Lipid and protein contributions to the membrane surface potential of vesicular stomatitis virus probed by a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin.
The surface potential of membranes of vesicular stomatitis virus and liposomes was determined by shift of ionization over a wide pH range of the membrane-inserted fluorophore, 4-heptadecyl-7-hydroxycoumarin. Incorporation into sonicated vesicles of negatively charged phosphatidylserine markedly increased the surface potential of uncharged phosphatidylcholine, but no significant effect on surface potential was produced by polar but uncharged glucocerebroside incorporated in phosphatidylcholine vesicles. The membrane of vesicular stomatitis virus was found to have a moderately high surface potential. Contributing to this viral membrane surface potential were glycoprotein spikes and phospholipid headgroups as determined by lowered charge after treatment of intact virions with thermolysin to remove glycoprotein or phospholipase C to remove phospholipid headgroups. The role of viral glycoprotein was confirmed by demonstrating increased surface charge of vesicles reconstituted with both viral glycoprotein and lipids compared with vesicles reconstituted with viral lipids alone. An unexpected finding was the large contribution to surface potential of cholesterol present in viral membrane. Increasing cholesterol concentration in virions by interaction with cholesterol-complexed serum lipoproteins resulted in a marked decrease in surface potential, whereas 75% depletion of virion cholesterol by interaction with sphingomyelin-complexed serum lipoproteins resulted in a significant increase in virion membrane surface potential. Although removal of glycoprotein spikes or depletion of cholesterol causes reduction in infectivity of vesicular stomatitis virus, no direct correlation could be found between alteration in surface charge and infectivity.
D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000032:analysis; D008563:Membrane Lipids / Q000502:physiology*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D014468:Umbelliferones; D014721:Vesicular stomatitis Indiana virus / Q000502:physiology*
D008081:Liposomes; D008563:Membrane Lipids; D008565:Membrane Proteins; D014468:Umbelliferones; C031827:4-heptadecylumbelliferone