Interaction of Single Viruslike Particles with Vesicles Containing Glycosphingolipids

Glycosphingolipids are involved in the first steps of virus-cell interaction, where they mediate specific recognition of the host cell membrane. We have employed total-internal-reflection fluorescence microscopy to explore the interaction kinetics between individual unlabeled noroviruslike particles, which are attached to a glycosphingolipid-containing lipid bilayer, and fluorescent vesicles containing different types and concentrations of glycosphingolipids. Under association equilibrium, the vesicle-binding rate is found to be kinetically limited, yielding information on the corresponding activation energy. The dissociation kinetics are logarithmic over a wide range of time. The latter is explained by the vesicle-size-related distribution of the dissociation activation energy. The biological, pharmaceutical, and diagnostic relevance of the study is briefly discussed.

Figure 1

Detection principle: (a) VLPs, immobilized on a supported lipid bilayer containing GSLs, interact with fluorescently labeled vesicles containing the GSL of interest. TIRF-based illumination is used to track surface-bound vesicles. (b) A typical TIRF image of surface-bound vesicles together with a kymograph and the intensity profile of a small image area containing a single vesicle. (c) Amount of newly arrived vesicles as a function of time. (d) Vesicle residence times for different time-lapse series. The data displayed here are for the 163 nm vesicles of type I.

Figure 2

Number of newly arrived vesicles as a function of dose $C_{\mathrm{ves}}t$ for (from top to bottom) vesicles of types I (163 nm, 5% H type 1, red curve), III (61 nm, 5% H type 1, blue curve), and II (163 nm, 2% H type 1, black curve). Control experiments were performed with fluorescent vesicles which did not contain GSL (163 nm, 0%, green curve) or in the absence of VLPs (163 nm, 10% H type 1, light blue curve). Both controls exhibit a hundredfold reduction in the association amount (the corresponding lines nearly coincide) compared to vesicles of type I. The measurements were executed with the time lapses of 0.5 s.

Figure 3

Number of vesicles (arbitrary units) that are still bound as a function of time for (a) (from top to bottom) vesicles of types I (163 nm, 5% H type 1, red circles) and IV (163 nm, 5% Lewis b, light blue diamonds) and (b) (from top to bottom) vesicles of types I (163 nm, 5% H type 1, red circles), III (61 nm, 5% H type 1, blue triangles), and II (163 nm, 2% H type 1, black squares). The time lapses are (a) 0.5 and (b) 2 s.

Figure 4

Logarithmic plots of dissociation rate for (from top to bottom) vesicles of types II (163 nm, 2% H type 1, black squares), III (61 nm, 5% H type 1, blue triangles), and I (163 nm, 5% H type 1, red circles) (time is in seconds). The data from the 0.5, 2, and 20 s time lapses have been merged and fitted linearly.