1. Volume 3, Issue 3, Pages 769-778 (March 2013)
Bat-Derived Influenza Hemagglutinin H17 Does Not Bind Canonical Avian or Human Receptors and Most Likely Uses a Unique Entry Mechanism 
Xiaoman Sun, Yi Shi, Xishan Lu, Jianhua He, Feng Gao, Jinghua Yan, Jianxun Qi, George F. Gao 
Cell Reports 
Volume 3, Issue 3, Pages (March 2013)
DOI: /j.celrep
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2. Cell Reports 2013 3, 769-778DOI: (10.1016/j.celrep.2013.01.025)
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3. Figure 1 H17 Protein Does Not Bind to Canonical SA Receptors
(A and B) SPR of H17 protein binding to α2,3-linked and α2,6-linked receptors at a series of concentrations from 0 to 100 μM. As a positive control, SPR of QH05-H5 protein binding to α2,3-linked receptor at a concentration of 2.5 μM was performed (dotted line in A).
(C) ELISA-based MDCK cell binding assay. H17 did not bind to MDCK cells, and as a positive control, the QH05-H5 protein bound to MDCK cells well.
(D) Glycan microarray analyses of the 1968 Hong Kong H3 protein (upper) and the H17 protein (lower). Binding to different types of glycans on the array is highlighted, where magenta represents Neu5Gc, blue represents α2,8-ligands, cyan represents α2,6-ligands, green represents α2,3-ligands, and yellow represents other glycans. The H3 protein displayed a good avidity to α2,6-ligands, but the H17 protein showed no obvious avidity to any of the glycans. Error bars represent SD of the mean.
See also Table S1.
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4. Figure 2
Biochemical and Biophysical Characterization of the Soluble H17 Protein
(A–D) Trypsin susceptibility assays of the soluble H17 protein (A), 1968 Hong Kong H3 protein (B), low-pH-incubated H17 protein (C), and H3 protein (D). The H17 protein can be digested into different HA1 fragments and one HA2 fragment at pH 8.0. As a representative of characterized HA proteins, the 1968 Hong Kong H3 protein can be digested into one HA1 fragment and one HA2 fragment. The low-pH-incubated (pH 5.0) H17 and H3 proteins can be digested into different HA1 fragments and one HA2 fragment, similar to the H17 protein at pH 8.0.
(E) Thermostability analyses of the H17 protein and other characterized HA proteins (09H1, H2, H3, H5, and H16). Temperature-dependent CD spectroscopic experiments revealed that the H17 protein has a much lower midpoint transition temperature (Tm = ∼40°C) than other known HA proteins (Tm = ∼50°C).
(F) Sedimentation-velocity analytical ultracentrifugation of H17 protein. The H17 protein exists as a trimer (∼180 kDa) in solution.
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5. Figure 3
Overall Crystal Structure of the H17 Protein and Comparison with Other Solved HA Subtype Structures
(A) Overall structure of the H17 protein. H17 adopts a typical HA trimer structure, containing a membrane-distal globular domain and membrane-proximal stem domain.
(B) Phylogenetic tree showing that H17 belongs to group 1.
(C and D) Comparison of the H17 monomer with other solved HA subtype structures (H1, light blue; H2, cyan; H3, magenta; H5, blue; H7, pink; H9, limon; H14, hot pink; H16, green; and H17, yellow). The interhelix loop of the H17 structure displays a similar conformation to the group 1 HAs (H1, H2, H5, H9, and H16), which is distinct from the group 2 HAs (H3, H7, and H14). The rigid body orientation of the globular domain in the H17 structure is similar to that of the H16 subtype, by means of superimposition through the long helix of HA2. They are located between other group 1 HAs and group 2 HAs.
See also Figures S1 and S2, and Tables S2 and S3.
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6. Figure 4
Structural Basis of the Lack of H17 Binding to Canonical SA Receptors
(A and B) Surface representations of the receptor binding cavity in typical H1 (PDB ID code: 3AL4) and H17 structures. The typical H1 subtype displays a clear shallow receptor binding cavity, whereas H17 does not have an obvious cavity. The bottoms of the cavity are marked in orange.
(C) Electrostatic potential maps of the receptor binding sites from the H17 structure. In the H17 structure, the receptor binding cavity is negatively charged.
(D) Cartoon diagram of the receptor binding site in the H17 structure. The key residues D136, Q190, H226, and D228 tightly link the 130-loop, 190-helix, and 220-loop together through a hydrogen bond and salt bridge network. The hydrogen bonds and salt bridges are shown in a dash line.
(E) Cartoon diagram of SA binding in the receptor binding site of the representative H3 structure. The SA forms three hydrogen bonds with the 130-loop, and the negatively charged carboxylate group forms a strong bond with the T/S136 residue.
(F) Model diagram of the putative SA binding site of the H17 structure. The negatively charged D136 residue has an electrostatic repulsion with the negatively charged carboxylate group of the SA, which impedes SA binding in H17.
See also Figure S3.
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7. Figure 5
Exposed Fusion Peptide in the Cleavage Site of the H17 Structure
(A) Surface diagram of the fusion peptide in the representative H3 structure. The fusion peptide inserts into the cavity near the cleavage site.
