Adaptation of novel H7N9 influenza A virus to human receptors

Abstract

The emergence of the novel H7N9 influenza A virus (IAV) has caused global concerns about the ability of this virus to spread between humans. Analysis of the receptor-binding properties of this virus using a recombinant protein approach in combination with fetuin-binding, glycan array and human tissue-binding assays demonstrates increased binding of H7 to both α2-6 and α2-8 sialosides as well as reduced binding to α2-3-linked SIAs compared to a closely related avian H7N9 virus from 2008. These differences could be attributed to substitutions Q226L and G186V. Analysis of the enzymatic activity of the neuraminidase N9 protein indicated a reduced sialidase activity, consistent with the reduced binding of H7 to α2-3 sialosides. However, the novel H7N9 virus still preferred binding to α2-3- over α2-6-linked SIAs and was not able to efficiently bind to epithelial cells of human trachea in contrast to seasonal IAV, consistent with its limited human-to-human transmission.

Figure 1. Fetuin binding of recombinant soluble trimeric HA proteins.

(A) Limiting dilutions of soluble HA trimers (sHA₃), complexed with horseradish peroxidase (HRP)-conjugated antibodies, were applied in the fetuin-binding assay. The optical density at 450 nm (OD 450) corresponds with binding of HA to fetuin. (B) Bar graph of the HA-fetuin binding at 0.5 μg HA protein. The mean values of at least two independent experiments performed in triplicate are shown Standard deviations are indicated, the asterisk (*) indicates a significant difference in binding between the human H7 (H7/Human) and the teal H7 (H7/Teal) protein (P < 0.001; One-way ANOVA followed by a Dunnett's multiple comparison test).

Figure 2. Activity and specificity of recombinant soluble tetrameric NA proteins.

(A) Binding of biotinylated peanut agglutinin (PNA), which correlates with the amount of sialic acids released from galactose, after incubation of fetuin with limiting dilutions of soluble NA tetramers (sNA₄). The specificity of the NA proteins was determined by measuring the binding of biotinylated MAL I, specific for α2-3 sialosides, (B) and SNA, specific for α2-6 sialosides (C) after incubation of fetuin with limiting dilutions of NA tetramers. The binding of PNA, MAL-I and SNA was detected using HRP-labeled streptavidin. The mean values of at least two independent experiments performed in triplicate are shown. Standard deviations are indicated, asterisks (*) indicate significant differences between the human N9 (N9/Human) and the teal N9 (N9/Teal) proteins (P < 0.001; One-way ANOVA followed by a Bonferroni test).

Figure 3. Fetuin binding of mutant recombinant soluble trimeric H7 proteins.

Fetuin binding of recombinant soluble trimeric HA (sHA₃) proteins carrying (A) single, (B) double or quadruple amino acid substitutions in H7/Teal or (C) single substitutions in H7/Human at 0.5 μg HA protein. The mean values of at least two independent experiments performed in triplicate are shown. Standard deviations are indicated and asterisks (*) and crosses (†) indicate significant differences in binding between (mutant) proteins and H7/Teal (A and B) or H7/Human (C), respectively (P < 0.001; One-way ANOVA followed by a Dunnett's multiple comparison test). Binding at different concentrations of HA is shown in supplementary Figure S4.

Figure 4. Glycan array analysis of H7 proteins.

H7/Human, H7/Teal and the indicated single and double amino acid substitution mutants were applied to the glycan array. Soluble trimeric HA proteins (10 ug) were pre-complexed with anti-Strep tag mouse monoclonal antibody and fluorescent secondary antibodies as described. Panels display binding to glycans carrying α2-6 linked (red), α2-8 linked (yellow) or α2-3 linked (blue) SIAs (full glycan array data will become available at www.functionalglycomics.org). Glycan numbers indicated on the X-axes correspond to the structures in supplementary table S1. Y-axes indicate relative fluorescence units. Glycans are sorted from left to right for highest binding to H7/human (α2-6 and α2-8 SIAs) or H7/teal (α2-3 SIAs) proteins. The mean values of an experiment performed in sixfold are shown. Crosses (†) indicate data sets (α2-6, α2-8, or α2-3 SIAs), the mean of which is significantly different from the mean of the data sets obtained with the H7/human protein (P < 0.001; One-way ANOVA followed by a Dunnett's multiple comparison test).

Figure 5. HA histochemistry.

HA histochemistry was performed by incubating human trachea and lung tissues with the indicated HA proteins (H1/Kentucky: H1 protein derived from a human seasonal influenza H1N1 virus; H5/VN04; H5 protein from an avian H5N1 virus) after precomplexing with Streptactin-HRP as described previously. Black arrowhead point to type II pneumocytes, while open arrowheads point to type I pneumocytes. URT: upper respiratory tract, LRT: lower respiratory tract. As a negative control, slides were incubated by Streptactin-HRP in the absence of HA (mock).