VIROSOMES IN VACCINE DEVELOPMENT: INDUCTION OF CYTOTOXIC T LYMPHOCYTE ACTIVITY WITH VIROSOME-ENCAPSULATED PROTEIN ANTIGENS

ABSTRACT

Virosomes are reconstituted viral envelopes which lack the genetic material but retain the cell entry and membrane fusion characteristics of the virus they are derived from. Thus, influenza virosomes are taken up by cells via receptor-mediated endocytosis, which directs the particles to the endosomal cell compartment. Subsequently, the virosomal membrane fuses with the endosomal membrane induced by the mildly acidic pH within the endosomes. This fusion process establishes continuity between the lumen of the virosome and the cell cytosol. Upon interaction of virosomes with antigen-presenting cells (APCs), protein antigens encapsulated within virosomes will be delivered to the cell cytosol, and thus, into the MHC class I presentation pathway. Indeed, virosome-mediated delivery of antigens in vivo results in efficient priming of a class I MHC-restricted cytotoxic T lymphocyte (CTL) response.     

Functional reconstitution of influenza virus envelopes.

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.     

Cellular Delivery of siRNA Mediated by Fusion-Active Virosomes

RNA interference is expected to have considerable potential for the development of novel specific therapeutic strategies. However, successful application of RNA interference in vivo will depend on the availability of efficient delivery systems for the introduction of small-interfering RNA (siRNA) into the appropriate target cells. This paper focuses on the use of reconstituted viral envelopes (“virosomes”), derived from influenza virus, as a carrier system for cellular delivery of siRNA. Complexed to cationic lipid, siRNA molecules could be efficiently encapsulated in influenza virosomes. Delivery to cultured cells was assessed on the basis of flow cytometry analysis using fluorescently labeled siRNA. Virosome-encapsulated siRNA directed against Green Fluorescent Protein (GFP) inhibited GFP fluorescence in cells transfected with a plasmid encoding GFP or in cells constitutively expressing GFP. Delivery of siRNA was dependent on the low-pH-induced membrane fusion activity of the virosomal hemagglutinin, supporting the notion that virosomes introduce their encapsulated siRNA into the cell cytosol through fusion of the virosomal membrane with the limiting membrane of cellular endosomes, after internalization of the virosomes by receptor-mediated endocytosis. It is concluded that virosomes represent a promising carrier system for cellular delivery of siRNA in vitro as well as in vivo.     

Cellular delivery of siRNA mediated by fusion-active virosomes

RNA interference is expected to have considerable potential for the development of novel specific therapeutic strategies. However, successful application of RNA interference in vivo will depend on the availability of efficient delivery systems for the introduction of small-interfering RNA (siRNA) into the appropriate target cells. This paper focuses on the use of reconstituted viral envelopes ("virosomes"), derived from influenza virus, as a carrier system for cellular delivery of siRNA. Complexed to cationic lipid, siRNA molecules could be efficiently encapsulated in influenza virosomes. Delivery to cultured cells was assessed on the basis of flow cytometry analysis using fluorescently labeled siRNA. Virosome-encapsulated siRNA directed against Green Fluorescent Protein (GFP) inhibited GFP fluorescence in cells transfected with a plasmid encoding GFP or in cells constitutively expressing GFP. Delivery of siRNA was dependent on the low-pH-induced membrane fusion activity of the virosomal hemagglutinin, supporting the notion that virosomes introduce their encapsulated siRNA into the cell cytosol through fusion of the virosomal membrane with the limiting membrane of cellular endosomes, after internalization of the virosomes by receptor-mediated endocytosis. It is concluded that virosomes represent a promising carrier system for cellular delivery of siRNA in vitro as well as in vivo.     

