Targeted liposomes for delivery of protein-based drugs into the cytoplasm of tumor cells

Our goal was to deliver therapeutically active macromolecules into the cytosol of target cells. First, attempts were made to prepare virosomes that specifically interact with OVCAR-3 cells (human ovarian cancer cells). Detergent solubilized influenza virus envelopes were reconstituted forming virosomes. Cell specificity was introduced by incorporating PEG-derivatized lipids with mAB 323/A3 (Fab' fragments) connected to their distal PEG end. These cell-specific, modified virosomes maintained their fusogenic activity when lowering the pH. Most importantly, antibody-mediated binding was a prerequisite for low-pH induced membrane fusion. However, basically, there are two problems with this approach: (1) these virosomes are quite leaky and (2) virosomes can be expected to be immunogenic. A solution to tackle leakage and potential immunogenicity of these site-specific liposomal structures is to use immuno-PEG-liposomes with a pH-dependent fusogen inside the liposome. The system that we designed to test this concept consisted of (1) the fusogenic di-peptide dINF-7, (2) the monoclonal antibody 425 connected to the distal end of PEG-PE (for site specific binding and endosomal uptake), (3) diphtheria toxin chain A (DTA, as carrier-dependent active compound) and phosphatidylcholine/cholesterol as 'bilayer backbone'. A series of tests were performed to show that selective binding and pH-dependent destabilization of (endosomal) membranes indeed occurred. To test the cytotoxic activity of these DTA loaded liposomes, OVCAR-3 cells were used for testing. OVCAR-3 cells express the epidermal growth factor receptor, which is the ligand for antibody 425. In vitro, these site specific and fusogenic liposomes showed a remarkable, cell specific cytotoxic effect.     

Functional characterization of an endosome-disruptive peptide and its application in cytosolic delivery of immunoliposome-entrapped proteins

Antibody-directed liposomes (immunoliposomes) are frequently used for targeted drug delivery. However, delivery of large biotherapeutic molecules (i.e. peptides, proteins, or nucleic acids) with immunoliposomes is often hampered by an inefficient cytosolic release of entrapped macromolecules after target cell binding and subsequent endocytosis of immunoliposomes. To enhance cytosolic drug delivery from immunoliposomes present inside endosomes, a pH-dependent fusogenic peptide (diINF-7) resembling the NH(2)-terminal domain of influenza virus hemagglutinin HA-2 subunit was used. Functional characterization of this dimeric peptide showed its ability to induce fusion between liposome membranes and leakage of liposome-entrapped compounds when exposed to low pH. In a second series of experiments, diINF-7 peptides were encapsulated in immunoliposomes to enhance the endosomal escape of diphtheria toxin A chain (DTA), which inhibits protein synthesis when delivered into the cytosol of target cells. Immunoliposomes targeted to the internalizing epidermal growth factor receptor on the surface of ovarian carcinoma cells (OVCAR-3) and containing encapsulated DTA did not show any cytotoxicity toward OVCAR-3 cells. Cytotoxicity was only observed when diINF-7 peptides and DTA were co-encapsulated in the immunoliposomes. Thus, diINF-7 peptides entrapped inside liposomes can greatly enhance cytosolic delivery of liposomal macromolecules by pH-dependent destabilization of endosomal membranes after cellular uptake of liposomes.     

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.     

Fusion of reconstituted influenza virus envelopes with liposomes mediated by streptavidin/biotin interactions

Reconstituted influenza virus envelopes (virosomes) containing the viral hemagglutinin (HA) represent an efficient fusogenic cellular delivery system. By interaction of HA with its natural receptors, sialylated lipids (gangliosides) or proteins, virosomes bind to cells and, following endocytic uptake, deliver their contents to the cytosol through fusion from within acidic endosomes. Here, we show that binding to sialic acid is not necessary for fusion. In the presence of streptavidin, virosomes containing a biotinylated lipid fused with liposomes lacking sialic acid if these liposomes also had a biotinylated lipid in their membranes. Moreover, fusion characteristics corresponded well with fusion of virosomes with ganglioside-containing liposomes.     

Interactions of human lymphoblasts with targeted vesicles containing Sendai virus envelope proteins

We have studied the internalization of targeted fusogenic liposome content to leukemic T cells (CEM) in vitro. We describe a method for the covalent coupling of T101 antibody to the surface of liposomes and the incorporation of fusogenic viral protein into the liposome membrane. Hygromycin B, an impermeant inhibitor of protein synthesis, was encapsulated in the targeted fusogenic liposomes and delivered directly to the cytoplasm of leukemic T cells by fusion between the two membranes. The cytotoxic effect was measured by [3H]thymidine incorporation. We show that CEM are rapidly and specifically killed by the drug encapsulated in the targeted fusogenic liposomes. This effect is due to the binding of the liposome by means of the antibody and then to the fusion of the liposome with the targeted cell membrane, mediated by F protein.     

