 24, 25, and 38). The G225D mutation (Fig. 5E) appeared to have little effect on the binding profile, in contrast to avian H1, where binding was not detected (Fig. 5B). The double mutant (E190D,G225D) did not bind to any glycan on the array (see Fig. 5F).
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49
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12144288130
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S. J. Gamblin et al., Science 303, 1838 (2004).
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50
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33645996480
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note
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190. Experiments are in progress to test this notion.
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51
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33645969376
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note
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The Q226L mutation eliminated binding to the microarray, except for two negatively charged α2-3 glycans [with either an extra sialic acid on the 6-position of a GalNAc (no. 20) or 6-sulfation on GlcNAc with a branched fucose (no. 25)]. The G2285 mutation did not have any significant effect compared with Viet04, except that sialosides with sulfation on the 6-position of the galactose, with or without branched fucosylation on the GlcNAc (nos. 12, 37) were tolerated. Stronger binding was observed for fucosylated glycans (nos. 26 to 29), and reduced binding was observed for sialosides-with β1-3 linkages between the galactose and GlcNAc/GalNAc (nos. 21 to 23) (Fig. 5H). In addition to 6′-sialyllactose (no. 49), as seen for Viet04, binding was observed for α2-6 biantennary structures (nos. 56 and 57). The double mutant (Q226L,G228S) showed reduced binding to α2-3 sialosides. Only sulfated and long-chain glycans were tolerated (nos. 16, 20, 24, 25, 35), but binding to α2-6 biantennary structures (nos. 56 and 57), as with the G2285 mutation, was also maintained.
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52
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0345734193
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54
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33645996800
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See glycan structure database (www.functionalglycomics. org).
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57
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1842585032
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M. N. Matrosovich, T. Y. Matrosovich, T. Gray, N. A. Roberts, H. D. Klenk, Proc. Natl. Acad. Sci. U.S.A. 101, 4620 (2004).
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59
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33645965985
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note
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Attenuated viruses with a 5227N mutation led to higher hemagglutinin inhibition titers in ferrets (60). Thus, enhanced binding to α2-3 ligands, especially to 6-sulfated GalNAc, could lead to an increased uptake into antigen-presenting cells and subsequent antibody production.
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60
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24644454633
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E. Hoffmann, A. S. Lipatov, R. J. Webby, E. A. Govorkova, R. G. Webster, Proc. Natl. Acad. Sci. U.S.A. 102, 12915 (2005).
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Hoffmann, E.1
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61
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note
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227.
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62
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Consortium for Functional Glycomics (www//functionalglycomics. org).
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63
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0037196586
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H. D. Klenk, R. Wagner, D. Heuer, T. Wolff, Virus Res. 82, 73 (2002).
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Klenk, H.D.1
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66
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33646014851
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W. L. Delano (2002); (www.pymol.org).
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Delano, W.L.1
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67
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33646010124
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note
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The work was supported in part by National Institute of Allergy and Infectious Diseases grant AI058113 (I.A.W., T.T., J.K.T.); National Institute of General Medical Sciences grants GM062116 (to J.C.P., I.A.W.) and GM060938 (to J.C.P.); and partial support from NIH grants to I.A.W. (CA55896 and AI42266). We thank P. Palese and L. Glaser (Mount Sinai School of Medicine, New York) for providing the full-length clone of A/Vietnam/1203/2004; the staff of the Advanced Light Source Beamline 8.2.2 for the beamline assistance; X. Dai, S. Ferguson, P. Carney, and J. Vanhnasy (The Scripps Research Institute) for expert technical assistance; and R. Stanfield and M. Elsliger (The Scripps Research Institute) for helpful discussions. This is publication 17916-MB from The Scripps Research Institute. Coordinates and structure factors have been deposited in the Protein Data Bank (code 2FK0) and will be released on publication.
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