Preservation of Influenza Virosome Structure and Function During Freeze-Drying and Storage

Virosomes derived from influenza virus are reconstituted viral envelopes, which retain the receptor-binding and cell entry properties of the native virus, but lack the viral genetic material. These virosomes are of interest because of their potential use as vaccines or cellular delivery systems. However, in aqueous dispersion influenza virosomes have a relatively poor stability. Although freeze-drying of the virosomes could improve their stability, a lyoprotectant is required to preserve the structure and function of the virosomes during the lyophilization process as well as during subsequent storage of the dry powder formulation. In this study, inulin, a medium-chain oligosaccharide, was identified as an effective stabilizer of influenza virosomes. When inulin was added to an aqueous virosomal dispersion, the vesicular structure of the virosomes, with spike proteins protruding from the virosomal surface, as well as their membrane fusion activity were completely preserved during freeze-drying. When the freeze-drying process was performed from dispersions lacking a lyoprotectant, both structure and fusogenic properties of the virosomes were lost. Moreover, it was shown that the immunogenicity of inulin-stabilized virosomes was preserved. For example, dry powder formulations of virosomes retained HA potency for at least 12 weeks at 20°C. Virosomes with encapsulated pDNA encoding for the eGFP reporter gene were also found to be stabilized by inulin. The fusion capacity and the transfection efficacy (determined in BHK-21 cells) could be preserved for 12 weeks during storage at 4°C. It is concluded that freeze-drying in the presence of inulin as a lyoprotectant completely preserves the structure and function of influenza virosomes.     

Virosomes in vaccine development: induction of cytotoxic T lymphocyte activity with virosome-encapsulated protein antigens

Virosomes are reconstituted viral envelopes which lack the genetic material but retain the cell entry and membrane fusion characteristics of the virus they are derived from. Thus, influenza virosomes are taken up by cells via receptor-mediated endocytosis, which directs the particles to the endosomal cell compartment. Subsequently, the virosomal membrane fuses with the endosomal membrane induced by the mildly acidic pH within the endosomes. This fusion process establishes continuity between the lumen of the virosome and the cell cytosol. Upon interaction of virosomes with antigen-presenting cells (APCs), protein antigens encapsulated within virosomes will be delivered to the cell cytosol, and thus, into the MHC class I presentation pathway. Indeed, virosome-mediated delivery of antigens in vivo results in efficient priming of a class I MHC-restricted cytotoxic T lymphocyte (CTL) response.     

Fusogenic properties of Sendai virosome envelopes in rat brain preparations

Sendai virosomes were characrerized with respect to their ability to bind to, fuse with, and introduce substances into several rat brain preparations. Encapsulation efficiency for Sendai virosomes was enhanced but binding to cerebral cortical P₂ preparations was attenuated by addition of bovine brain phosphatidylcholine during reconstitution. A higher percentage of Sendai virosomes than phosphatidylcholine liposomes appeared to bind to, fuse with and subsequently deliver [¹⁴C]sucrose into osmotically labile pools of the P₂ preparation. Fusogenic activity was estimated by measuring dequenching of fluorescently labelled N-NBD-phosphatidylethanolamine. More virosomally encapsulated [¹⁴C]sucrose was bound to the P₂ fraction than introduced into osmotically labile organelles, and the fraction of vesicles undergoing fusion was intermediate between these two values. Non-encapsulated [¹⁴C]sucrose did not bind to and was not taken up by the P₂ fraction in a quantifiable manner. Virosomal envelopes also bound to primary cultures of rat brain neurons and glia in an apparently saturable manner. Addition of increasing amounts of the adenoassociated virus-derived vector pJDT95 increased encapsulation efficiency, and virosomes reconstituted in the presence of 60 g DNA retained most of their binding activity (5.4% of total label) compared to those containing [¹⁴C]sucrose alone (8.4%). These data indicate that Sendai virosomes may be useful in the delivery of substances into brain-derived tissues, potentially for the modulation of gene expression and neurotransmission.     

Fusogenic activity of reconstituted newcastle disease virus envelopes: a role for the hemagglutinin-neuraminidase protein in the fusion process

Enveloped viruses, such as newcastle disease virus (NDV), make their entry into the host cell by membrane fusion. In the case of NDV, the fusion step requires both transmembrane hemagglutinin-neuraminidase (HN) and fusion (F) viral envelope glycoproteins. The HN protein should show fusion promotion activity. To date, the nature of HN-F interactions is a controversial issue. In this work, we aim to clarify the role of the HN glycoprotein in the membrane fusion step. Four types of reconstituted detergent-free NDV envelopes were used, on differing in their envelope protein contents. Fusion of the different virosomes and erythrocyte ghosts was monitored using the octadecyl rhodamine B chloride assay. Only the reconstituted envelopes having the F protein, even in the absence of HN protein, displayed residual fusion activity. Treatment of such virosomes with denaturing agents affecting the F protein abolished fusion, indicating that the fusion detected was viral protein-dependent. Interestingly, the rate of fusion in the reconstituted systems was similar to that of intact viruses in the presence of the inhibitor of HN sialidase activity 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. The results show that the residual fusion activity detected in the reconstituted systems was exclusively due to F protein activity, with no contribution from the fusion promotion activity of HN protein.     

