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. 2016 Oct 31:6:36216.
doi: 10.1038/srep36216.

Glycosylation changes in the globular head of H3N2 influenza hemagglutinin modulate receptor binding without affecting virus virulence

Irina V Alymova  1 , Ian A York  1 , Gillian M Air  2 , John F Cipollo  3 , Shelly Gulati  2 , Tatiana Baranovich  4 , Amrita Kumar  1   5 , Hui Zeng  1 , Shane Gansebom  1 , Jonathan A McCullers  4   6
Affiliations

Glycosylation changes in the globular head of H3N2 influenza hemagglutinin modulate receptor binding without affecting virus virulence

Irina V Alymova et al. Sci Rep. .

Abstract

Since the emergence of human H3N2 influenza A viruses in the pandemic of 1968, these viruses have become established as strains of moderate severity. A decline in virulence has been accompanied by glycan accumulation on the hemagglutinin globular head, and hemagglutinin receptor binding has changed from recognition of a broad spectrum of glycan receptors to a narrower spectrum. The relationship between increased glycosylation, binding changes, and reduction in H3N2 virulence is not clear. We evaluated the effect of hemagglutinin glycosylation on receptor binding and virulence of engineered H3N2 viruses. We demonstrate that low-binding virus is as virulent as higher binding counterparts, suggesting that H3N2 infection does not require either recognition of a wide variety of, or high avidity binding to, receptors. Among the few glycans recognized with low-binding virus, there were two structures that were bound by the vast majority of H3N2 viruses isolated between 1968 and 2012. We suggest that these two structures support physiologically relevant binding of H3N2 hemagglutinin and that this physiologically relevant binding has not changed since the 1968 pandemic. Therefore binding changes did not contribute to reduced severity of seasonal H3N2 viruses. This work will help direct the search for factors enhancing influenza virulence.