A three-step strategy for targeting drug carriers to human ovarian carcinoma cells in vitro.

To improve tumor-to-tissue ratios of anticancer agents in radioimmunotherapy, a three-step targeting approach was used to deliver biotinylated liposomes to human ovarian cancer cells (NIH:OVCAR-3, SK-OV-3) in vitro. Targeting was based upon the use of two antibodies specific for the CA-125 antigen that is highly expressed on NIH:OVCAR-3 cells but not expressed on SK-OV-3 cells. Briefly, the approach consists of prelabeling target cells with biotinylated anti-CA-125 antibody and FITC-labeled streptavidin (SAv) prior to administration of biotinylated liposomes containing a marker dye for visualization by confocal laser scanning microscopy (CLSM). In addition, the two anti-CA-125 antibodies (B27.1 and B43.13) were labeled with FITC and incubated with ovarian cancer cells at 37 degrees C from 30 min to 24 h to study binding and uptake kinetics. Shedding kinetics of bound antibody from tumor cells was performed using radiolabeled B27.1. Results demonstrated that both B27.1 and B43.13 specifically bound to the cell surface of OVCAR-3 cells but not to SK-OV-3 cells. Biotinylation, FITC-labeling and radiolabeling of the antibodies did not compromise immunoreactivity. Less than 6% of the bound B27.1 was shed from tumor cells by 4 h following incubation, and the antibody-antigen complex resided predominantly on the cell surface by 4 h at 37 degrees C with slow internalization by 12-24 h. Biotinylated, conventional liposomes were specifically and effectively delivered to OVCAR-3 cells prelabeled with biotinylated B27.1 and SAv. The slow internalization and shedding properties of these antibodies are useful for multistep pretargeting methods. Thus, a modified targeting strategy, utilizing a bispecific antibody and liposomes, may be feasible for radioimmunoliposomal therapy of ovarian cancer.     

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.     

Transferrin-modified liposomes equipped with a pH-sensitive fusogenic peptide: an artificial viral-like delivery system

Liposomes are one of the most promising systems for selective cellular targeting via introduction of specific ligands for cell-surface receptors. After being taken up by the cells, these liposomes usually follow intracellular pathways of receptor-mediated endocytosis. Control of intracellular trafficking is required for optimized drug delivery. In this study, we elucidated the intracellular fate of transferrin-modified liposomes and succeeded in altering it by introducing the pH-sensitive fusogenic peptide, GALA (WEAALAEALAEALAEHLAEALAEALEALAA). Transferrins that are chemically attached to a liposomal surface (Tf-L) were internalized via receptor-mediated endocytosis more slowly than unmodified transferrins. In contrast to the recyclable nature of transferrin, liposome-attached transferrins together with encapsulated rhodamines were retained in vesicular compartments. When GALA was introduced into liposomal membranes using a cholesteryl moiety for anchoring (Chol-GALA), rhodamines were efficiently released and diffused into the cytosol. The addition of GALA to the Tf-L-containing medium or the encapsulation of GALA in Tf-L did not induce similar effects. These results clearly indicate that GALA must be present on the surface of liposomes to exert its function. In vitro energy transfer and dynamic light scattering experiments suggested that the endosomal escape of the encapsulates in Tf-L equipped with Chol-GALA can be attributed to pH-dependent membrane fusion. With GALA present on the surface, intracellular trafficking of liposomes after receptor-mediated endocytosis could be successfully controlled.     

Membrane fusogenic high-density lipoprotein nanoparticles

Membrane fusion under mildly acidic pH occurs naturally during viral infection in cells and has been exploited in the field of nanoparticle-mediated drug delivery to circumvent endosomal entrapment of the cargo. Herein, we aimed to confer virus-like fusogenic activity to HDL in the form of a ca. 10-nm disc comprising a discoidal lipid bilayer and two copies of a lipid-binding protein at the edge. A series of HDL mutants were prepared with a mixture of three lipids and a cell-penetrating peptide (TAT, penetratin, or Arg8) fused to the protein. In a lipid-mixing assay with anionic liposomes at pH 5.5, one HDL mutant showed the fusogenic activity higher than known fusogenic liposomes. In live mammalian cells, this HDL mutant showed high plasma membrane-binding activity in the presence of serum independent of pH. In the absence of serum, a mildly acidic pH dependency for binding to the plasma membrane and the subsequent lipid mixing between them was observed for this mutant. We propose a novel strategy to develop HDL-based drug carriers by taking advantage of the HDL lipid/protein composite structure.     