Preservation of influenza virosome structure and function during freeze-drying and storage

Virosomes derived from influenza virus are reconstituted viral envelopes, which retain the receptor-binding and cell entry properties of the native virus, but lack the viral genetic material. These virosomes are of interest because of their potential use as vaccines or cellular delivery systems. However, in aqueous dispersion influenza virosomes have a relatively poor stability. Although freeze-drying of the virosomes could improve their stability, a lyoprotectant is required to preserve the structure and function of the virosomes during the lyophilization process as well as during subsequent storage of the dry powder formulation. In this study, inulin, a medium-chain oligosaccharide, was identified as an effective stabilizer of influenza virosomes. When inulin was added to an aqueous virosomal dispersion, the vesicular structure of the virosomes, with spike proteins protruding from the virosomal surface, as well as their membrane fusion activity were completely preserved during freeze-drying. When the freeze-drying process was performed from dispersions lacking a lyoprotectant, both structure and fusogenic properties of the virosomes were lost. Moreover, it was shown that the immunogenicity of inulin-stabilized virosomes was preserved. For example, dry powder formulations of virosomes retained HA potency for at least 12 weeks at 20 degrees C. Virosomes with encapsulated pDNA encoding for the eGFP reporter gene were also found to be stabilized by inulin. The fusion capacity and the transfection efficacy (determined in BHK-21 cells) could be preserved for 12 weeks during storage at 4 degrees C. It is concluded that freeze-drying in the presence of inulin as a lyoprotectant completely preserves the structure and function of influenza virosomes.     

Interactions of Semliki Forest virus spike glycoprotein rosettes and vesicles with cultured cells

Semliki Forest virus (SFV)-derived spike glycoprotein rosettes (soluble octameric complexes), virosomes (lipid vesicles with viral spike glycoproteins), and liposomes (protein-free lipid vesicles) have been used to investigate the interaction of subviral particles with BHK-21 cells. Cell surface binding, internalization, degradation, and low pH- dependent membrane fusion were quantitatively determined. Electron microscopy was used to visualize the interactions. Virosomes and rosettes, but not liposomes, bound to cells. Binding occurred preferentially to microvilli and was inhibited by added SFV; it increased with decreasing pH but was, in all cases, less efficient than intact virus. At 37 degrees C the cell surface-bound rosettes and virosomes were internalized via coated pits and coated vesicles. After a lag period of 45 min the protein components of the internalized ligands were degraded and appeared, as acid-soluble activity, in the medium. The uptake of rosettes and virosomes was found to be similar to the adsorptive endocytosis of SFV except that their average residence times on the cell surface were longer. The rosettes and the liposomes did not show low pH-induced membrane fusion activity. The virosomes, however, irrespective of the lipid compositions used, displayed hemolytic activity at mildly acidic pH and were able to fuse with the plasma membrane of cells with an efficiency of 0.25 that observed with intact viruses. Cell-cell fusion activity was not observed with any of the subviral components. The results indicated that subviral components possess some of the entry properties of the intact virus.     

