Dynamic Changes during Acid-Induced Activation of Influenza Hemagglutinin

Abstract

SUMMARY Influenza hemagglutinin (HA) mediates virus attachment to host cells and fusion of the viral and endosomal membranes during entry. While high-resolution structures are available for the pre-fusion HA ectodomain and the post-fusion HA2 subunit, the sequence of conformational changes during HA activation has eluded structural characterization. Here we apply hydrogen-deuterium exchange with mass spectrometry to examine changes in structural dynamics of the HA ectodomain at various stages of activation, as well as to compare the soluble ectodomain with intact HA on virions. At pH conditions approaching activation (pH 6.0–5.5) HA exhibits increased dynamics at the fusion peptide and neighboring regions, while the interface between receptor-binding subunits (HA1) becomes stabilized. In contrast to many activation models, these data suggest that HA responds to endosomal acidification by releasing the fusion peptide prior to HA1 uncaging and the spring-loaded refolding of HA2. This staged process may facilitate efficient HA-mediated fusion.

Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion

“…A previous cryo-ET study of influenza virus at low pH found evidence that HA can adopt a conformation in which HA1 domains remain associated but the stem of the spike shows differences consistent with the fusion peptide subdomain of HA2 being released (25). Structural mass spectrometry studies from our lab also indicated that as the pH approaches 5.5, the fusion peptide and associated regions in the stem become significantly more dynamic, suggesting they may be at least transiently released, while the HA1-HA1 interface remains well-ordered (23). Based on those previously reported studies, it thus appears that HA can adopt a type of fusion peptide-primed state.…”

“…For X31 influenza virus, this activity peaks as the pH approaches 5.0 (24). While in vitro fusion assays often employ an abrupt pH drop to 5.0 where fusion activity is high, during endocytosis the virus experiences stages of acidification (49), and priming of HA and virus takes place under more-elevated pH conditions found in early to maturing endosomes (8,23,24,31). We sought to determine whether the relative populations of the fusion contact types shifted as the pH was lowered toward more fusion-optimal conditions.…”

“…7C), most of the HA spikes on the virus surface retained well-defined densities, similar to that of the prefusion trimer. Recent EM reports (25), hydrogen/ deuterium exchange data (23), and epitope mapping using monoclonal antibodies (24) have revealed an HA state in which the HA1-HA1 receptor-binding domains remain clasped together but the fusion peptide region gains conformational freedom. Cryo-ET images show that these closely resemble the prefusion conforma- tion.…”

“…The kinetics data for populations of fusion states indicate that the fusion commences with the formation of HA-mediated bridging contacts (type I) between influenza virus and liposomes (8,51,52). This occurs even in the absence of sialic acid receptor, as the fusion peptides become accessible for membrane binding at the earliest stages of HA activation but prior to HA undergoing the full extent of its spring-loaded conformational change (8,23,24).…”

“…Acidic pH induces a sequence of conformational changes in HA that lead to deployment of the amphipathic fusion peptide at the N terminus of the HA2 fusion subunit (21,(23)(24)(25). Fusion peptide release enables HA to bind to target host membranes prior to the full refolding of HA2 into the postfusion hairpin structure, which has been described crystallographically (26,27).…”