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.     

Degradation of exogenous membrane proteins implanted into the plasma membrane of cultured hepatoma cells

A method for implanting exogenous membrane proteins into recipient hepatoma cells is described. Red cell band 3 and Sendai virus envelope proteins HN and F were extracted from their respective sources and purified by centrifugation to equilibrium through sucrose step gradients in the presence of octyl-beta-D-glucopyranoside. 0.05-0.15 micron vesicles were formed by adding lipid to combined detergent solubilized, isolated membrane proteins and removing detergent by dialysis. The vesicles were hybrid band 3-Sendai envelope vesicles and not a mixture of two distinct vesicle types as judged by (1) the ability of Sendai specific antibody to immunoprecipitate greater than 99% of band 3 from vesicle suspensions and (2) comigration of band 3 and Sendai envelope proteins on isopyknic sucrose density gradients. The hybrid vesicles (virosomes) were not fusogenic but did bind to cultured hepatoma cells in the cold. Subsequent treatment of virosomes absorbed onto cultured cells with polyethylene glycol resulted in a stable association of 2-10% of added band 3 and Sendai envelope proteins with the cells. Efficient transfer of virosome-associated band 3 to the cells was dependent on both lipid and Sendai envelope proteins. Fluid phase marker transfer, immunofluorescence, and protease digestion experiments demonstrate that the majority of the virosomes were implanted into recipient hepatoma membranes and not simply adsorbed onto their surface or immediately endocytosed. The hybrid membrane protein-viral envelope vesicles thus offer an efficient means for insertion of foreign proteins into the membranes of recipient cultured cells.     

Monophosphoryl Lipid A‐Adjuvanted Virosomes with Ni‐Chelating Lipids for Attachment of Conserved Viral Proteins as Cross‐Protective Influenza Vaccine

Induction of CD8⁺ cytotoxic T cells (CTLs) to conserved internal influenza antigens, such as nucleoprotein (NP), is a promising strategy for the development of cross‐protective influenza vaccines. However, influenza NP protein alone cannot induce CTL immunity due to its low capacity to activate antigen‐presenting cells (APCs) and get access to the MHC class I antigen processing pathway. To facilitate the generation of NP‐specific CTL immunity the authors develop a novel influenza vaccine consisting of virosomes with the Toll‐like receptor 4 (TLR4) ligand monophosphoryl lipid A (MPLA) and the metal‐ion‐chelating lipid DOGS‐NTA‐Ni incorporated in the membrane. In vitro, virosomes with incorporated MPLA induce stronger activation of APCs than unadjuvanted virosomes. Virosomes modified with DOGS‐NTA‐Ni show high conjugation efficacy for his‐tagged proteins and facilitate efficient uptake of conjugated proteins by APCs. Immunization of mice with MPLA‐adjuvanted virosomes with attached NP results in priming of NP‐specific CTLs while MPLA‐adjuvanted virosomes with admixed NP are inefficient in priming CTLs. Both vaccines induce equally high titers of NP‐specific antibodies. When challenged with heterosubtypic influenza virus, mice immunized with virosomes with attached or admixed NP are protected from severe weight loss. Yet, unexpectedly, they show more weight loss and more severe disease symptoms than mice immunized with MPLA‐virosomes without NP. Taken together, these results indicate that virosomes with conjugated antigen and adjuvant incorporated in the membrane are effective in priming of CTLs and eliciting antigen‐specific antibody responses in vivo. However, for protection from influenza infection NP‐specific immunity appears not to be advantageous.     

Reconstitution of the fusogenic activity of vesicular stomatitis virus.

Enveloped virus glycoproteins exhibit membrane fusion activity. We have analysed whether the G protein of vesicular stomatitis virus, reconstituted into liposomes, is able to fuse nucleated cells in a pH-dependent fashion. Proteoliposomes produced by octylglucoside dialysis did not exhibit cell fusion activity of the G protein. However, by making use of n-dodecyl octaethylene monoether (C12E8) as the solubilizing agent and by removal of the detergent in two steps, we were able to produce fusogenic G protein liposomes. These G protein liposomes fuse to the BHK-21 cell surface at pH 5.7-6.0 with an efficiency of fusion comparable with that of the parent virus. Physical and chemical analysis revealed that the fusogenic liposomes exhibited a protein to lipid weight ratio of 0.67 and showed an average diameter of 130 nm.     

