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

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. T he influenza virus envelope protein, hemagglutinin (HA), is organized as a noncovalently associated homotrimer on the viral surface. Each monomer of HA is posttranslationally cleaved into HA1 and HA2 subunits that are disulfide linked. The HA trimer consists of a large membrane-distal, globular domain formed only by HA1 and an elongated membrane-proximal stem domain comprised of HA2 and the N-and C-terminal segments of HA1. HA1 mediates virus binding to cell surface sialic acid receptors to initiate viral e...

Mechanisms of influenza viral membrane fusion

“…Fusion assays on HA mutants have revealed several salt bridges and hydrogen bonds at the subunit interfaces that are weakened upon protonation of one of the participating residues [28]. Among these are the highly conserved His184 at the HA1-HA1 interface [52] and His205 in a pandemic 2009 H1N1 strain [53]. Both the loss of specific stabilizing contacts and an increased net charge on the subunits could contribute to the dissociation of HA1 [46].…”

Generation of a protective murine monoclonal antibody against the stem of influenza hemagglutinins from group 1 viruses and identification of resistance mutations against it

“…Whole viral particles and purified HA proteins have been used to demonstrate acidic pHinduced HA conformation changes [29,[33][34][35][36][37][38]. The H1 HA-pseudoviruses, made in serumfree medium in the absence of human airway trypsin-like protease (HAT), were used for MAb binding.…”

“…After immunoprecipitation, the protein G beads were washed four times with 1% NP40-PBS. The immunoprecipitation samples were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by Western blot using rabbit antiserum against H1 HA2 subunit [38].…”

The role of fusion activity of influenza A viruses in their biological properties

“…Th e globular head formed by HA1 subunits is, at neutral pH, positively charged, while HA2 has a negative charge (Huang et al, 2002;. Th e intermolecular bonds at the interface between HA1 and HA2 chains contribute to the stability of infl uenza HA in the acidic environment, which depends on the composition and properties of aa (Wang et al, 2015). Th ose aa that are preferentially protonated at low pH, play the most important role in the 3-D structure of HA.…”

“…HA conformation is infl uenced by the ionizable amino acid residues at the critical sites of HA trimer, which are the fusion peptide (interacting with the fusion peptide pocket), coiled-coil regions of HA2, and the site of interaction of HA1 and HA2 subunit surfaces (Wang et al, 2015). Th e globular head formed by HA1 subunits is, at neutral pH, positively charged, while HA2 has a negative charge (Huang et al, 2002;.…”