Influenza Hemagglutinin-Mediated Membrane Fusion: Influence of Receptor Binding on the Lag Phase Preceding Fusion

Abstract

Fusion of influenza virus with liposomes is triggered by low pH, resulting in a conformational change in the fusion protein (HA) and the insertion of fusion peptides from HA into the liposomal membrane. Fusion does not take place immediately after insertion but is preceded by a lag phase, the duration of which, as we have found previously, depends on the presence of ganglioside receptors in the liposomal membrane [Stegmann, T., White, J. M., & Helenius, A. (1990) EMBO J. 9, 4231-4241]. Here we have investigated why that is the case. Surprisingly, the 2-4-fold shorter lag phase observed with phosphatidylcholine (PC)/phosphatidylethanolamine (PE)/ganglioside liposomes was not due to slower or more readily reversible binding of the virus to PC/PE liposomes lacking receptors. Nevertheless, using liposomes with various glycolipids as targets, it was found that specific HA-receptor interactions were required for a shorter lag, and not just the negative charge of the gangliosides, or the presence of ceramide lipid tails in the liposomal membrane. Receptor binding also did not facilitate the conformational change in HA. Surprisingly, however, it was found that after an incubation of the virus at low pH in the absence of target membranes at 0 degrees C for several minutes, the binding and fusion activity of virus using PC/PE liposomes, but not PC/PE/ganglioside liposomes as targets, was decreased. The population of virus that did still bind to and fuse with the PC/PE liposomes after low pH preincubation did so after a significantly increased lag time.(ABSTRACT TRUNCATED AT 250 WORDS)

Fusion of reconstituted influenza virus envelopes with liposomes mediated by streptavidin/biotin interactions

“…Thus, the notion that the interaction between HA and a sialic-acid-containing receptor has a fundamental effect on the fusion process does not seem tenable. Rather, our present findings support previous conclusions of Stegmann et al [14,17] that gangliosides do not influence the low-pH-dependent conformational change of HA or the characteristics of the membrane merger itself.…”

“…These observations have implications for the mechanism of HA-mediated fusion. Sialic-acid-containing receptors, such as gangliosides, have been shown to enhance the rate [14,17] and extent [17] of influenza virus fusion in liposomal model systems, by increasing prefusion binding of the virus to the target membrane at neutral pH. In the absence of gangliosides, fusion is also observed, but the rates and extents are relatively low [14,17] (see also Fig.…”

“…All fusion curves were corrected for this value. This slow fusion can be accounted for by hydrophobic membrane association of the H A fusion peptides [13,14] that are exposed upon acidification [15]. The possibility of specifically targeting influenza virosomes was investigated by co-reconstituting biotinPE in the virosomes and measuring fusion of these virosomes with liposomes lacking gangliosides but containing biotinPE, in the presence of streptavidin (Fig.…”

“…1). In addition to enhancing the extent of prefusion binding, it has been concluded that interaction of HA with gangliosides also facilitates correct insertion of the HA fusion peptides into the target membrane [14,18]. This may result in higher rates of membrane fusion and, at temperatures below 37°C, in a detectable reduction of lag times preceding the actual onset of fusion [14].…”

Tight Binding of Influenza Virus Hemagglutinin to Its Receptor Interferes with Fusion Pore Dilation

“…From these observations, we concluded that HA-receptor binding that is too tight interferes with the enlargement of fusion pores. Many studies have been published concerning the relationship between HA binding and fusion activities (1,7,12,13,15,20,21,23), and most indicate that the binding of HA to receptors is necessary for an efficient fusion reaction. The question is whether this concept is inconsistent with our conclusions from the present study.…”