FIG. 5.

VP5* selectively permeabilizes liposomes. Purified LUVs containing either CF (70 mM) or FITC-dextran 4000 (FD-4) (30 mM) were incubated in 1.6 ml of TN buffer at 37°C for 30 min with nickel affinity-purified VP5* (4 μg, 44 nM) or melittin (0.03 nM) in duplicate (31, 42). (A) To measure CF release, fluorescence dequenching of samples was measured in a fluorimeter at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. The total CF content of each sample was assessed by the addition of Triton X-100 (0.125%) and quantitated as described in Materials and Methods. (B) FD-4 is not present at self-quenching concentrations, and release was determined after centrifuging samples in a Centricon-100 microseparator (Amicon) and measuring released FD-4 in the effluent using a Perkin-Elmer Luminescence Spectrometer (LS-5B) at 520 nm (490-nm excitation). Liposome-associated FD-4 was assayed after resuspension in 1.6 ml of TN buffer plus 0.125% Triton X-100. Released FD-4 is expressed as a percentage of the total fluorescence (the sum of the effluent and the retentate). This experiment was repeated twice with similar results.