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, 12 (1), e1005411
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An Open Receptor-Binding Cavity of Hemagglutinin-Esterase-Fusion Glycoprotein From Newly-Identified Influenza D Virus: Basis for Its Broad Cell Tropism

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An Open Receptor-Binding Cavity of Hemagglutinin-Esterase-Fusion Glycoprotein From Newly-Identified Influenza D Virus: Basis for Its Broad Cell Tropism

Hao Song et al. PLoS Pathog.

Abstract

Influenza viruses cause seasonal flu each year and pandemics or epidemic sporadically, posing a major threat to public health. Recently, a new influenza D virus (IDV) was isolated from pigs and cattle. Here, we reveal that the IDV utilizes 9-O-acetylated sialic acids as its receptor for virus entry. Then, we determined the crystal structures of hemagglutinin-esterase-fusion glycoprotein (HEF) of IDV both in its free form and in complex with the receptor and enzymatic substrate analogs. The IDV HEF shows an extremely similar structural fold as the human-infecting influenza C virus (ICV) HEF. However, IDV HEF has an open receptor-binding cavity to accommodate diverse extended glycan moieties. This structural difference provides an explanation for the phenomenon that the IDV has a broad cell tropism. As IDV HEF is structurally and functionally similar to ICV HEF, our findings highlight the potential threat of the virus to public health.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Receptor specificity of the IDV HEF protein.
(A) Glycan microarray analyses of the IDV enzymatically inactive HEF (HEF-mut) protein using the CFG glycan microarray. 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, red represents Neu5,9Ac2 and yellow represents other glycans. The HEF-mut protein displayed a good avidity to Neu5,9Ac2 ligands. The structure formulas of the top representative are shown. Error bars represent the standard deviation (SD) of the mean value. Percent coefficient of variation (%CV) = 100×standard deviation/mean. A %CV of less than 50% indicates data reliability. (B) Glycan microarray analysis of IDV HEF-mut proteins using a more extended paired array. The library of glycans tested was designed to test the influence on protein binding of (1) glycosidic linkage of 9-O-Ac-Sia (α2–3 versus α2–6) and (2) modifications at C5 (N-acetylation, 5-N-Ac, versus N-glycosylation, 5-N-Gc). Data presented as mean ± SD.
Fig 2
Fig 2. Hemagglutination assay of the IDV and ICV HEF proteins at 25°C or 4°C.
Receptor binding activity of HEF protein was assessed by hemagglutination assay with chicken erythrocytes. Serial dilutions of purified HEF proteins (100 μg/mL to 0.1 μg/mL per well) were mixed with washed chicken erythrocytes and incubated to analyze the receptor binding and cross-linking of chicken erythrocytes at two different temperatures 25°C (A) or 4°C (B). The H5 HA protein was used as positive control. Positive hemagglutination results formed a uniform reddish color across the well, whereas negative results appeared as dots in the center of round-bottomed plates due to erythrocytes sedimentation.
Fig 3
Fig 3. Receptor binding of the IDV and ICV HEF proteins using MDCK cell based ELISA or solid-phase lectin-binding assay towards BSM.
(A) Receptor binding activity of HEF protein was assayed by MDCK cell based ELISA. After MDCK cell were fixed and blocked, different concentrations of His-tagged HEF or H5 HA proteins (10 μg/mL, 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL, 100 μg/mL) was incubated for detection by ELISA method. (B) Binding of two-fold serial dilutions (starting at 100 μg/mL) of HEF protein was assayed in a solid-phase lectin-binding assay towards BSM. Mean values were determined from three duplicates and are presented with SDs indicated by error bars.
Fig 4
Fig 4. Esterase activity assay of HEF proteins.
