Membrane fusion is a common stage of diverse cell biological processes, including exocytosis, protein trafficking, fertilization, and entry of enveloped viruses into host cells. However, even for the best-characterized fusion process, which is mediated by influenza virus hemagglutinin (HA), the mechanism remains unknown. At the time of fusion, membranes are packed with fusogenic proteins, but we do not know whether each of these proteins serves as an independent fusion machine, whether adjacent individual proteins interact with each other in the plane of the membrane, or whether proteins outside the contact zone are involved in fusion. To address these questions experimentally and to characterize the role of HA-HA interactions, we investigated the effects of the surface density of HA on the efficiency of HA activation and fusion. Based on the results of these studies and an analysis of the literature, we have proposed a new mechanism of protein-mediated fusion.

Synchronized Activation and Unfolding of Influenza Virus Hemagglutinins in Multimeric Fusion Machine
Markovic, Leikina, Zhukovsky, Chernomordik; in collaboration with Zimmerberg
Many authors have hypothesized that fusion is mediated by a multiprotein machine that somehow delivers the conformational energy of multiple proteins to the intermediates of membrane fusion. If this is correct, it is appropriate to ask how these proteins synchronize their refolding to minimize dissipation of the energy. We found that the proximity of other HAs affects triggering of the conformational change in an individual HA trimer. We modified the surface density of the HA of the Japan and X31 strains of influenza and assayed the transition of HA from its initial to its low pH conformation, measured as both the development of HA susceptibility to S-S reduction and the digestion of the exposed fusion peptide by thermolysin. Conformational change in HA was also detected functionally as inactivation of HA by low pH pretreatment in the absence of a target membrane. As expected, Japan HA–membranes retained fusogenic activity after longer low-pH incubations than did X31 HA–membranes. Our results suggest that the difference reflects slow activation rather than inactivation, as formerly thought. More important, we show that in both slow- and fast-activating strains, the percentage of activated HA increases with the increase in HA density, indicating that HA activation involves positive inter-trimer cooperativity. We propose that the spreading of the activation among adjacent HA trimers leads to the synchronized release of their conformational energy and is the mechanism by which multiple fusion proteins coordinate their activity at the fusion site (Markovic et al., 2001).

Reversible Stages of the Low-pH–Triggered Conformational Change in Influenza Virus Hemagglutinin
Leikina, Ramos, Markovic, Chernomordik; in collaboration with Zimmerberg
The refolding of the prototypic fusogenic protein hemagglutinin at the pH of fusion has often been thought of as a concerted and irreversible discharge of a loaded spring, with no distinct intermediates between the initial and final conformations. However, in our new study, we found that hemagglutinin refolding involves reversible conformations with a lifetime of minutes. After reneutralization, low pH–activated hemagglutinin returns from the conformations, wherein both the fusion peptide and the kinked loop of the HA2 subunit are exposed even though the HA1 subunits have not yet dissociated, to a structure that is functionally, biochemically, and immunologically indistin-guishable from the initial structure. The rate of the transition from reversible conformations to irreversible refolding depends on pH and the presence of target membrane. Importantly, recovery of the initial conformation is blocked by the interactions between adjacent hemag-glutinin trimers.

To explain the positive cooperativity of HA activation at low pH (see above), we hypothesized that individual HA trimers first establish a reversible, activated conformation. Our new work confirms our prediction experi-mentally and identifies an early reversible form of low pH–activated HA from which HA can revert to the initial conformation, if there are no adjacent trimers with which to interact. Inter-trimer interaction shifts HA restructuring toward irreversible stages. We propose that the existence of this relatively long-lived interme-diate state before the major refolding of HA is of importance for coupling between HA refolding and membrane fusion. We hypothesize that, at low pH, HA starts to flicker between its initial conformation and an early “primed” reversible state, with most of the time spent in the initial conformation. The delay before the discharge of most of the conformational energy of HA gives the adjacent activated trimers in the contact region time to interact and to synchronize their discharge.

While pathways of diverse membrane fusion reactions appear to have common membrane intermediates, the structures of the specialized fusion proteins can be rather dissimilar. However, reversible stages of refolding of activated fusion protein identified in our work for the fusion protein of influenza virus have been discussed in the literature for some other viruses, including tick-borne encephalitis, rabies virus, and HIV. By analogy with HA-mediated fusion, we hypothesize that different viral fusion reactions and intracellular fusion involve a distinct reversible stage of refolding of fusogenic proteins that allows adjacent trigger-activated proteins to assemble at the contact site. Subsequent concerted discharge of most of the conformational energy of these proteins drives membrane fusion (Leikina et al., 2002).

The Protein Coat in Membrane Fusion: Lessons from Fission
Chernomordik; in collaboration with Kozlov
Multiple cell biological processes involve two opposite rearrangements of membrane configuration referred to as fusion and fission. While membrane intermediates in protein-mediated fusion have been studied in some detail, the global force that drives sequential stages of fusion reaction, from early local intermediates to an expanding fusion pore, remains unknown. Neither of the published hypothetical mechanisms of protein-mediated fusion has addressed the question of how fusion proteins generate the membrane tension necessary for expansion of the fusion pore. A local protein machine is unable to generate tension in a membrane part larger than the initial fusion site. To generate a membrane stress that drives expansion of the fusion pore, the fusion machine must act on a large area of the membrane. This consideration, along with the established role of interactions between low-pH–activated HA in fusion, has motivated us to search for a mechanism that is based on fusion protein aggregation.

Fusion proceeds via stages that are analogous but oppositely directed to membrane budding-off and fission driven by protein coats. The energy needed for membrane budding and for fission of the membrane neck in the best-characterized budding-fission reactions, including intracellular fission and exit of enveloped viruses from host cells, is apparently produced by self-assembly of the coat proteins at the membrane surface. On the basis of the analogy between fusion and fission, we propose that an interconnected coat, formed by membrane-bound activated fusion proteins surrounding the membrane contact zone, generates the driving force for fusion. This fusion protein coat has a strongly curved intrinsic shape of opposite curvature to that of the protein coat driving fission. Since the rigidity of the protein coat is most likely much greater than that of the lipid bilayer, to adopt the intrinsic shape and thus to relieve the internal elastic stresses, the protein coat spontaneously bends out of the initial shape of the membrane surface. The bending produces elastic stresses in the underlying lipid bilayer and drives its fusion with the apposing membrane. The hypothesis that fusion proteins outside the contact zone participate in the generation of the driving force for fusion offers a new interpreta-tion for a number of known features of the fusion reaction mediated by the prototype fusion protein, influenza hemagglutinin, and might bring new insights into mechanisms of other fusion reactions (Kozlov and Chernomordik, 2002).