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Stability of HA2 Prefusion Structure and pH-Induced Conformational Changes in the HA2 Domain of H3N2 Hemagglutinin.

ABSTRACT: Influenza hemagglutinin is the fusion protein that mediates fusion of the viral and host membranes through a large conformational change upon acidification in the developing endosome. The "spring-loaded" model has long been used to describe the mechanism of hemagglutinin and other type 1 viral glycoproteins. This model postulates a metastable conformation of the HA2 subunit, caged from adopting a lower-free energy conformation by the HA1 subunit. Here, using a combination of biochemical and spectroscopic methods, we study a truncated construct of HA2 (HA2*, lacking the transmembrane domain) recombinantly expressed in Escherichia coli as a model for HA2 without the influence of HA1. Our data show that HA2* folds into a conformation like that of HA2 in full length HA and forms trimers. Upon acidification, HA2* undergoes a conformational change that is consistent with the change from pre- to postfusion HA2 in HA. This conformational change is fast and occurs on a time scale that is not consistent with aggregation. These results suggest that the prefusion conformation of HA2 is stable and the change to the postfusion conformation is due to protonation of HA2 itself and not merely uncaging by HA1.

SUBMITTER: Eller MW

PROVIDER: S-EPMC8485334 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UNLABELLED: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.

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Stability of HA2 Prefusion Structure and pH-Induced Conformational Changes in the HA2 Domain of H3N2 Hemagglutinin.

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