Structural intermediates in influenza haemagglutinin-mediated fusion

Fusion pore formation in the haemagglutinin (HA)-mediated fusion is a culmi nation of a multistep process, which involves low-pH triggered refolding of HA and rearrangement of mem brane lipid bilayers. This rearrangement was a rrested or slowed down by either altering lipid composition of the membrane s, or lowering the density of HA, and/or temperature. The results suggest t hat fusion starts with the lateral assembly of activated HA into multimeric complexes surrounding future fusion sites, The next fusion stage involves hemifusion, i.e. merger of only contacting membrane monolayers. Lysophospha tidylcholine reversibly arrests fusion prior to this hemifusion stage. In t he normal fusion pathway, hemifusion is transient and is not accompanied by any measurable transfer of lipid probes between the membranes. A temperatu re of 4 degrees C stabilizes this 'restricted hemifusion' intermediate. The restriction of lipid flow through the restricted hemifusion site is HA-dep endent and can be released by partial cleaving of low pH-forms of HA with m ild proteinase K treatment. Lipid effects indicate that fusion proceeds thr ough two different lipid-involving intermediates, which are characterized b y two opposite curvatures of the lipid monolayer, Hemifusion involves forma tion of a stalk, a local bent connection between the outer membrane monolay ers. Fusion pore formation apparently involves bending of the inner membran e monolayers, which come together in hemifusion. To couple low pH-induced r efolding of HA with lipid rearrangements, it is proposed that the extension of the alpha-helical coiled coil of HA pulls fusion peptides inserted into the HA-expressing membrane and locally bends the membrane into a saddle-li ke shape. Elastic energy drives self-assembly of these HA-containing membra ne elements into a ring-like complex and causes the bulging of the host mem brane into a dimple growing towards the target membrane. Bending stresses i n the lipidic top of the dimple facilitate membrane fusion.