Gating Movements of Colicin A and Colicin Ia Are Different

The effect of temperature on fusion of Sendai virus with target membranes and mobility of the viral glycoproteins was studied with fluorescence methods. When intact virus was used, the fusion threshold temperature (20–22°C) was not altered regardless of the different types of target membranes. Viral glycoprotein mobility in the intact virus increased with temperature, particularly sharply at the fusion threshold temperature. This effect was suppressed by the presence of erythrocyte ghosts and/or dextran sulfate in the virus suspension. In these cases also, no change in the fusion threshold temperature was observed. On the other hand, reconstituted viral envelopes (virosomes) bearing viral glycoproteins but lacking matrix proteins were capable of fusing with erythrocyte ghosts even at temperatures lower than the fusion threshold temperature and no fusion threshold temperature was observed over the range of 10–40°C. The mobility of viral glycoproteins on virosomes was much greater and virtually temperature-independent. The intact virus treated with an actin-affector, jasplakinolide, reduced the extent of fusion with erythrocyte ghosts and the mobility of viral glycoproteins, while the treatment of virosomes with the same drug did not affect the extent of fusion of virosomes with erythrocyte ghosts and the mobility of the glycoproteins. These results suggest that viral matrix proteins including actins affect viral glycoprotein mobility and may be responsible for the temperature threshold phenomenon observed in Sendai virus fusion.This revised version was published online in August 2005 with a corrected cover date.     

pH-dependent fusion of reconstituted vesicular stomatitis virus envelopes with Vero cells. Measurement by dequenching of fluorescence

Reconstituted vesicular stomatitis virus (VSV) envelopes were formed by solubilization of the viral envelope with Triton X-100 followed by removal of detergent by direct addition of SM2 biobeads. We provide direct demonstration of fusion of reconstituted VSV with cells using fluorescent lipid and aqueous probes incorporated into the VSV virosomes during reconstitution. We show a direct comparison of the kinetics and pH profile of fusion with cells between reconstituted VSV and fluorescently labeled intact virus. With this preparation it is now possible to gain additional information about the role of cooperativity in viral protein-mediated fusion, and to permit construction of efficient vehicles for delivery of drugs and other materials into cells.     

Use of a dialyzable short-chain phospholipid for efficient solubilization and reconstitution of influenza virus envelopes

Virosomes are reconstituted viral envelopes that can serve as vaccines and as vehicles for cellular delivery of various macromolecules. To further advance the use of virosomes, we developed a novel dialysis procedure for the reconstitution of influenza virus membranes that is easily applicable to industrial production and compatible with encapsulation of a variety of compounds. This procedure relies on the use of 1,2-dicaproyl-sn-glycero-3-phosphocholine (DCPC) as a solubilizing agent. DCPC is a short-chain lecithin with detergent-like properties and with a critical micelle concentration of 14 mM. DCPC effectively dissolved the influenza virus membranes after which the nucleocapsids could be removed by ultracentrifugation. The solubilized membrane components were reconstituted either by removal of DCPC by dialysis or by a procedure involving initial dilution of the solubilized membrane components followed by dialysis. Both protocols resulted in removal of 99.9% of DCPC and simultaneous formation of virosomes. Analysis of the virosome preparations by equilibrium sucrose density gradient centrifugation revealed co-migration of phospholipid and protein for virosomes produced by either method. Moreover, both virosome preparations showed morphological and fusogenic characteristics similar to native influenza virus. Size, homogeneity and spike density of the virosomes varied with the two different reconstitution procedures employed. The recovery of viral membrane proteins and phospholipids in the virosomes was found to be higher for the dilution/dialysis procedure than for the simple dialysis protocol. This novel procedure for the production of virosomes is straightforward and robust and allows further exploitation of virosomes as vaccines or as drug delivery vehicles not only in academia, but also in industrial settings.     

Targeting influenza virosomes to ovarian carcinoma cells.

Reconstituted influenza virus envelopes (virosomes) containing the viral hemagglutinin (HA) have attracted attention as delivery vesicles for cytosolic drug delivery as they possess membrane fusion activity. Here, we show that influenza virosomes can be targeted towards ovarian carcinoma cells (OVCAR-3) with preservation of fusion activity. This was achieved by incorporating poly(ethylene glycol) (PEG)-derivatized lipids into the virosome membrane. This PEG layer serves as shield to prevent interaction of HA with ubiquitous sialic acid residues and as spatial anchor for antibody attachment. Coupling of Fab' fragments of mAb 323/A3 (anti-epithelial glycoprotein-2) to the distal ends of PEG lipids resulted in specific binding of virosomes to OVCAR-3 cells. These antibody-redirected virosomes fused with membranes of OVCAR-3 cells in a pH-dependent fashion.     

