The HA2 subunit of influenza hemagglutinin inserts into the target membrane prior to fusion.

The interaction between influenza virus and target membrane lipids during membrane fusion was studied with hydrophobic photoactivatable probes. Two probes, the newly synthesized bisphospholipid diphosphatidylethanolamine trifluoromethyl [3H]phenyl diazirine and the phospholipid analogue 1-palmitoyl-2(11-[4-[3-(trifluoromethyl)diazirinyl]phenyl]-[2-3H]- undecanoyl]-sn-glycero-3-phosphocholine (Harter, C., Bächi, T., Semenza, G., and Brunner , J. (1988) Biochemistry 27, 1856-1864), were used. Both labeled the HA2 subunit of the virus at low pH. By measuring virus-liposome interactions at 0 degrees C, it could be demonstrated that HA2 was inserted into the target membrane prior to fusion. As we have recently demonstrated, at this temperature, exposure of the fusion peptide of HA2 takes place within 15 s after acidification, but fusion does not start for 4 min (Stegmann, T., White, J. M., and Helenius, A. (1990) EMBO J. 9, 4231-4241). HA2 was labeled at least 2 min before fusion. No labeling of the HA1 subunit was seen. These data indicate that fusion is triggered by a direct interaction of the HA2 subunit of a kinetic intermediate form of HA with the lipids of the target membrane. Most likely, it is the fusion peptide of HA2 that is inserted into the target membrane. Just before fusion, HA is thus an integral membrane protein in both membranes. In contrast, the bromelain-derived ectodomain of HA was labeled by 1-palmitoyl-2(11-[4-[3-(trifluoromethyl)diazirinyl]phenyl]- [2-3H]undecanoyl)-sn-glycerol-3-phosphocholine at low pH but not by diphosphatidylethanolamine trifluoromethyl [3H]phenyl diazirine. This indicates that insertion of the fusion peptide of the bromelain-derived ectodomain of HA into a membrane differs from that of viral HA during fusion.