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. 2013 Aug;87(15):8756-66.
doi: 10.1128/JVI.00593-13. Epub 2013 Jun 5.

N-linked glycosylation of the hemagglutinin protein influences virulence and antigenicity of the 1918 pandemic and seasonal H1N1 influenza A viruses

Xiangjie Sun  1 , Akila Jayaraman, Pavithra Maniprasad, Rahul Raman, Katherine V Houser, Claudia Pappas, Hui Zeng, Ram Sasisekharan, Jacqueline M Katz, Terrence M Tumpey
Affiliations

N-linked glycosylation of the hemagglutinin protein influences virulence and antigenicity of the 1918 pandemic and seasonal H1N1 influenza A viruses

Xiangjie Sun et al. J Virol. 2013 Aug.

Abstract

The hemagglutinin (HA) protein is a major virulence determinant for the 1918 pandemic influenza virus; however, it encodes no known virulence-associated determinants. In comparison to seasonal influenza viruses of lesser virulence, the 1918 H1N1 virus has fewer glycosylation sequons on the HA globular head region. Using site-directed mutagenesis, we found that a 1918 HA recombinant virus, of high virulence, could be significantly attenuated in mice by adding two additional glycosylation sites (asparagine [Asn] 71 and Asn 286) on the side of the HA head. The 1918 HA recombinant virus was further attenuated by introducing two additional glycosylation sites on the top of the HA head at Asn 142 and Asn 172. In a reciprocal experimental approach, deletion of HA glycosylation sites (Asn 142 and Asn 177, but not Asn 71 and Asn 104) from a seasonal influenza H1N1 virus, A/Solomon Islands/2006 (SI/06), led to increased virulence in mice. The addition of glycosylation sites to 1918 HA and removal of glycosylation sites from SI/06 HA imposed constraints on the theoretical structure surrounding the glycan receptor binding sites, which in turn led to distinct glycan receptor binding properties. The modification of glycosylation sites for the 1918 and SI/06 viruses also caused changes in viral antigenicity based on cross-reactive hemagglutinin inhibition antibody titers with antisera from mice infected with wild-type or glycan mutant viruses. These results demonstrate that glycosylation patterns of the 1918 and seasonal H1N1 viruses directly contribute to differences in virulence and are partially responsible for their distinct antigenicity.

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Figures

Fig 1
Fig 1
The glycosylation patterns of selective strains of human H1N1 viruses. Ribbon diagrams of monomeric H1 HA structures were illustrated with the PyMol program using 1918 SC HA0 as the template (PDB entry, 1RD8). The italicized numbers inside brackets are based on H1 HA crystal structure (1RUZ) numbering. The antigenic sites Sa, Sb, Ca, and Cb are colored in magenta, red, green, and blue, respectively. The potential glycosylation sites on the HA of 1918 SC, Texas/91, and SI/06 virus are shown in yellow circles, with the corresponding Asn labeled.
Fig 2
Fig 2
The effect of mutating glycosylation sites on the HA molecular weight and growth kinetics of SI/06 and 1918 HA recombinant viruses. The surface expression of WT or glycan mutant forms of SI/06 HA (A) and 1918 HA (B) was examined by labeling surface proteins with sulfo-NHS-SS-biotin followed by immunoprecipitation with NeutrAvidin beads. The surface-expressed HAs were detected by Western blotting with anti-H1 HA antibody. Calu-3 cells were infected with the recombinant viruses bearing WT or glycan mutant HAs from SI/06 (C) or 1918 SC (D) virus at an MOI of 0.01 for 1 h, and the infected cell supernatants were collected at the indicated time points for viral titer determination. Error bars represent the standard deviation (SD) of the mean from three independent experiments. An asterisk indicates that the titers of the mutant viruses were significantly different from those of the WT virus at the indicated time points by two-way analysis of variance (ANOVA) test.
Fig 3
Fig 3
Glycan mutant virus infection in mice. Groups of BALB/c were inoculated i.n. with 103 PFU of the indicated 1918 (A) or SI/06 (B) recombinant viruses. Whole-lung tissues were isolated on days 3 (n = 5) and 5 (n = 5) p.i., and viral titers were determined by plaque assay in MDCK cells (expressed as mean log10 PFU/ml ± SD) (A) or in eggs (expressed as mean log10 EID50/ml ± SD) (B). The asterisks indicate the lung titers were significantly different (P < 0.05) from those of the corresponding wild-type virus group by two-way ANOVA statistical analysis.
Fig 4
Fig 4
Structure of 1918 and SI/06 HA1 proteins. (A) 1918 HA1 with glycosylated sites 71, 104, 142, 172, and 286 in complex with human receptor. Shown in the figure is the glycan receptor binding site of 1918 HA, with key amino acids labeled (according to the numbering in the X-ray crystal structure) and their side chains shown. Glu 131 and Lys 133 side chains are shown at 50% transparency to highlight the change in interresidue interaction network when Glu 131 was mutated to Thr to add glycosylation site 142 in 1918 HA. The human receptor is shown, with the carbon atom colored orange. A basic trimannosyl core structure of an N-linked glycan is shown at the glycosylation sites 71 (58), 104 (91), 142 (129), 172 (158), and 286 (272). Among these sites, the glycosylation sites that were added through mutagenesis and their N-linked glycans are shown by the carbon atom colored yellow. (B) Structure of SI/06 HA HA1 with glycosylated sites 71, 104, 142, and 177 in complex with avian receptor. Shown in the figure is the glycan receptor binding site of SI/06 HA, where key amino acids are labeled (according to the numbering in the X-ray crystal structure) and their side chains are shown. The avian receptor is shown by the carbon atom colored cyan. A basic trimannosyl core structure of N-linked glycan is shown at the glycosylation sites 71 (58), 104 (91), 142 (129), and 177 (163), with the carbon atom colored yellow.
Fig 5
Fig 5
Dose-dependent glycan array binding of SI/06 and 1918 HA recombinant viruses. Dose-dependent binding of 1918 HA (A and B) or SI/06 HA (C and D) recombinant viruses representative of α2–3 and α2–6 sialylated glycans on the glycan array. The y axis shows percentage of maximum binding signal intensities.
Fig 6
Fig 6
HI reactivity of antisera against SI/06-HA-WT, 1918-HA-WT, and glycan mutant viruses. HI assays were performed against both homologous and heterologous viruses, and the HI titer against homologous virus was normalized to 100% (indicated by the arrows). An HI titer of 160 or higher against homologous virus was obtained for all antisera collected. The average HI titers from three repeats are shown in the graph, and the error bars represent SD.
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