A possible involvement of virus-associated protease in the fusion of Sendai virus envelopes with human erythrocytes

A proteolytic activity is shown to be associated with relatively purified preparations of intact Sendai virus particles or with their reconstituted envelopes which are vesicles containing mainly the viral glycoproteins. Intact Sendai virus as well as reconstituted Sendai virus envelopes have been shown to be able to hydrolyze various protein molecules such as the human erythrocyte membrane polypeptide designated as band 3 and soluble polypeptides such as histone and insulin B-chain. The results of the present work raise the possibility that a direct correlation exists between the virus-associated proteolytic activity and the ability of the virions to lyse cells, to fuse with their membranes, and to promote cell-cell fusion. Inhibitors of proteolytic enzymes such as phenylmethylsulfonyl fluoride, tosyllysinechloromethylketone and tosylamidephenylethylchloromethylketone, or combinations thereof, inhibit the virus-associated proteolytic activity concomitantly with inhibition of its hemolytic and fusogenic activities. Electron microscopic studies showed that the various inhibitors did not affect the binding ability of the virus preparations. The possible involvement of a protease in the process of virus-membrane fusion is discussed.     

Use of a dialyzable short-chain phospholipid for efficient solubilization and reconstitution of influenza virus envelopes

Virosomes are reconstituted viral envelopes that can serve as vaccines and as vehicles for cellular delivery of various macromolecules. To further advance the use of virosomes, we developed a novel dialysis procedure for the reconstitution of influenza virus membranes that is easily applicable to industrial production and compatible with encapsulation of a variety of compounds. This procedure relies on the use of 1,2-dicaproyl-sn-glycero-3-phosphocholine (DCPC) as a solubilizing agent. DCPC is a short-chain lecithin with detergent-like properties and with a critical micelle concentration of 14 mM. DCPC effectively dissolved the influenza virus membranes after which the nucleocapsids could be removed by ultracentrifugation. The solubilized membrane components were reconstituted either by removal of DCPC by dialysis or by a procedure involving initial dilution of the solubilized membrane components followed by dialysis. Both protocols resulted in removal of 99.9% of DCPC and simultaneous formation of virosomes. Analysis of the virosome preparations by equilibrium sucrose density gradient centrifugation revealed co-migration of phospholipid and protein for virosomes produced by either method. Moreover, both virosome preparations showed morphological and fusogenic characteristics similar to native influenza virus. Size, homogeneity and spike density of the virosomes varied with the two different reconstitution procedures employed. The recovery of viral membrane proteins and phospholipids in the virosomes was found to be higher for the dilution/dialysis procedure than for the simple dialysis protocol. This novel procedure for the production of virosomes is straightforward and robust and allows further exploitation of virosomes as vaccines or as drug delivery vehicles not only in academia, but also in industrial settings.     

Animal viruses are able to fuse with prokaryotic cells. Fusion between Sendai or influenza virions and Mycoplasma

Sendai and influenza virions are able to fuse with mycoplasmata. Virus-Mycoplasma fusion was demonstrated by the use of fluorescently labeled intact virions and fluorescence dequenching, as well as by electron microscopy. A high degree of fusion was observed upon incubation of both virions with Mycoplasma gallisepticum or Mycoplasma capricolum. Significantly less virus-cell fusion was observed with Acholeplasma laidlawii, whose membrane contains relatively low amounts of cholesterol. The requirement of cholesterol for allowing virus-Mycoplasma fusion was also demonstrated by showing that a low degree of fusion was obtained with M. capricolum, whose cholesterol content was decreased by modifying its growth medium. Fluorescence dequenching was not observed by incubating unfusogenic virions with mycoplasmata. Sendai virions were rendered nonfusogenic by treatment with trypsin, phenylmethylsulfonyl fluoride, or dithiothreitol, whereas influenza virions were made nonfusogenic by treatment with glutaraldehyde, ammonium hydroxide, high temperatures, or incubation at low pH. Practically no fusion was observed using influenza virions bearing uncleaved hemagglutinin. Trypsinization of influenza virions bearing uncleaved hemagglutinin greatly stimulated their ability to fuse with Mycoplasma cells. Similarly to intact virus particles, also reconstituted virus envelopes, bearing the two viral glycoproteins, fused with M. capricolum. However, membrane vesicles, bearing only the viral binding (HN) or fusion (F) glycoproteins, failed to fuse with mycoplasmata. Fusion between animal enveloped virions and prokaryotic cells was thus demonstrated.     

