Formation of helix bundles has been proposed as a general mechanism for viral and cellular membrane fusion reactions. Class I viral fusion proteins, including HIV Env and influenza hemagglutinin (HA), form six-helix bundles in their fusogenic forms. The HIV Env six-helix bundle extends to the membrane proximal end of the protein, where it is poised to pull the fusing membranes together. In contrast, the HA six-helix bundle is located at the membrane distal end of the protein. It is followed by a C-terminal 'leash' that packs into the grooves and extends to the membrane proximal end of the coiled-coil. Here, we describe the ability of C-terminal leash mutants to change conformation and induce fusion. Our data indicate that packing of the C-terminal leash into the grooves of the coiled-coil is necessary for HA to mediate the lipid mixing stage of fusion, and that hydrophobic membrane proximal leash residues secure this interaction. Therefore, HA employs a 'leash in the groove,' rather than a helix-bundle, mechanism of membrane fusion.

Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-helix "spring-loaded" conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded conformational change and two additional mutants with double proline substitutions in this region. All double mutants were fusion inactive. We analyzed one double mutant, F63P/F70P, as an example. We observed that F63P/F70P undergoes key low-pH-induced conformational changes and binds tightly to target membranes. However, limited proteolysis and electron microscopy observations showed that the mutant forms a coiled coil that is only ϳ50% the length of the wild type, suggesting that it is splayed in its N-terminal half. This work further supports the hypothesis that the spring-loaded conformational change is necessary for fusion. Our data also indicate that the spring-loaded conformational change has another role beyond presenting the fusion peptide to the target membrane.

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