Specificity of wild type and mutant H7 HAs on glycan arrays and binding to chicken and human trachea epithelium.

<p>Glycan binding analyses of Sh2 H7N9 HA wild type and several mutants that confer human-type receptor binding: G228S, K193T G228S, V186K K193T G228S, V186G K193T G228S, with human Cal/04/09 2009 pandemic H1N1 HA as a control. (A) ELISA-like assay using sialoside polymers. The mean signal and standard error were calculated from six independent replicates; white open circles represent α2–3 linked sialylated di-LacNAc (3’SLNLN), black closed circles represent α2–6 linked sialylated di-LacNAc (6’SLNLN), and non-sialylated di-LacNAc (LNLN) are represented in asterisks. (B) The glycan array mean signal and standard error were calculated from six independent replicates; α2–3 linked sialosides are shown in white bars (glycans 11 to 79 on the x axis) and α2–6 linked sialosides in black (glycans 80 to 135). Glycans 1 to 10 are non-sialylated controls (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006390#ppat.1006390.s001" target="_blank">S1 Table</a>). (C) Tissue binding to either chicken or human tracheal sections is observed by HRP-staining. The sialoside array, ELISA-like assay, and tissue binding experiments are representative of three independent assays performed with different batches of HA proteins.</p

Avidity of Sh2 (WT) and Sh2 V186K-K193T-G228S variant HA for N-linked glycan receptors assessed by glycan ELISA.

<p>Sh2 (upper panels) binds strongly to avian-type (α2–3) receptors (left, white open shapes) with weaker binding to human-type (α2–6) receptors (right, black closed shapes). Sh2 V186K-K193T-G228S (lower panels) shows vastly reduced avidity for avian N-glycans and increased selectivity for extended glycan receptors to human receptors. Assays are conducted with biantennary, N-linked glycans (N) with one to four LacNAc (LN, Galβ1-4GlcNAc) repeats terminated with sialic acid (S) in α2–3 or α2–6 linkage (SLN_1-4-N). An asialo, mono-LacNAc (LacNAc-biotin, LN-L) was used as a negative binding control.</p

Melting curve of recombinant HA obtained by DSC to determine the thermostability of Sh2, Sh2 V186K K193T G228S, and A/KY/07.

<p>The raw data are depicted in the solid line, while the fitted curve, from which the <i>T</i>_m was derived, is depicted with a dotted line. (A) A summary of the peaks observed during the DSC experiments for each recombinant HA, (B) Sh2, (C) A/KY/07 and (D) Sh2 V186K K193T G228S.</p

H7 Sh2 mutant combinations that also bind to human-type receptors.

<p>Glycan binding analyses of Sh2 H7N9 mutant HAs, V186N G226S (A) and V186N N224K G228S (B). The mean signal and standard error were calculated from six independent replicates on both the PAA (left column) and the sialoside array (right column). Tissue binding to either chicken or human tracheal sections is observed by HRP-staining (right column). In the PAA array, white open circles represent α2–3 linked sialylated di-LacNAc (3’SLNLN), black closed circles represent α2–6 linked sialylated di-LacNAc (6’SLNLN), and non-sialylated di-LacNAc (LNLN) are represented in asterisks. In the sialoside array α2–3 linked sialosides are shown in white bars (glycans 11 to 79 on the x axis) and α2–6 linked sialosides in black (glycans 80 to 135). Glycans 1 to 10 are non-sialylated controls (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006390#ppat.1006390.s001" target="_blank">S1 Table</a>). The sialoside array, ELISA-like assay and tissue binding experiments shown are representative of three independent assays performed with different batches of HA proteins.</p

Amino acid variation in the receptor binding pocket of influenza HAs and impact of K193T mutation on receptor conformation.

<p>(A) Variation at HA positions that are known to mediate the switch in receptor binding specificity for human H1, H2 and H3 pandemic viruses and corresponding avian viruses of H1, H2, H3 and H5 subtypes in comparison with human H7N9. Red indicates amino acids involved in either human- or avian-type receptor specificity, blue indicates amino-acid positions that are mutated to the amino acids found in human H3N2 and H2N2 viruses. (B) Projection of the receptor glycan from the binding pocket. The receptor analog 6’SLNLN (α2–6 linked sialylated di-LacNAc; NeuAcα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc) is modeled in the WT H7 with K193 (dark gray), and the mutant H7 with V186K K193T G228S (light gray). In the WT, K193 causes the receptor to project further away from the 190 helix. Symbols in the sugar rings are the conventions for the Symbol Nomenclature For Glycans (SNFG) where sialic acid is the purple cubic diamond, galactose is the yellow sphere and GlcNAc is the blue cube.</p

SDS-PAGE of A/bat/Peru/10 HA0 (lanes 1 to 4) and its mature HA (lanes 5 to 8) in trypsin susceptibility assay.

