Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 89 (20), 10602-11

Intermonomer Interactions in Hemagglutinin Subunits HA1 and HA2 Affecting Hemagglutinin Stability and Influenza Virus Infectivity

Affiliations

Intermonomer Interactions in Hemagglutinin Subunits HA1 and HA2 Affecting Hemagglutinin Stability and Influenza Virus Infectivity

Wei Wang et al. J Virol.

Abstract

Influenza virus hemagglutinin (HA) mediates virus entry by binding to cell surface receptors and fusing the viral and endosomal membranes following uptake by endocytosis. The acidic environment of endosomes triggers a large-scale conformational change in the transmembrane subunit of HA (HA2) involving a loop (B loop)-to-helix transition, which releases the fusion peptide at the HA2 N terminus from an interior pocket within the HA trimer. Subsequent insertion of the fusion peptide into the endosomal membrane initiates fusion. The acid stability of HA is influenced by residues in the fusion peptide, fusion peptide pocket, coiled-coil regions of HA2, and interactions between the surface (HA1) and HA2 subunits, but details are not fully understood and vary among strains. Current evidence suggests that the HA from the circulating pandemic 2009 H1N1 influenza A virus [A(H1N1)pdm09] is less stable than the HAs from other seasonal influenza virus strains. Here we show that residue 205 in HA1 and residue 399 in the B loop of HA2 (residue 72, HA2 numbering) in different monomers of the trimeric A(H1N1)pdm09 HA are involved in functionally important intermolecular interactions and that a conserved histidine in this pair helps regulate HA stability. An arginine-lysine pair at this location destabilizes HA at acidic pH and mediates fusion at a higher pH, while a glutamate-lysine pair enhances HA stability and requires a lower pH to induce fusion. Our findings identify key residues in HA1 and HA2 that interact to help regulate H1N1 HA stability and virus infectivity.

Importance: Influenza virus hemagglutinin (HA) is the principal antigen in inactivated influenza vaccines and the target of protective antibodies. However, the influenza A virus HA is highly variable, necessitating frequent vaccine changes to match circulating strains. Sequence changes in HA affect not only antigenicity but also HA stability, which has important implications for vaccine production, as well as viral adaptation to hosts. HA from the pandemic 2009 H1N1 influenza A virus is less stable than other recent seasonal influenza virus HAs, but the molecular interactions that contribute to HA stability are not fully understood. Here we identify molecular interactions between specific residues in the surface and transmembrane subunits of HA that help regulate the HA conformational changes needed for HA stability and virus entry. These findings contribute to our understanding of the molecular mechanisms controlling HA function and antigen stability.

