Fusion activity of influenza virus PR8/34 correlates with a temperature-induced conformational change within the hemagglutinin ectodomain detected by photochemical labeling.

Fusion of influenza viruses with membranes is catalyzed by the viral spike protein hemagglutinin (HA). Under mildly acidic conditions (approximately pH 5) this protein undergoes a conformational change that triggers the exposure of the "fusion peptide", the hydrophobic N-terminal segment of the HA2 polypeptide chain. Insertion of this segment into the target membrane (or viral membrane?) is likely to represent a key step along the fusion pathway, but the details are far from being clear. The photoreactive phospholipid 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl] [2-3H]undecanoyl]-sn-glycero-3-phosphocholine ([3H]PTPC/11), inserted into the bilayer of large unilamellar vesicles (LUVs), allowed us to investigate both the interaction of viruses with the vesicles under "prefusion" conditions (pH 5; 0 degrees C) and the fusion process itself occurring at elevated temperatures (greater than 15-20 degrees C) only. Despite the observed binding of viruses to LUVs at pH 5 and 0 degrees C, labeling of HA2 was very weak (less than 0.002% of the radioactivity originally present). In contrast, fusion could be readily monitored by the covalent labeling of that polypeptide chain. We have studied also the effect of temperature on the acid-induced (pH 5) interaction of bromelain-solubilized HA (BHA) with vesicles. Labeling of the BHA2 polypeptide chain was found to show a remarkable correlation with the temperature dependence of the fusion activity of whole viruses. A temperature-induced structural change appears to be critical for both the interaction of BHA with membranes and the expression of fusion activity of intact viruses.