Abstract
Virosomes were prepared by the insertion of vesicular stomatitis virus glycoprotein, a pH-sensitive fusion protein, into preformed liposomes. The fusogenic activity of these virosomes was characterized in cell-free fusion assays using liposomal targets. Fusion was monitored by concentration-dependent changes in the efficiency of resonance energy transfer between N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine and N-(4-nitrobenzo-2-oxa-1,3-diazol)-phosphatidylethanolamine and by electron microscopy. The fusogenic activity was dependent on the presence of vesicular stomatitis virus glycoprotein, was pH-sensitive, and had a pH threshold of activation similar to that of the native virus. The extent of fusion was dependent upon the lipid composition of the vesicles. This technique will allow vesicles prepared by any method to be made fusogenic.
Highlights
From the Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524
The fusogenic activitythoemf witha method of protecting the molecules from degradathese virosomes was characterized in cell-free fauss-iontion while promoting their introduction into the cytoplasm of says using liposomal targets
Virosomes composed of DOPC: Cho1:N-NBD-PE:N-Rh-PE (68:30:1:1) andcontaining 0.1 mol % of VSV-G with respect to total lipid were incubated with a 10-fold molar excess of D0PC:Chol (70:30) targets at 37 "C
Summary
PE), and N-(lissamine rhodamine B sulfonyl)-phosphatidylethanol- Asymmetric Labeling of Virosomes--Virosomes were labeled excluamine (N-Rh-PE)were purchased from Avanti Biochemicals(Pelham, sively onthe inner leaflet by the method of McIntyre and Sleight (33). The visible virus band copy were fixedand stainedby a modificationof the method of Williams was removed from the gradient, pelleted to remove the sucrose, and et al (35).2Briefly, liposomes, virosomes,or fusion products were futed resuspended in 10 rn HEPES, 0.9% (w/v) NaC1, pH 7.4. Acid, 0.1 M sodium phosphate, pH 7.3 They were pelleted in an Purification of VSV-&Lipid- and detergent-free VSV-G was ob- Eppendorf centrifuge for 30 min, washed, postfixed in 1%(w/v) OsO,, tained according tothe method of Petri and Wagner (24). The supernatant, containing detergent, lipid, and VSV-G, was layered onto a 15-30% sucrose (w/v) gradient, 60 rn octyl glucoside, 10 rn HEPES, 0.9% (w/v) NaCI, pH 7.4,and centrifuged for 24 h at 250,000 xg,, (43,000rpm) in aBeckman SW55Ti rotor. Octyl glucoside detergent dialysis liposomes were prepared by a modification of the method
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