Solubilization and reconstitution of vesicular stomatitis virus envelope using octylglucoside.

Reconstituted vesicular stomatitis virus envelopes or virosomes are formed by detergent removal from solubilized intact virus. We have monitored the solubilization process of the intact vesicular stomatitis virus by the nonionic surfactant octylglucoside at various initial virus concentrations by employing turbidity measurements. This allowed us to determine the phase boundaries between the membrane and the mixed micelles domains. We have also characterized the lipid and protein content of the solubilized material and of the reconstituted envelope. Both G and M proteins and all of the lipids of the envelope were extracted by octylglucoside and recovered in the reconstituted envelope. Fusion activity of the virosomes tested either on Vero cells or on liposomes showed kinetics and pH dependence similar to those of the intact virus.     

Reconstitution and fusogenic properties of Sendai virus envelopes

Sendai virus membranes were reconstituted by detergent dialysis, using the non-ionic detergents Triton X-100 and octyl glucoside. Membrane reassembly was determined by measuring the surface-density-dependent efficiency of resonance energy transfer between two fluorescent phospholipid analogues, which were co-reconstituted with the viral envelopes. The functional incorporation of the viral proteins was established by monitoring the ability of the reconstitution products to fuse with erythrocyte membranes, utilizing assays based on either resonance energy transfer or on relief of fluorescence selfquenching. The persistent adherence of residual Triton X-100 with the reconstituted membrane was revealed by an artificial detergent-effect on the resonance energy transfer efficiency and the occurrence of hemolysis of human erythrocytes under conditions where fusion does not occur. Properly reconstituted Sendai virus envelopes were obtained with octyl glucoside. The fusion activity of the viral envelopes was dependent on the initial concentration of octyl glucoside used to disrupt the virus and the rate of detergent removal. Rapid removal of detergent by dialysis against large volumes of dialysis buffer (ratio 1:850) or by gel filtration produced reconstituted membranes capable of inducing hemagglutination but significant fusion activity was not detected. By decreasing the volume ratio of dialysate versus dialysis buffer to 1:250 or 1:25, fusogenic viral envelopes were obtained. The initial fusion kinetics of the reconstituted viral membrane and the parent virus were different in that both the onset and the initial rate of fusion of the reconstituted membranes were faster, whereas the extents to which both particles eventually fused with the target membrane were similar. The differences in the initial fusion kinetics lead us to suggest that the details of the fusion mechanism between Sendai virus and the target membrane involve factors other than the mere presence of glycoproteins F and HN in the viral bilayer. Finally, the results also indicate that determination of the viral fusion activity in a direct manner, rather than by an indirect assay, such as hemolysis, is imperative for a proper evaluation of the functional properties retained upon viral reconstitution.     

Reconstitution of functional influenza virus envelopes and fusion with membranes and liposomes lacking virus receptors.

Reconstituted influenza virus envelopes were obtained following solubilization of intact virions with Triton X-100. Quantitative determination revealed that the hemolytic and fusogenic activities of the envelopes prepared by the present method were close or identical to those expressed by intact virions. Hemolysis as well as virus-membrane fusion occurred only at low pH values, while both activities were negligible at neutral pH values. Fusion of intact virions as well as reconstituted envelopes with erythrocyte membranes--and also with liposomes--was determined by the use of fluorescently labeled viral envelopes and fluorescence dequenching measurements. Fusion with liposomes did not require the presence of specific virus receptors, namely sialoglycolipids. Under hypotonic conditions, influenza virions or their reconstituted envelopes were able to fuse with erythrocyte membranes from which virus receptors had been removed by treatment with neuraminidase and pronase. Inactivated intact virions or reconstituted envelopes, namely, envelopes treated with hydroxylamine or glutaraldehyde or incubated at low pH or 85 degrees C, neither caused hemolysis nor possessed fusogenic activity. Fluorescence dequenching measurements showed that only fusion with liposomes composed of neutral phospholipids and containing cholesterol reflected the viral fusogenic activity needed for infection.