Delay of influenza hemagglutinin refolding into a fusion-competent conformation by receptor binding: A hypothesis

Two subunits of influenza hemagglutinin (HA), HA1 and HA2, represent one of the best-characterized membrane fusion machines. While a low pH conformation of HA2 mediates the actual fusion, HA1 establishes a specific connection between the viral and cell membranes via binding to the sialic acid-containing receptors. Here we propose that HA1 may also be involved in modulating the kinetics of HA refolding. We hypothesized that binding of the HA1 subunit to its receptor restricts the major refolding of the low pH-activated HA to a fusion-competent conformation and, in the absence of fusion, to an HA-inactivated state. Dissociation of the HA1-receptor connection was considered to be a slow kinetic step. To verify this hypothesis, we first analyzed a simple kinetic scheme accounting for the stages of dissociation of the HA1/receptor bonds, inactivation and fusion, and formulated experimentally testable predictions. Second, we verified these predictions by measuring the extent of fusion between HA-expressing cells and red blood cells. Three experimental approaches based on 1) the temporal inhibition of fusion by lysophosphatidylcholine, 2) rapid dissociation of the HA1-receptor connections by neuraminidase treatment, and 3) substitution of membrane-anchored receptors by a water-soluble sialyllactose all provided support for the proposed role of the release of HA1-receptor connections. Possible biological implications of this stage in HA refolding and membrane fusion are being discussed.