Functional reconstitution of influenza virus envelopes.

Abstract

We have examined several procedures for the reconstitution of influenza virus envelopes, based on detergent removal from solubilized viral membranes. With octylglucoside, no functionally active virosomes are formed, irrespective of the rate of detergent removal: in the final preparation the viral spike proteins appear predominantly as rosettes. Protein incorporation in reconstituted vesicles is improved when a method based on reverse‐phase evaporation of octylglucoside‐solubilized viral membranes in an ether/water system is employed. However, the resulting vesicles do not fuse with biological membranes, but exhibit only a non‐physiological fusion reaction with negatively charged liposomes. Functional reconstitution of viral envelopes is achieved after solubilization with octaethyleneglycol mono(n‐dodecyl)ether (C12E8), and subsequent detergent removal with Bio‐Beads SM‐2. The spike protein molecules are quantitatively incorporated in a single population of virosomes of uniform buoyant density and appear on both sides of the membrane. The virosomes display hemagglutination activity and a strictly pH‐dependent hemolytic activity. The virosomes fuse with erythrocyte ghosts, as revealed by a fluorescence resonance energy transfer assay. The rate and the pH dependence of fusion are essentially the same as those of the intact virus. The virosomes also fuse with cultured cells, either at the level of the endosomal membrane or directly with the cellular plasma membrane upon a brief exposure to low pH.

Gene transfer mediated by fusion protein hemagglutinin reconstituted in cationic lipid vesicles

“…17 The different virosome preparations with and without DODAC were incubated for 30 min at pH 4.5, 37°C. Following low pH incubation the virosome suspensions were neutralized and tested for fusion activity.…”

“…Previous studies have shown that the membrane fusion mediated by reconstituted virosomes closely resembles that of the intact virus. 17,19,20 Cationic lipids were incorporated into virosomes to attach plasmid DNA to the lipid bilayer surface by electrostatic interaction. Exogenous lipids (up to 30 mol% EPC and/or DODAC) were successfully incorporated into the virosomal membrane without affecting the acidic pH-induced fusion properties with target membranes (Figure 1).…”

“…21 Virosomes were prepared from the virus by a procedure described previously. 17,18 Virus (the equivalent of about 1.5 mol membrane phospholipid) was sedimented at 4°C by centrifugation. The pelleted virus was resuspended and solubilized by the addition of 0.…”

“…17,18 These virosomes show pH-induced fusion with target membranes. The application of influenza virosomes as a fusigenic carrier system for the delivery of membrane impermeable water-soluble compounds into target cells has been evaluated.…”

Reconstituted influenza virus envelopes as an efficient carrier system for cellular delivery of small-interfering RNAs

“…26 Virosomes are prepared from membrane-enveloped viruses, in particular influenza virus, by solubilization of the viral membrane with a suitable detergent, removal of the nucleocapsids by ultracentrifugation and reconstitution of the viral envelope through extraction of the detergent. [27][28][29] Vesicles reconstituted in this manner consist of the viral lipids and the viral glycoproteins HA and NA and resemble the native virus particles in size and morphology. Moreover, influenza virosomes bind to sialic acid residues on the cell membrane and fuse with artificial and cellular target membranes in a strictly pH-dependent manner.…”