(B, D, and F) Model diagrams of different conformations of the fusion peptide in the H3, H17, and HEF structures. The black arrow represents the direction of the fusion peptide. The dashed lines represent the residues that are not seen. Residues G1, L2, F3, and G4 are omitted in the H17 structure due to poor electrostatic mapping. In HEF structure, the omitted residues are I1, F2, and G3.
(C) Surface diagram of the fusion peptide in the H17 structure. The fusion peptide is exposed away from the cavity.
(E) Surface diagram of the fusion peptide in the HEF structures of influenza C viruses. The fusion peptide is partially exposed away from the cavity.
See also Figures S4 and S5.
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8. Figure 6
Conserved Hydrophobic Groove in H17 Protein Reveals the Structure Basis of Binding with the Broad Neutralizing Antibody FI6
(A and B) Surface representation of the F subdomains of 09H1 HA (A) and H17 HA (B) with selected side chains that contribute to the conserved hydrophobic groove. The approximate boundaries of the hydrophobic grooves are indicated by the black lines. Although the residues contributing to the hydrophobic groove are moderately different between 09H1 and H17, similar hydrophobic grooves guarantee the binding potential by the antibody FI6.
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9. Figure 7
Structural Comparison of the Ig-like Fold Elements among Measles Virus H Protein, N10, and H17
(A) Cartoon diagram of the complex structure of measles virus H (MV-H) protein and its receptor SLAM. The Ig-like SLAM molecule binds the H proteins mainly through the interaction between two β sheets.
(B) Cartoon diagram of the structure of the bat influenza N10 molecule. N10 has a similar Ig-like fold, which might provide the β sheet to interact with a protein receptor.
(C) Cartoon diagram of the structure of the H17 protein. H17 has similar Ig-like fold in the globular domain, which might provide the β sheet to interact with a protein receptor.
(D–F) The Ig-like fold elements from MV-H, N10, and H17 are picked up and shown alone. Both the N10 and H17 molecules have a β sheet similar to the MV-H protein.
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10. Figure S1
SDS-PAGE of the H17 Crystals and N-terminal Amino Acid Sequencing of the HA2 Band, Related to Figure 3
(A) The SDS-PAGE of H17 crystals shows that there are two bands, HA1 and HA2, confirming the H17 protein had undergone proteolytic processing in the crystal form.
(B–F) Mass spectrometry maps of the first five N-terminal amino acids of the HA2 band. The maps show the first five amino acids are GLFGA.
(G) Standard mass spectrometry map of different amino acids.
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11. Figure S2
Detailed Comparison of the Interhelix Loops among Solved HA Structures, Related to Figure 3
H1, light blue; H2, cyan; H3, magenta; H5, blue; H7, pink; H9, limon; H14, hot pink; H16, green; and H17, yellow. According to the conformation of the top of the interhelix loop, the HA structure can be classified into two groups, group 1 and group 2, which is consistent with the phylogenetic tree. The group 1 HAs have a higher top conformation than the group 2 HAs. Otherwise, the middle of the interhelix loop adopts different conformations among different HA subtypes. The different conformations of the interhelix loop can affect the orientations of the globular domain.
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12. Figure S3
Sequence Alignment of the Receptor Binding Site in Different Influenza A Virus Subtypes, Related to Figure 4
(A–C) The three secondary elements (130-loop (A), 190-helix (B) and 220-loop (C)) are included. Compared with other known 16 subtypes, the H17 has special amino acids in positions 136, 226 and 228 that are critical for sialic acid binding. The sequences are derived from the following virus strains for each subtype: H1:A/California/04/2009(H1N1);H2:A/Singapore/1/1957(H2N2);H3:A/Aichi/2/1968(H3N2); H4:A/duck/Czechoslovakia/1956(H4N6);H5:A/VietNam/1203/2004(H5N1);H6:A/chicken/California/431/2000(H6N2);H7:A/turkey/Italy/8458/2002(H7N3);H8:A/turkey/Ontario/6118/1968(H8N4); H9:A/Swine/Hong Kong/9/98(H9N2);H10:A/chicken/Germany/N/1949(H10N7); H11:A/duck/England/1/1956(H11N6);H12:A/duck/Alberta/60/1976(H12N5);H13:A/gull/Maryland/704/1977(H13N6); H14:A/mallard/Astrakhan/263/1982(H14N5);H15:A/shearwater/West Australia/2576/79(H15N9);H16:A/black-headed gull/Sweden/2/99(H16N3);H17:A/little yellow-shouldered bat/Guatemala/060/2010(H17N10);
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13. Figure S4
Electron Density of the Fusion Peptide in H17 Structure, Related to Figure 5
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14. Figure S5
Comparison of the Trimerization Modes of H17 (Green) and H3 (Magenta), Related to Figure 5
(A and B) H17 and H3 have different trimerization scales, which are shown by the different sizes of the triangles formed by the carbon atoms of the R76 residue from the HA2 chain.
(C) The trimerization of H17 is more contorted compared to H3. The F3 residue of the fusion peptide forms a hydrophobic core in the center of the H3 trimer.
(D) Narrower cavity in H17 (green). Due to contorted trimerization mode, the cavity is narrower than that in H3 (magenta), and the cavity is formed by the long helices of the HA2 subunits from different HA monomers.
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