<p>A/bat/Peru/10 HA with a monobasic cleavage site was expressed in its HA0 form in a baculovirus expression system. Lanes 1 and 2 show A/bat/Peru/10 native HA0 at pH 8.0 and pH 4.9, respectively, while lanes 3 and 4 show the equivalent reducing gel of HA0 treated with trypsin at pH 8.0 and pH 4.9, respectively. Similarly, lanes 5 and 6 show A/bat/Peru/10 mature HA at pH 8.0 and pH 4.9, respectively, while lanes 7 and 8 show the equivalent reducing gel of mature HA treated with trypsin at pH 8.0 and pH 4.9, respectively.</p

Crystal structures of A/bat/Peru/10 HA.

<p>(A) Overall structure of A/bat/Peru/10 HA. The H18 HA trimer consists of three identical monomers with one RBS per monomer. HA1 is highlighted in green and HA2 in cyan. N-linked glycans observed in the electron density maps are shown with yellow carbons. (B) The A/bat/Peru/10 HA putative RBS (in crystal 1, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003657#ppat.1003657.s014" target="_blank">Table S6</a>) in ribbon representation with the side chains of key binding residues shown. The three highly conserved residues (W153, H183, and Y195) in HAs are colored with green carbon atoms, whereas nine residues that are conserved in A/bat/Peru/10 H18 and two H17 HAs from bat influenza viruses A/little yellow-shouldered bat/Guatemala/164/2009 (H17N10) (GU09-164) and A/little yellow-shouldered bat/Guatemala/060/2010 (H17N10) (GU10-060) are labeled in red. E190 and G225 are also conserved, especially in avian influenza A viruses. (C) Molecular surface of the putative RBS of A/bat/Peru/10 HA compared to the RBS of 2009 H1 HA from A/California/04/2009 (H1N1) (PDB code 3UBQ). A canonical sialic acid is modeled in the HA for comparison as observed in other HA structures. The RBS of A/bat/Peru/10 HA is shallower and wider than 2009 H1 HA with no space for the glycerol moiety of sialic acid (indicated by the red arrow). For comparison, figures (B) and (C) are generated in the same orientation.</p

Detection of influenza A virus and antibody in bats from Peru (2010).

<p>Detection of influenza A virus and antibody in bats from Peru (2010).</p

Important functional motifs in non-bat influenza A internal proteins are largely conserved in bat influenza proteins.

†<p>Letters written as subscripts denote amino acid substitutions in bat viruses compared to the consensus non-bat amino acid sequence.</p

Crystal structures of A/bat/Peru/10 NAL.

<p>(A) Overall structure of A/bat/Peru/10 NAL with a conserved calcium binding site. The N11 NAL tetramer is viewed from above the viral surface, and consists of four identical monomers with C4 symmetry. One monomer is colored in six different colors to illustrate the canonical <i>β</i>-propeller shape of six four-stranded, anti-parallel <i>β</i>-sheets. The putative active site is located on the membrane-distal surface (on top of the molecule). The observed N-linked glycosylation sites are shown with attached carbohydrates. A single calcium ion is shown in red spheres. (B) The A/bat/Peru/10 NAL putative active site (crystal form 1, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003657#ppat.1003657.s014" target="_blank">Table S6</a>) with the conserved catalytic and active site residues in other NAs shown as well as other polar and charged residues. The six residues (R118, W178, S179, R224, E276 and E425) conserved in all influenza NAs are colored with green carbon atoms in contrast to other putative active site residues in yellow carbon atoms, whereas eight residues that are conserved in two bat influenza N10 and N11 NAL proteins are labeled in red. (C) Molecular surface of the active site of A/bat/Peru/10 N11 NAL and 1918 N1 NA from A/Brevig Mission/1/18 (H1N1) (PDB code 3BEQ). A canonical sialic acid is modeled in A/bat/Peru/10 NAL as in other NA structures and appears to collide with the NA putative active site around the glycerol moiety (as indicated by the red arrow). The putative active site pocket of A/bat/Peru/10 NAL is much wider than 1918 N1 NA. For comparison, figures (B) and (C) are generated in the same orientation.</p