The ectodomain of HA2 of influenza virus promotes rapid pH dependent membrane fusion

To better understand the roles of different regions of influenza hemaggluti nin in membrane fusion, we have studied the fusion properties of large unil amellar vesicles in the presence of constructs comprising the 127 amino aci d ectodomain of the HA2 fragment (FHA2) as well as mutated forms of FHA2 co ntaining single amino acid substitutions, the 95 amino acid truncated form of FHA2 lacking the N-terminal fusion peptide (SHA2), the 20 amino acid N-t erminal fusion peptide and the ten amino acid peptide corresponding to the kinked loop region of FHA2. The 100 nm liposomes were made from dioleoylpho sphatidylethanolamine, dioleoylphosphatidylcholine and cholesterol in equim olar ratio. At pH 5 a high rate of lipid mixing was observed with FHA2 pres ent, even at very low molar concentrations, whereas much lower rates were o bserved using the shorter constructs: SHA2, the fusion peptide, and the loo p peptide. Concentrations of FHA2 which promoted extensive lipid mixing als o induced leakage of aqueous contents. Marked effects of FHA2 were also obs erved with liposomes of egg phosphatidylcholine. All of the changes observe d with the liposomes were highly pH-dependent, with only negligible changes occurring at pH 7. The results demonstrate the potent action of FHA2 in pr omoting lipid mixing and demonstrate the contribution of other regions of t he ectodomain of FHA2, in addition to the fusion peptide, to the mechanism of acceleration of membrane fusion. The results also indicate that the pH d ependence of fusion is not due solely to changes in the interactions betwee n the HA1 and HA2 subunits. Thus, the "spring loaded energy" is not require d to bring about the apposition of the two membranes, considering that FHA2 is already in its thermostable conformation. The acidic amino acid residue s in the kinked loop region appear to play a particularly important role in the pH-dependent fusion process as demonstrated by the marked loss of lipi d mixing activity of mutant forms of FHA2. (C) 1999 Academic Press.