pH-dependent fusion induced by vesicular stomatitis virus glycoprotein reconstituted into phospholipid vesicles.
Purified G-protein from vesicular stomatitis virus was reconstituted into egg phosphatidylcholine vesicles by detergent dialysis of octyl glucoside. A homogeneous population of reconstituted vesicles could be obtained, provided the protein to lipid ratio was high (about 0.3 mol % protein) and the detergent removal was slow. The reconstituted vesicles were assayed for fusion activity using electron microscopy and fluorescence energy transfer. The fusion activity mediated by the viral envelope protein was dependent upon pH, temperature, and target membrane lipid composition. Incubation of reconstituted vesicles at low pH with small unilamellar vesicles containing negatively charged lipids resulted in the appearance of large cochleate structures, as shown by electron microscopy using negative stain. This process did not cause leakage of a vesicle-encapsulated aqueous marker. The rate of fusion was pH-dependent with a pK of about 4 and the apparent energy of activation for the fusion was 16 +/- 1 kcal/mol. G-protein-mediated fusion showed a large preference for target membranes which contain phosphatidylserine or phosphatidic acid. Inclusion of 36% cholesterol in any of the lipid compositions had no effect on the rate of fusion. These reconstituted vesicles provide a system to study the mechanism of pH-dependent fusion induced by a viral spike protein.
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D005960:Glucosides; D006863:Hydrogen-Ion Concentration; D007668:Kidney; D007700:Kinetics; D008081:Liposomes; D008562:Membrane Glycoproteins; D008854:Microscopy, Electron; D010713:Phosphatidylcholines; D014721:Vesicular stomatitis Indiana virus / Q000502:physiology* / Q000648:ultrastructure; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
C028391:G protein, vesicular stomatitis virus; D005960:Glucosides; D008081:Liposomes; D008562:Membrane Glycoproteins; D010713:Phosphatidylcholines; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C018619:octyl-beta-D-glucoside