title: Cellular cytoplasmic delivery of a polypeptide toxin by reconstituted influenza virus envelopes (virosomes).
abstract: In this paper it is demonstrated that reconstituted influenza virus envelopes (virosomes) fuse efficiently with membranes of the endosomal cell compartment of cultured cells, after internalization through receptor-mediated endocytosis. As a consequence, molecules, encapsulated in the virosomal interior, are transferred to the cell cytoplasm. This process was monitored on the basis of delivery of subunit A of diphtheria toxin (DTA), initially encapsulated in the virosomal lumen. Virosome-mediated cytoplasmic delivery of DTA resulted in a virtually complete inhibition of cellular protein synthesis. DTA delivery was blocked by factors inhibiting the pH-dependent fusion activity of viral hemagglutinin, i.e., 20 mM NH4Cl, preincubation of the virosomes at low pH, and anti-hemagglutinin antibodies. Quantitation of the extent of virosome-mediated delivery of biologically active DTA demonstrated that a lower bound of approximately 10% of the virosomes entering the cells deposited their aqueous contents into the cytosol. This is in good agreement with the final extent of virosome fusion of 40%, as determined by a fluorescence lipid mixing assay based on the dilution of pyrene-labeled phosphatidylcholine from the virosomal into the endosomal membrane. At an added concentration of 1 microM virosomal lipid per 50,000 cells, depending on the condition of the experiment, a minimum of 4000 virosomes per cell were found to be internalized. Virosomes could also be induced to fuse with the cell plasma membrane by a transient lowering of the extracellular pH, as detected by appearance of virosome-encapsulated DTA in the cytoplasm.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Physiological Chemistry, University of Groningen, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Bron
authors.forename: A
authors.initials: A
authors.lastname: Ortiz
authors.forename: J
authors.initials: J
authors.lastname: Wilschut
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002642:Chick Embryo; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D004167:Diphtheria Toxin / Q000378:metabolism* / Q000633:toxicity; D004337:Drug Carriers; D009980:Influenza A virus / Q000502:physiology*; D007668:Kidney; D007700:Kinetics; D010713:Phosphatidylcholines; D011500:Protein Synthesis Inhibitors / Q000378:metabolism / Q000633:toxicity; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000378:metabolism*
chemical_list: D004167:Diphtheria Toxin; D004337:Drug Carriers; D010713:Phosphatidylcholines; D011500:Protein Synthesis Inhibitors; D014759:Viral Envelope Proteins