A conserved histidine in Group-1 influenza subtype hemagglutinin proteins is essential for membrane fusion activity

Abstract

Influenza A viruses enter host cells through the endocytic pathway, where acidification triggers conformational changes of the viral hemagglutinin (HA) to drive membrane fusion. During this process, the HA fusion peptide is extruded from its buried position in the neutral pH structure and targeted to the endosomal membrane. Conserved ionizable residues near the fusion peptide may play a role in initiating these structural rearrangements. We targeted highly conserved histidine residues in this region, at HA1 position 17 of Group-2 HA subtypes and HA2 position 111 of Group-1 HA subtypes, to determine their role in fusion activity. WT and mutant HA proteins representing several subtypes were expressed and characterized, revealing that His 111 is essential for HA functional activity of Group-1 subtypes, supporting continued efforts to target this region of the HA structure for vaccination strategies and the design of antiviral compounds.

Keywords: Fusion; Hemagglutinin; Influenza; Virus entry.

Figure 1.

(A) The H3 subtype monomer is shown with the HA1 displayed in blue, and HA2 shown in red. The gray circles highlight the receptor binding domain (top) and fusion peptide pocket (bottom). The structural locations of the HA1 17 and HA2 111 residues are indicated by black arrows. The lower right of the panel shows the residues specific to group 1 and group 2 clades which are highly conserved within each group. The blue arrow indicates the location of HA2 His 111 (Thr in Group-2 HAs), and the red arrow shows the location of HA1 His 17 (Tyr in Group-1 HAs). The other residues identified are all in the HA2 subunit (from top – K51, R/H106, E/Q105, D109, and D112). The HA2 N-terminal fusion peptide is noted as FP. (B) The phylogenetic tree of the panel of 16 avian-origin HA subtypes found in avian reservoirs which are separated into five clades (color-coded) and can be segregated into two groups, Group-1 and Group-2.

Figure 2.

Surface expression of WT and mutant HAs by metabolic labelling and biotinylation of HA expressing cells. SDS-Page analysis, where lanes “T” represent total HA protein and “SE” represent surface expressed HA protein. (A) Gel images for human and avian subtypes corresponding to Group-1 are shown. (B) Gel images for human and avian subtypes corresponding to Group-2 are shown. (C) Quantitative representation of surface expressed mutants relative to the WT surface expression level. Error bars represent the standard deviation following three independent experiments.

Figure 3.

Proteolytic cleavage potential of WT and mutant HAs. Radiolabeled HA-expressing cells were treated with or without trypsin as indicated by + and − and lysates were immunoprecipitated with an HA-specific antibody and resolved using SDS-PAGE. (A) Gel images for both human and avian Group-1 HA subtypes. (B) Gel images for both human and avian Group-2 HA subtypes. (C) Quantitation of the trypsin cleavage of mutant HA subtypes relative to the WT HA.

Figure 4.

The pH of fusion as detected by the formation of syncytia. Photomicrographs of syncytia formation by avian and human Group-1 (A) and Group-2 (B) HA subtypes. BHK cells expressing HA were treated with trypsin and pH adjusted in 0.1 pH unit increments. The pH of fusion is shown as the highest pH at which syncytia were observed adjacent to cell monolayers the next increment lower, where fusion was significantly reduced by comparison.

Figure 5.

The pH of fusion as determined by the quantitative luciferase-based reporter assay. The pH of fusion is represented by the point at which relative luminescence units are able to be detected, indicating fusion of the two cell populations and expression of luciferase. (A) Group-1 HA human and avian subtypes. Notably in this group, no fusion was detected of the H111A mutants. (B) Group-2 HA human and avian subtypes.

Figure 6.

Conformational change of A/Japan/305/57 WT and mutant HA1 Y17H, HA2 H111A and HA2 H111T HAs. Enzyme-linked immunosorbent assay was performed as follows: Expressed HAs were trypsin treated followed by incubation at neutral pH (7.0) or low pH (4.4). (A) HA reactivity using H2 subtype polyclonal antibody. (B) HA reactivity using monoclonal antibody 2/9.

Figure 7.

Structural depiction of HA2 position 111 region comparing Group-1 and Group-2 HAs, represented by H2 and H3 subtypes respectively. A full trimer viewed from the side is shown in the middle to indicate the region represented in the panels (area between hatched lines). Panels A and B represent views down the 3-fold axis of symmetry (top view) for H2 and H3 subtype HAs, and panels C and D show magnified views of these structures from the same orientation represented in the HA trimer at the center of the figure. Fusion peptide residues are shown in navy and group-specific residues HA1 17 and HA2 111, as well as highly conserved HA2 residues Trp21 and Tyr22, are shown in red.