Membrane rearrangements and protein refolding in biological fusion intermediates

Child Health And Human Development

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Abstract

Two subunits of influenza hemagglutinin (HA), HA1 and HA2, represent one of the best-characterized membrane fusion machine. 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. We proposed 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 a 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 extents of fusion between HA-expressing cells and red blood cells. Three experimental approaches based on i) the temporal inhibition of fusion by lysophosphatidylcholine; ii) rapid dissociation of the HA1-receptor connections by neuraminidase treatment; and iii) substitution of membrane-anchored receptors by a water-soluble sialyllactose, all provided support for our hypothesis. Unlocking of HA1-receptor connection follows an early low pH-induced activation of HA such as exposure of the HA fusion peptide and precedes a major refolding of HA leading to fusion or inactivation. We suggest that activated HA molecules can build up functional fusion complexes while still being locked. This would prevent premature inactivation of HA and thus increase fusion efficiency. Alternatively, slowing down the major refolding of HA by HA1-receptor connection may allow more time for the fusion peptide insertion into the right membrane and in the right orientation. Well-timed release of connections between HA1 and receptor molecules would also facilitate the final expansion of a fusion pore. This study proposes a mechanism by which receptor can control the time course of protein refolding to a fusion-competent conformation.Along with these studies on the conformational changes in the fusion protein, we have continued characterization of the membrane intermediates in the fusion pathway. Fusion mediated by HA is commonly detected as lipid and content mixing between fusing cells. Lowering the surface density of fusion-competent HA inhibited these advanced fusion phenotypes and allowed us to identify an early stage of fusion at physiological temperature. While lipid flow between membranes was restricted, the contacting membrane monolayers were apparently transiently connected as detected by the transformation of this fusion intermediate into complete fusion following treatments known to destabilize hemifusion diaphragms. These reversible connections disappeared within 10-20 min after low pH application indicating that after the energy released by HA refolding dissipated, the final low pH conformation of HA did not support membrane merger. While the dynamic character and the lack of lipid mixing at 37C distinguish the newly identified fusion intermediate from the 4C-arrested intermediate described earlier, both intermediates apparently belong to the same family of restricted hemifusion (RH) structures. Since the formation of transient RH structures at physiological temperatures was as fast as fusion pore opening and required less HA, we hypothesize that fusion starts with the formation of multiple RH sites, only a few of which then evolve to become expanding fusion pores.