SUMMARYWe have studied the interactions of synthetic peptides corresponding to the sequence of the amino terminus of the HA2 subunit of influenza virus haemagglutinin with artificial lipid membranes. The peptides could fuse cholesterol-free liposomes at neutral as well as acid pH; however, liposomes containing cholesterol could only be fused below pH 6. The fusion process caused leakage of aqueous liposomal contents. Peptides with amino acid substitutions had fusion properties similar to whole haemagglutinin molecules with the corresponding sequence changes. Non-fusogenic peptides still interacted with the membrane but did not cause leakage of liposomal contents. A correlation between the ~-helical content of peptide and its fusogenicity was noted, but this was not absolute. The results reported here support suggestions for a role of the amino terminus of HA2 in virus-endosome fusion.
INTRODUCTIONInfluenza virus haemagglutinin (HA) mediates the fusion of viral and endosomal membranes which is required to initiate infection of cells (reviewed by White et al., 1983;Wharton, 1987). At the low pH of the endosome (between 5 and 6) a conformational change occurs in HA, which is a prerequisite for fusion activity. A number of studies have shown that this change involves a decrease in subunit contacts within the HA trimer (Skehel et al., 1982;Daniels et al., 1983bDaniels et al., , 1985Ruigrok et al., 1986) and the exposure of the highly conserved hydrophobic amino terminus of HA2 (Skehel et al., 1982; Daniels et al., 1983Daniels et al., a, 1985Doms et al., 1985). It has been proposed that this hydrophobic region interacts with lipid membranes during the fusion process and support for this proposition comes from the observation that aggregation of bromelainreleased HA (BHA), which is specifically induced at the pH of fusion, involves the HA2 aminoterminal region (Daniels et aL, 1983a; R. W. H. Ruigrok et al., unpublished results). Also, variant HAs with amino acid substitutions in the amino-terminal region of HA2 constructed by site-directed mutagenesis have fusion and membrane association characteristics different to those of wild-type HA (Gething et al., 1986). In a recently published report, Lear & De Grado (1987) have shown that the peptide corresponding to the amino terminus of HA2 B/Lee/40 is capable of fusing liposomes at neutral pH. In addition, Murata et al. (1987) have demonstrated pH-dependent fusion of liposomes by a peptide corresponding to the amino terminus of HA2 of A/PR/8/34 virus. In the study reported here we synthesized a peptide with the sequence of the 23 amino-terminal residues of HA2 of X31 (H3N2) which was capable of interacting with and fusing membranes. Using specifically modified analogues of this region, we also showed that interaction with membranes was not sufficient for fusion, that the fusion characteristics of peptides were affected by the lipid composition of the liposomes, and that peptides with sequences equivalent to the amino-terminal regions of variants had fusion properties simila...