Glycan receptor binding of the influenza A virus H7N9 hemagglutinin

Abstract

The advent of H7N9 in early 2013 is of concern for a number of reasons, including its capability to infect humans, the lack of clarity in the etiology of infection, and because the human population does not have pre-existing immunity to the H7 subtype. Earlier sequence analyses of H7N9 hemagglutinin (HA) point to amino acid changes that predicted human receptor binding and impinge on the antigenic characteristics of the HA. Here, we report that the H7N9 HA shows limited binding to human receptors; however, should a single amino acid mutation occur, this would result in structural changes within the receptor binding site that allow for extensive binding to human receptors present in the upper respiratory tract. Furthermore, a subset of the H7N9 HA sequences demarcating coevolving amino acids appears to be in the antigenic regions of H7, which, in turn, could impact effectiveness of the current WHO-recommended prepandemic H7 vaccines.

Figure 1. Structural HA-glycan receptor complexes

A, shows structural model of Anh13 H7 HA RBS in complex with avian receptor. The avian receptor is shown as a stick at 40% transparency with carbon atom colored in blue. The loop and helix regions used to define molecular features of the HA RBS are shown. The side chains of the key amino acids in these regions that make contact with the glycan receptor are shown. The side chain of Q at 226 position observed in all other H7 HAs (prior to H7N9 outbreak) is shown as stick with 40% transparency (carbon atom colored green). The networks of inter-residue interaction contacts are shown as two-dimensional maps near the corresponding residue positions. The map comprises of circular nodes representing the amino acid positions (which are labeled above the nodes) and colored according to the degree of inter-residue contact (lightest shade of red indicating lowest contact and darkest shade of red indicating highest contact). B, shows the structural model of Anh13 H7 HA RBS in complex with human receptor (shown as a stick with 40% transparency and carbon atom colored in orange). C, shows Aichi68 H3 HA RBS – human receptor complex as observed in the X-ray co-crystal structure (PDB ID: 2YPG). The inter-residue interaction network of the key RBS residues is shown similar to what was shown in A. See also Figure S1. Note the similarities in the network of residues in 130-, 140- and 220-loop between the H3 and H7 HA (expanded in Table 1). On the other hand the network involving residues in the 190 helix are quite different and this difference is brought about by the amino acid differences and also the Gly in H7 HA versus Ser in H3 HA in the 228 position. D, shows the structural model of the G228→S mutant of Anh13 H7 HA RBS in complex with human receptor. Note that network involving residues in 190 helix in the mutant is more similar to that observed in H3 HA than the wild-type. The inter-residue contacts networks that are different between the mutant and wild-type HA are shown in red dotted circle.

Figure 2. Staining of human trachea with wild-type and G228S A/Anhui/1/13 hemagglutinin

Human Paraffinized tissue sections were stained with recombinant HAs expressed and purified from 293 F cells. Specific staining by recombinant HA (in green), is also demarked by white arrows. The recombinant HAs were precomplexed with primary anti-His and Alexa fluor 488 tagged (green) secondary antibodies (for multivalent presentation) before adding to the tissue sections. The wild-type protein did not stain the trachea (A) as intensely as the G228S mutant HA (B). The G228S HA showed intense staining of the apical surface of the trachea (marked by white arrow). One key feature of the G228S protein is the staining of the submucosal gland (C) and the goblet cells (D) in the human trachea. The staining to goblet cells is similar to staining by other human-adapted influenza A virus HA (Figure 3). The tissue was counterstained with propidium iodide (PI) shown in red. Images A, B and C were captured at 25X magnification and Image D was captured at 63X magnification. See also Figure S2.