Role of hemagglutinin surface density in the initial stages of influenza virus fusion: Lack of evidence for cooperativity

Membrane fusion mediated by influenza virus hemagglutinin (HA) is believed to proceed via the cooperative action of multiple HA trimers. To determine the minimal number of HA trimers required to trigger fusion, and to assess the importance of cooperativity between these HA trimers, we have generated virosomes containing coreconstituted HAs derived from two strains of virus with different pH dependencies for fusion, X-47 (optimal fusion at pH 5.1; threshold at pH 5.6) and A/Shangdong (optimal fusion at pH 5.6; threshold at pn 6.0), and measured fusion of these virosomes with erythrocyte ghosts by a fluorescence lipid mixing assay. Virosomes with different X-47-to-A/Sh angdong HA ratios, at a constant HA-to-lipid ratio, showed comparable ghost -binding activities, and the low-pH-induced conformational change of A/Shan gdong HA did not affect the fusion activity of X-47 HA. The initial rate of fusion of these virosomes at pH 5.7 increased directly proportional to the surface density of A/Shangdong HA, and a single A/Shangdong trimer per vir osome appeared to suffice to induce fusion. The reciprocal of the lag time before the onset of fusion was directly proportional to the surface density of fusion-competent HA. These results support the notion that there is no cooperativity between HA trimers during influenza virus fusion.