(A) Receptor destroying enzyme activity towards BSM. Coated BSM was treated with two-fold serial dilutions (starting at 100 ng/mL) of IDV enzymatically-active HEF proteins for 1 h at 37°C and 9-O-Ac-Sia content was detected by solid-phase lectin-binding assay with IDV HEF-mut protein. Decrease in signal as compared to untreated BSM is plotted in percentages. (B-D) Esterase kinetic analysis of HEF proteins against pNPA at 37°C (B), 25°C (C) and 4°C (D). The reaction velocity (OD min-1μM-1 protein) is shown based on substrate conversion. PNPA substrate was used at a final concentration of 0–4mM. Both IDV(red cycle) and ICV HEF (cyan square) display obvious enzymatic activities, whereas IDV (green rhombus) and ICV HEF-mut (blue square) lack sialidase activity. BSA (magenta triangle) was used as a negative control. Mean values were determined from three duplicates and are presented with SDs indicated by error bars.
Fig 5
Fig 5. Staining of human, swine and bovine trachea with either ICV or IDV HEF protein.
Paraffinized tissue sections were stained with recombinant HEF-mut protein derived from the baculovirus expression system. Specific staining by recombinant protein (green) is indicated by white arrows. Nuclei were counterstained with DAPI (blue).
Fig 6
Fig 6. Overall crystal structure of the IDV HEF protein.
(A) Ribbon representation of the trimers of ICV and IDV HEF. For ICV HEF (PDB code: 1FLC), the trimer is colored gray; For IDV HEF, one HEF protomer is colored by domain separately: fusion domain (F1, F2, F3, red), receptor domain (R, blue) and esterase domain (E1, E', E2, green) and the others are colored gray. Fusion peptides are shown in yellow. N-linked glycans are highlighted in sticks (magenta) and disulfide linkages are shown in orange. Linear order of the sequence segments in HEF colored by domains. The scissor indicates the HEF cleavage site. (B) Superposition of IDV HEF and ICV HEF subdomains are shown with RMSD values and colored by domains.
Fig 7
Fig 7. The variable receptor-binding site in the IDV HEF.
(A) Comparison of the receptor-binding sites of ICV (light blue) and IDV (orange) HEFs. A salt bridge interaction is formed between the cationic K235 (blue) of 230-helix and the anionic D269 (red) of 270-loop, shown in a dashed line. (B and C) The surface presentations of the salt bridge interaction in ICV HEF (B) and absence of the salt bridge in IDV HEF (C), resulting in an open channel. (D-F) The surface presentations of ICV HEF binding to 9-N-Ac-Sia (D) and IDV HEF-mut binding to 9-O-Ac-3'SLN (E) or 9-O-Ac-3'SleC (F).
Fig 8
Fig 8. Detailed Interactions of the Receptor-Binding Site in the IDV HEF.
Molecular interactions of the ICV HEF binding to 9-N-Ac-Sia (A) and IDV HEF-mut binding to 9-O-Ac-3'SLN (B) or 9-O-Ac-3'SleC (C). The secondary elements of the HEF receptor-binding site (170-loop, 230-helix, 270-loop and 290-loop) are labeled and shown in cartoon representation. Selected residues and receptor analogs are labeled and shown in sticks. The water molecules mediating hydrogen-bonding interactions are shown as spheres.
Fig 9
Fig 9. The esterase pocket of the IDV HEF.
(A) Superposition of IDV HEF (green), IDV HEF-mut (orange), ICV HEF (gray) and BCoV HE (cyan) esterase sites. Residue labeling refers to IDV HEF. (B) Surface representation of the esterase pocket of IDV HEF. S57 is covalently modified by the addition of a dimethylarsenic group (cacodylate ion, CAC). The 2Fo-Fc electron density map for this dimethylarsenic modified serine contoured at 1.0 sigma is represented in blue. (C) Surface representation of ICV HEF esterase pocket with substrate analog 9-N-Ac-Sia. (D) Surface representation of IDV HEF esterase pocket with substrate analog 9-N-Ac-Sia in presence of arsenic modification of S57. The 2Fo-Fc electron density map for 9-N-Ac-Sia and the dimethylarsenic modified S57 contoured at 1.0 sigma is represented in blue.
Fig 10
Fig 10. Phylogenetic trees of influenza virus HA/HEF glycoproteins and the related HE or HN proteins.
Maximum-likelihood analysis in combination with 1000 bootstrap replicates was used to derive trees. Evolutionary analyses were conducted in MEGA6 [40].
Fig 11
Fig 11. Exposed fusion peptide in the cleavage Site of IDV HEF.
(A) Surface diagram of the fusion peptide in the ICV HEF structure. The fusion peptide is partially exposed away from the cavity. (B) Surface diagram of the fusion peptide in the IDV HEF structure. The fusion peptide is exposed away from the cavity.

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