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Figures

Figure 1
Figure 1. Evolution of H3 N-linked glycosylation from 1968–2013.
N-linked glycosylation sites present on the HA trimer of pandemic H3N2 A/Hong Kong/1/1968 (HK68) (crystal structure PDB 4FNK7) are shown in red. Glycosylation sites added to H3 during 1968–2013 are colored in blue. Numbers indicate the start of an N-linked glycosylation sequon (Asn) (numbering based on the mature HA molecule after cleavage of the 16-amino acid signal but ignoring the HA1-HA2 cleavage site; residue 483 is HA2 154 in PDB 4FNK). A green oval shows the location of the receptor binding site (RBS).
Figure 2
Figure 2. Binding of rgHK68 viruses to sialic acids on a glycan array.
(A) Binding of rgHK68, rgHK68 + 2, and rgHK68 + 4 viruses to the 61 sialylated glycans that showed binding (see the Methods section for standard experimental conditions). The average from four replicates for each glycan is shown. The glycan numbers (indicated on the horizontal axis) refer to those on version 5.1 of the Consortium for Functional Glycomics printed array. Details of glycan structures can be found at www.functionalglycomics.org; structures for selected glycans are shown in Table 1. (B) Total numbers and binding intensities, and proportion of Neu5AC α2,3- and Neu5Acα2,6-linked selected glycans bound by rgHK68 viruses at standard conditions. (C) Total binding intensities of rgHK68 viruses to selected glycans at doses ranging from 2,550 HAU to 25,500 HAU (indicated on the horizontal axis). Binding intensities shown in relative fluorescence units (RFU; indicated on the vertical axis).
Figure 3
Figure 3. Elution of rgHK68 viruses from red blood cells.
In HA assays, the rgHK68, rgHK68 + 2, and rgHK68 + 4 were diluted to provide 16 HAU with 0.5% chicken, or human, or turkey, or 0.75% guinea pig RBC after 1 hour at 4 °C. Then the plates were shifted to 37 °C, and elution of viruses from RBC was recorded after 4, 8, and 20 hours of incubation.
Figure 4
Figure 4. Catalytic activities of rgHK68 viruses.
The activity of each viral NA was measured by a standard fluorometric assay and proportioned to the amount of NA or NP in the sample. The NA activities (expressed as NA/NA and NA/NP ratios) of rgHK68 + 2 and rgHK68 + 4 mutants are normalized to those of rgHK68. Error bars indicate the standard deviations (SD) of the mean of the results from three independent experiments. An asterisk indicates a significant difference (p < 0.05) by unpaired Student’s t-test compared with rgHK68 and rgHK68 + 2.
Figure 5
Figure 5. Growth kinetics of rgHK68 viruses in epithelial cell lines.
Cell monolayers were infected with rgHK68, or rgHK68 + 2, or rgHK68 + 4 at MOI 0.01 (A and C) or 1.0 (B) at 37 °C (A, B and C) or at 32 °C (C). Viruses’ growth in Calu-3 cells (C) was examined in the absence or presence of mucin (as indicated). Virus titers were determined for apical culture supernatant fluids at the times indicated on the x-axis. Error bars indicate the SD of the mean of the results from three independent experiments (MDCK, A549, and Vero cell lines) or from three cultures (NHBE and Calu-3 cell lines).
Figure 6
Figure 6. Cell pathology induced by rgHK68 viruses.
Monolayers of Calu-3 cells grown in liquid-covered conditions were infected with three viruses at MOI 0.001 and 32 °C. (A) Viral growth kinetics. Error bars indicate the SD of the mean of the results from three culture replicates. (B) Confocal images of Calu-3 cells stained with antibodies specific for ZO-1 protein (green) 36 hours post-infection. Cell nuclei were visualized with DAPI (blue). Bar, 10 mm. Left column: ZO-1 staining; middle column: higher scale of the ZO-1 region outlined by a box in the left column; right column: ZO-1 staining merged with DAPI. Control: uninfected cell monolayers.
Figure 7
Figure 7. Pathogenicity of rgHK68 viruses in mice.
(A) Virus lung titers and (B) weight loss in BALB/c mice infected with 105.0 PFU per mouse of rgHK68 or rgHK68 + 2 or rgHK68 + 4. (A) Growth kinetics of rgHK68 viruses in lungs of mice (n = 3–5 per time point). The mean ± SD are shown. (B) Mice (n = 10) were monitored individually for weight loss; results are presented as a mean percentage of starting weight ± SD. (A,B) An asterisk indicates a significant difference (p < 0.05) by ANOVA compared with the results for the mice infected with rgHK68 + 2 or rgHK68 + 4. A double asterisk shows a significant difference compared to the results from rgHK68 + 2-infected group.
Figure 8
Figure 8. Binding of H3N2 influenza A viruses to receptors of human RT.
The heat map of relative binding percent of viruses used in our previous and current studies to human RT-associated glycans on version 5.1 of the Consortium for Functional Glycomics printed array is shown, and color-coded from 100 (the highest binding; dark brown) to 0 (the lowest binding; white). Glycans on the horizontal axis are ordered according to α2,6-, α2,3-, or dual α2,3-/α2,6-linkage, and refer to the numbers of the glycans on the printed array. Structures for glycan numbers: 464 - Neu5Acα2-6Galβ1-4GlcNAcβ1-4Mana1-6(GlcNAcβ1-4) (Neu5Acα2-6Galβ1-4GlcNAcβ1-4(Neu5Acα2-6Galβ1-4GlcNAcβ1-2)Mana1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp21; 465 - Neu5Acα2-6Galβ1-4GlcNAcβ1-6(Neu5Acα2-6Galβ1-4GlcNAcβ1-2)Mana1-6(GlcNAcβ1-4)(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Mana1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp21; 461 - Neu5Acα2-3Galβ1-4GlcNAcβ1-6(Neu5Acα2-3Galβ1-4GlcNAcβ1-2)Mana1-6(GlcNAβb1-4)(Neu5Acα2-3Galβ1-4GlcNAcβ1-2Mana1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp21; 460 - Neu5Acα2-3Galβ1-4GlcNAcβ1-4 Mana1-6(GlcNAcβ1-4) (Neu5Acα2-3Galβ1-4GlcNAcβ1-4(Neu5Acα2-3Galβ1-4GlcNAcβ1-2)Mana1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp21; 474 - Neu5Acα2-3Galβ1-3GlcNAcβ1-6(Neu5Acα2-3Galβ1-4GlcNAcβ1-2)Mana1-6(Neu5Acα2-3Galβ1-3GlcNAcβ1-2Mana1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp19; 482 - Neu5Acα2-6Galβ1-4GlcNAcβ1-2Mana1-6(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Mana1-3)Manβ1-4GlcNAcβ1-4(Fuca1-6)GlcNAcβ-Sp24; 483 - Neu5Acα2-3Galβ1-4GlcNAcβ1-2Mana1-6(Neu5Acα2-3Galβ1-4GlcNAcβ1-2Mana1-3)Manβ1-4GlcNAcβ1-4(Fuca1-6)GlcNAcβ-Sp24; 57 - Neu5Acα2-6Galβ1-4GlcNAcβ1-2Mana1-6(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Mana1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp24; 55 - Neu5Acα2-6Galβ1-4GlcNAcβ1-2Mana1-6(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Mana1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12; 56 - Neu5Acα2-6Galβ1-4GlcNAcβ1-2Mana1-6(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Man-a1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp21; 326 - Neu5Acα2-3Galβ1-4GlcNAcβ1-2Mana1-6(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Mana1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12; 318 - Neu5Acα2-6Galβ1-4GlcNAcβ1-2Mana1-6(Neu5Acα2-3Galβ1-4GlcNAcβ1-2Mana1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12; 325 - Neu5Acα2-3Galβ1-4GlcNAcβ1-2Mana1-6(Neu5Acα2-3Galβ1-4GlcNAcβ1-2Mana1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12; 396 - Neu5Acα2-3Galβ1-3GlcNAcβ1-2Mana1-6(Neu5Acα2-3Galβ1-3GlcNAcβ1-2Mana1-3)Manβ1-4GlcNAcβ1-4GlcNAc-Sp19; 319 - Galβ1-4GlcNAcβ1-2Mana1-6(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Mana1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12; 301 - Neu5Acα2-6Galβ1-4GlcNAcβ1-2Mana1-6(Galβ1-4GlcNAcβ1-2Mana1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12; 332 - Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-GlcNAcβ-Sp0; 258 - Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcb-Sp0; 317 - Neu5Acα2-3Galβ1-4GlcNAcβ1-6(Neu5Acα2-3Galβ1-3)GalNAcα-Sp14; 271 - Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-Sp0; 602 - Neu5Acα2-6Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAcα-Sp14; 288 - Neu5Acα2-3Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAcα-Sp14; 247 - Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ-Sp0; 261 - Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-Sp0; 243 - Neu5Acα2-6(Neu5Acα2-3Galβ1-3)GalNAcα-Sp8; 244 - Neu5Acα2-6(Neu5Acα2-3Galβ1-3)GalNAcα-Sp14; 242 - Neu5Acα2-3Galβ1-3(6S)GalNAcα-Sp8; 135 - Neu5Acα2-6(Galβ1-3)GalNAcα-Sp8; 136 - Neu5Acα2-6(Galβ1-3)GalNAcα-Sp14; 223 - Neu5Acα2-3Galβ1-3GalNAcα-Sp8; 224 - Neu5Acα2-3Galβ1-3GalNAcα-Sp14.
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