Figures

FIG 1
FIG 1
Intermonomer interactions between residue 205 in HA1 and residue 72 in HA2. (A) Alignment of NCD, Bris, and Mex HA1 amino acid residues 200 to 250 and HA2 amino acid residues 350 to 400 (HA2 residues 23 to 73). The residues in the 205-72 pair are marked with blue and red ovals, respectively. (B) (Left) The 205-72 pair position in the HA trimer complex based on the structure with PDB accession number 3M6S; (right) proposed residue interactions of R-H, R-K, H-H, and H-K in the 205-72 pair.
FIG 2
FIG 2
R-K and E-K pairs at the 205-72 pair in HA decrease viral infectivity. (A and B) The HA pseudovirus infectivities of wild-type (WT), NCD, Bris, and Mex HAs containing R-H, H-H, H-K, R-K, and E-K pairs at the 205-72 pair (A) and of chimeric Mex-NCD and Mex-Bris containing the R-H, H-H, H-K, R-K, and E-K pairs at the 205-72 pair (B) are compared. Data are shown as the mean and standard deviation from three independent experiments. *, t test, P < 0.05. (Bottom) The levels of mature HA (HA2) in pseudoviruses shown by Western blotting were detected with rabbit antisera against the HA2 C helix. (C) Influenza viruses with Bris HA generated by reverse genetics (rg) and recovered from the supernatants of transfected cells (left) or after a single passage on MDCK cells (right). Infectivity was scored by quantifying the HAU on turkey red blood cells. The relative levels of HA in viruses recovered from the supernatants shown by Western blotting were detected with rabbit antisera against the HA2 C helix. The data shown are representative of those from two independent experiments.
FIG 3
FIG 3
The R-K pair at the 205-72 pair destabilizes the HA prefusion structure at fusogenic pH. HA pseudoviruses were treated at acidic pH and digested with TPCK-trypsin. Nonreducing Western blots of TPCK-trypsin-digested Bris (A) and chimeric Mex.HA1-Bris.HA2 (B) HA pseudoviruses are shown. (Bottom) The undigested HA contents shown in the Western blots at different pHs were quantified. Full-length undigested HA (HA0) and HA fragments (disulfide-bonded HA1 and HA2 proteolytic fragments) were detected with rabbit antisera against the HA2 C helix.
FIG 4
FIG 4
Effects of the 205-72 pair on HA sensitivity to acidic pH in HA-mediated cell-cell fusion. The fusion indexes of each HA containing different residues in 205-72 pair-mediated cell-cell fusion are compared. To obtain the fusion index, the fusions in each fusion curve (response to pH) were normalized to the maximum fusion for that curve. (A) Comparison of wild-type Bris H-K, R-K, and E-K HA sensitivity to pH. (B) Comparison of wild-type Bris H-K, A-K, Q-K, Y-K, D-K, and F-K HA sensitivity to pH. Fusion data are shown as the means from three independent experiments.
FIG 5
FIG 5
The 205-72 pair is conserved in H1 HAs. (A) Different 205-72 pair contents in seasonal human, swine, and avian influenza virus HAs. All available seasonal H1 virus HA sequences in the database were counted. (B) Different 205-72 pair contents in the HAs of A(H1N1)pdm09 influenza virus isolated from human, swine, and avian sources.
FIG 6
FIG 6
A histidine in the 205-72 pair is important for HA stability and virus infectivity. (A) HA pseudoviruses were treated with acidic pH and digested with TPCK-trypsin. Nonreducing Western blots were used to compare TPCK-trypsin-digested Bris HA pseudoviruses containing A-K, Q-K, Y-K, D-K, and F-K pairs. Full-length HA (HA0) and HA fragments (disulfide-bonded HA1 and HA2 proteolytic fragments) were detected with rabbit antisera against the HA2 C helix. (B) The contents of the undigested HA in panel A were quantified at different pHs. (C) Infectivities of HA pseudoviruses containing the A-K, Q-K, Y-K, D-K, and F-K pairs. (Bottom) The levels of mature HA (HA2) in pseudoviruses shown by Western blotting were detected with rabbit antisera against the HA2 C helix. Data are shown as the mean and standard deviation from three independent experiments. *, t test, P < 0.05.
FIG 7
FIG 7
Intermonomer interactions between HA1 and HA2. (A) The intermonomer interactions between HA1 residue 205 and HA2 residue 72 are modeled for the A-K, Q-K, Y-K, and F-K pairs using the structure with PDB accession number 3M6S. The HA1 residues (A, Q, Y, and F) at position 205 are shown in green. The K residue at residue 72 of HA2 is shown in red. (B and C) Different rotational perspectives of the local environment surrounding the intermonomer interface between HA1 residue 205 and HA2 residue 72 of different HA subtypes (H1, PDB accession number 1RU7; H2, PDB accession number 3QQB; H3, PDB accession number 2HMG; H5, PDB accession number 2IBX; H6, PDB accession number 4XKF). For clarity, only residues 198 to 213 in HA1 are shown and illustrated as purple ribbons. Positions 72 and 205 from the H1 subtype are shown in yellow. For other subtypes, residue 72 of HA2 is shown in red, while residue 205 of HA1 is shown in blue. The blue dotted lines indicate the direction between C alpha carbons for the respective residue pairs. (D and E) Alternate interfaces between HA1 and HA2 are shown for subtype H3 (PDB accession number 2HMG) (D), and alternate interfaces between two adjacent HA2 monomers and HA1 are shown for subtype H6 (PDB accession number 4XKF) (E). Residue positions are indicated. Magenta, HA1; green, HA2.

Similar articles

See all similar articles

Cited by 8 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

LinkOut - more resources

Feedback