Heterogeneity of early intermediates in cell-liposome fusion mediated by influenza hemagglutinin

Abstract

To explore early intermediates in membrane fusion mediated by influenza virus hemagglutinin (HA) and their dependence on the composition of the target membrane, we studied lipid mixing between HA-expressing cells and liposomes containing phosphatidylcholine (PC) with different hydrocarbon chains. For all tested compositions, our results indicate the existence of at least two types of intermediates, which differ in their lifetimes. The composition of the target membrane affects the stability of fusion intermediates at a stage before lipid mixing. For less fusogenic distearoyl PC-containing liposomes at 4 degrees C, some of the intermediates inactivate, and no intermediates advance to lipid mixing. Fusion intermediates that formed for the more fusogenic dioleoyl PC-containing liposomes did not inactivate and even yielded partial lipid mixing at 4 degrees C. Thus, a more fusogenic target membrane effectively blocks nonproductive release of the conformational energy of HA. Even for the same liposome composition, HA forms two types of fusion intermediates, dissimilar in their stability and propensity to fuse. This diversity of fusion intermediates emphasizes the importance of local membrane composition and local protein concentration in fusion of heterogeneous biological membranes.

FIGURE 1

Lipid mixing at 4°C between HA-cells and DOPC-LS (curves 1 and 3), HA-cells and DSPC-LS (curves 2 and 4), recorded at pH 4.9 (curves 1 and 2; pH was lowered to 4.9 at the onset of measurements) and pH 7.4 (curves 3 and 4).

FIGURE 2

(A) Lipid mixing at 37°C at neutral pH between HA-cells and DSPC-LS recorded without low-pH application (curve 1) and after the following durations of incubation at 4°C at pH 4.9: 1 min (curve 2), 3 min (curve 3), 5 min (curve 4), 10 min (curve 5), 60 min (curve 6). (B) Lipid mixing at 37°C at neutral pH between HA-cells and SOPC-LS recorded without low-pH application (curve 1) and after the following durations of incubation at 4°C at pH 4.9: 1 min (curve 2), 5 min (curve 3), 10 min (curve 4), 20 min (curve 5). (C) Lipid mixing at 37°C at neutral pH between HA-cells and DSPC-LS, normalized to the level of fluorescence observed 10 min after raising the temperature and recorded after the following durations of incubation at 4°C at pH 4.9: 1 min, 2 min, 3 min, 5 min, 10 min, 30 min, 60 min, 90 min (curves 1–8).

FIGURE 3

(A) Lipid mixing at 37°C at neutral pH between HA-cells and DOPC-LS recorded without low-pH application (curve 1) and after the following durations of incubation at 4°C at pH 4.9: 1 min (curve 2), 15 min (curve 3), 30 min (curve 4), and 60 min (curve 5). (B) Lipid mixing at 37°C at neutral pH between HA-cells and DSPC/CL-LS recorded without low-pH application (curve 1) and after the following durations of incubation at 4°C at pH 4.9: 2 min (curve 2), 5 min (curve 3), 15 min (curve 4), 60 min (curve 5).

FIGURE 4

Lipid mixing at 37°C at neutral pH between HA-cells and liposomes is dependent on the duration of incubation at 4°C at pH 4.9. The percentage of fluorescence dequenching of the lipid probe observed 4.5 min after raising the temperature is plotted as function of the duration of incubation at 4°C at pH 4.9, normalized to the level of fluorescence after incubation at pH 4.9 for 1 min. Liposomes are DOPC-LS (○), DSPC/CL-LS (•), SOPC-LS (□), and DSPC-LS (▪).

FIGURE 5

Lipid mixing between HA-cells and bound RBCs is dependent on the duration of incubation of the cell pairs at pH 5.2 at 4°C. After this incubation, pH of the medium was returned to neutral, and simultaneously the temperature was raised to 37°C. The extent of fusion was measured 10 min after the low-pH application. Bars are mean ± SE, n ≥ 3.

FIGURE 6

Lipid mixing at 37°C at neutral pH between HA-cells and liposomes is dependent on the duration of incubation at 4°C at pH 4.9. The percentage of fluorescence dequenching of the lipid probe observed 10 min after raising the temperature is plotted as a function of the duration of incubation at 4°C at pH 4.9. Dequenching for ganglioside-containing DSPC-LS (•), dequenching for ganglioside-free DSPC-LS (▵), ratio of dequenching extents for ganglioside-containing and ganglioside-free DSPC-LS (□).

FIGURE 7

Lipid mixing between virus and DOPC-LS (A) or DSPC-LS (B) was recorded at pH 4.9 at 37°C after R18-labeled viral particles with bound liposomes were preincubated at pH 4.9 at 4°C for 1 min, 5 min, and 10 min (A) and 5 min, 15 min, and 45 min (B). Recordings shown in blue and black represent experiments without pretreatment (blue) and the experiments where lipid mixing was detected at 4°C (black). Note that lipid mixing in the later experiments proceeded much more slowly and thus is presented with a different time scale.