Minimal aggregate size and minimal fusion unit for the first fusion pore of influenza hemagglutinin-mediated membrane fusion

The data of Melikyan et al, (J. Gen. Physiol. 106:783, 1995) for the time r equired for the first measurable step of fusion, the formation of the first flickering conductivity pore between influenza hemagglutinin (HA) expressi ng cells and planar bilayers, has been analyzed using a new mass action kin etic model. The analysis incorporates a rigorous distinction between the mi nimum number of HA trimers aggregated at the nascent fusion site (which is denoted the minimal aggregate size) and the number of those trimers that mu st to undergo a slow essential conformational change before the first fusio n pore could form (which is denoted the minimal fusion unit). At least eigh t (and likely more) HA trimers aggregated at the nascent fusion site. Remar kably, of these eight (or more) HAs, only two or three must undergo the ess ential conformational change slowly before the first fusion pore can form. Whether the conformational change of these first two or three HAs are suffi cient for the first fusion pore to form or whether the remaining HAs within the aggregate must rapidly transform in a cooperative manner cannot be det ermined kinetically. Remarkably, the fitted halftime for the essential HA c onformational change is roughly 10(4) s, which is two orders of magnitude s lower than the observed halftime for fusion. This is because the HAs refold with distributed kinetics and because the conductance assay monitored the very first aggregate to succeed in forming a first fusion pore from an ense mble of hundreds or thousands (depending upon the cell line) of fusogenic H A aggregates within the area of apposition between the cell and the planar bilayer. Furthermore, the average rate constant for this essential conforma tional change was at least 10(7) times slower than expected for a simple co iled coil conformational change, suggesting that there is either a high fre e energy barrier to fusion and/or very many nonfusogenic conformations in t he refolding landscape. Current models for HA-mediated fusion are examined in light of these new constraints on the early structure and evolution of t he nascent fusion site. None completely comply with the data.