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. 2013 Jan 22;110(4):1458-63.
doi: 10.1073/pnas.1218509110. Epub 2013 Jan 7.

Hemagglutinin homologue from H17N10 bat influenza virus exhibits divergent receptor-binding and pH-dependent fusion activities

Xueyong Zhu  1 , Wenli Yu, Ryan McBride, Yan Li, Li-Mei Chen, Ruben O Donis, Suxiang Tong, James C Paulson, Ian A Wilson
Affiliations

Hemagglutinin homologue from H17N10 bat influenza virus exhibits divergent receptor-binding and pH-dependent fusion activities

Xueyong Zhu et al. Proc Natl Acad Sci U S A. .

Abstract

Bat influenza virus H17N10 represents a distinct lineage of influenza A viruses with gene segments coding for proteins that are homologs of the surface antigens, hemagglutinin (HA) and neuraminidase (NA). Our recent study of the N10 NA homolog revealed an NA-like structure, but with a highly divergent putative active site exhibiting little or no NA activity, and provided strong motivation for performing equivalent structural and functional analyses of the H17 HA protein. The overall structure of the H17 HA homolog from A/little yellow-shouldered bat/Guatemala/060/2010 at 3.18 Å resolution is very similar to other influenza HAs, with a putative receptor-binding site containing some conserved aromatic residues that form the base of the sialic acid binding site. However, the rest of the H17 receptor-binding site differs substantially from the other HA subtypes, including substitution of other conserved residues associated with receptor binding. Significantly, electrostatic potential analyses reveal that this putative receptor-binding site is highly acidic, making it unfavorable to bind any negatively charged sialylated receptors, consistent with the recombinant H17 protein exhibiting no detectable binding to sialylated glycans. Furthermore, the fusion mechanism is also distinct; trypsin digestion with recombinant H17 protein, when exposed to pH 4.0, did not degrade the HA1 and HA2, in contrast to other HAs. These distinct structural features and functional differences suggest that the H17 HA behaves very differently compared with other influenza HAs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Crystal structure of GU10-060 HA2-47G HA. (A) Overview of the H17 HA trimer. For clarity, one of the monomers is highlighted in magenta (HA1) and cyan (HA2). The receptor-binding site (RBS) in influenza A and B HAs is highlighted in blue and designated here as the HA putative RBS because no binding activity has yet been found for H17 HA. The HA1/HA2 single Arg cleavage site is highlighted in green. Carbohydrate observed at HA1 Asn117 in the electron density map is colored yellow, and other asparagines, 23 and 289 of HA1, and 145 and 154 of HA2, that code for potential N-glycosylation sites are also labeled. The GU10-060 HA2 A47G mutation was made to stabilize an ectodomain trimer during baculovirus expression; the HA2 47 position is on the trimer interface and far from the RBS. (B) Putative RBS of the H17 HA with key side chains shown in sticks. The four residues that are conserved in the H17 HA and other HAs are colored with green carbon atoms, whereas other nonconserved putative RBS residues are colored with yellow carbon atoms.
Fig. 2.
Fig. 2.
Stereoview of structural comparison of the putative RBS of H17 HA (in green side chains and yellow Cα carbons) with the H1 HA from A/California/04/2009 (H1N1) (in gray; PDB ID code 3UBQ) after superimposing the RBS subdomain. The overall structures are all superimposable, including Trp153, His183, and Tyr195, as well as Leu194, in the base of the RBS. Other key residues are substituted between H17 HA and H1 HA, and are labeled in green for H17 HA and in gray for H1 HA.
Fig. 3.
Fig. 3.
Electrostatic potential surface around putative RBS of different HAs. Electrostatic surface potentials were calculated using the APBS program (28). Negatively charged regions are red, positively charged regions are blue, and neutral regions are white (−10–10 KbT/ec potential range). The coordinates used in this figure are as follows. Group 1 HAs: H17, GU10-060 HA2-47G HA; H1 (PDB ID code 3UBQ), H2 (PDB ID code 3KU6), H5 (PDB ID code 2FK0), and H9 (PDB ID code 1JSD). Group 2 HAs: H3 (PDB ID code 2HMG), H7 (PDB ID code 1TI8), and H14 (PDB ID code 3EYJ), as well as influenza B HA (PDB ID code 3BT6). The putative RBS of the H17 HA is unusual in its strong negative charge.
Fig. 4.
Fig. 4.
Reducing SDS/PAGE of trypsin-digested GU10-060 HA2-47G HA0 to HA1 and HA2 subdomains at different pHs. Lanes 1, 2, and 3 show GU10-060 HA0 trypsin-digested after exposure to pH 4.0, 4.9, and 8.0, respectively, which cleaves HA0 only to HA1 and HA2 (note: HA2 has different glycan forms). Trypsin did not degrade the HA exposed to low pH, as observed in other HA subtypes. Lane 4 shows uncleaved H17 HA2-47G HA0. These results suggest that GU10-060 HA requires processing to the HA1/HA2 subunits to generate the putative fusion peptide, but low-pH–induced conformational changes, which render HA susceptible to extensive degradation, are not apparent.
Fig. 5.
Fig. 5.
Structural comparison around the HA1/HA2 cleavage site of H17 HA with other HAs. (A) GU10-060 HA HA0 cleavage site with HA1 colored green and HA2 colored cyan. Due to flexibility of the cleavage loop, the residues corresponding to HA1 325–329 and HA2 1–5 are not modeled. Overlay around the cleavage loops of H17 HA0 (in green) with H3 HA0 (in brown; PDB ID code 1HA0) (B) and H1 HA0 (in gray; PDB ID code 1RD8) (C). The conformation of modeled putative fusion peptide of H17 is more similar to H3 HA0 than to H1 HA0. For comparison, Fig. 5 A–C are generated in the same orientation.
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