CELLULAR CYTOPLASMIC DELIVERY OF A POLYPEPTIDE TOXIN BY RECONSTITUTED INFLUENZA-VIRUS ENVELOPES (VIROSOMES)

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 mu M 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. The results demonstrate that virosome-mediated delivery of polypeptide toxin molecules can be used as a sensitive means to study the interactions of enveloped viruses with host cells. In addition, virosomes may be used as versatile vehicles for the delivery of water-soluble molecules to the cytoplasm of cultured cells.