Targeted Liposomes Bearing Sendai for Influenza Envelope Glycoproteins as a Potential Carrier for Protein Molecules and Genes

Abstract

Infection of animal cells by enveloped viruses is mediated by a membrane fusion step. Sendai virus particles introduce nucleoprotein molecules into recipient cells via fusion with the plasma membrane of the cells, while influenza viruses accomplish this via fusion with membranes of endosomes following receptor-mediated endocytosis of virus particles. Virus-membrane fusion can be observed on a quantitative basis using fluorescently labeled virus particles (bearing octadecylrhodamine B-chloride, R₁₈) and the fluorescent dequenching method. Treatment of either Sendai or influenza virus particles with the non-ionic detergent Triton X-100 results in solubilization of the virus envelopes’ glycoproteins and phospholipids. Removal of the detergent leads to the formation of fusogenic reconstituted viral envelopes. Macromolecules such as SV40-DNA or Ricin A (the A subunit of the plant toxin Ricin) can be enclosed within reconstituted viral envelopes if added to the reconstitution system. With the aid of cross-linking reagents, specific non-viral binding proteins such as anti-membrane antibodies or polypeptide hormones can be attached to the viral envelopes, resulting in the formation of “targeted” fusogenic envelopes. Using these methods, reconstituted Sendai virus envelopes bearing insulin molecules and loaded with either SV40-DNA or Ricin A molecules were able to introduce their content into cultured cells from which virus receptors were removed by treatment with neuraminidase. Fusion-mediated microinjection of SV40-DNA and Ricin A was inferred from the appearance of SV40-T-antigen and the inhibitions of protein synthesis in recipient cells, respectively. Free insulin molecules completely inhibited fusion-mediated microinjection, indicating that virus-cell binding was promoted by virus-attached insulin molecules.