Liposome mediated antigen delivery leads to induction of CD8+ T lymphocyte and antibody responses against the V3 loop region of HIV gp120

There is general consensus that the use of whole viruses for the development of a vaccine against human immunodeficiency virus (HIV) would be unsafe. While currently available nonreplicating vaccines, composed of synthetic peptides or purified subunit antigens, can help in tricking the humoral immune responses, they fail to incite the other major arm of the immune defense system, i.e., cell mediated immunity (CMI). To overcome the difficulty in generating CMI, we have entrapped an immunodominant HIV envelope glycoprotein peptide in liposomes made up of fusogenic lipids isolated from Escherichia coli. We have established the role of fusogenic liposomes in stimulation of HIV-specific CD8+ cytotoxic T lymphocytes. Interestingly, the same liposomes elicit strong HIV-specific antibody production as well. Moreover, untoward manifestations such as skin damage or antibody production against lipid components were also not observed. Thus, E. coli lipid liposomes (escheriosomes) could prove to be a potent candidate vaccine, capable of eliciting both humoral and cell mediated immune responses against HIV infection.     

Delivery of protein antigens to the immune system by fusion-active virosomes: a comparison with liposomes and ISCOMs

The induction of effective cellular and humoral immune responses against protein antigens is of major importance in vaccination strategies against infectious diseases and cancer. Immunization with protein alone in general does not result in efficient induction of cytotoxic T lymphocyte (CTL) and antibody responses. Numerous other immunization strategies have been explored. In this review we will discuss a number of lipid-based antigen delivery systems suitable for the induction of CTL responses. These systems comprise reconstituted virus envelopes (virosomes), liposomes, and immune-stimulating complexes (ISCOMs). We will concentrate on delivery of the protein antigen ovalbumin (OVA) since extensive studies with this antigen have been performed for all of the systems discussed, allowing direct comparison of antigen delivery efficiency. Stimulation of CTL activity requires processing of the antigen in the cytosol of antigen-presenting cells (APCs) and presentation of antigenic peptides on surface major histocompatibility class I complexes (MHC class I). In vitro, the ability of antigen delivery systems to induce MHC class I presentation indeed correlates with their capacity to deliver antigen to the cytosol of cells. This capacity appears to be less important for the induction of cytotoxic T lymphocytes in vivo. Instead, other properties of the antigen delivery system like activation of APCs and induction of T helper cells play a more prominent role. Fusion-active virosomes appear to be a very potent system for induction of CTL activity, most likely since virosomes combine efficient delivery of antigen with general stimulation of the immune system.     

Antitumor activity of a thioether-linked immunotoxin: OVB3-PE

A thioether-linked immunotoxin was made between Pseudomonas exotoxin and the monoclonal antibody OVB3. This conjugate, OVB3-PE, was cytotoxic for the human ovarium cancer cell line OVCAR-3 (ID of 2.5 x 10(-12) M) and it was therefore tested for antitumor activity in a nude mouse model of ovarian cancer. This model employs the injection of a lethal number of OVCAR-3 cells into the peritoneal cavity of nude mice. When 0.2-1 micrograms of OVB3-PE was injected intraperitoneally on three successive days beginning 3-5 days after OVCAR-3 cell implantation, the survival of the tumor-bearing mice was increased 2-4-fold compared to that of untreated control mice. Median survival times for control mice ranged from 44 to 50 days while survival times of 150 days or greater were seen in mice treated with OVB3-PE. When OVB3-PE administration was delayed until 2-4 weeks after tumor cell implantation, OVB3-PE treatment also showed antitumor activity, but the duration of survival was less than with the early treatments. OVB3-PE was also cytotoxic for MCF-7 breast carcinoma cells, HT-29 colon carcinoma cells, and A431 epidermoid carcinoma cells.     

Sendai virus fusion protein mediates simultaneous induction of MHC class I/II-dependent mucosal and systemic immune responses via the nasopharyngeal-associated lymphoreticular tissue immune system

Nasal administration of Ags using a novel hybrid Ag delivery vehicle composed of envelope glycoproteins of Sendai virus on the surface of liposome membranes (fusogenic liposome) efficiently delivered Ags to Ag-sampling M cells in nasopharyngeal-associated lymphoreticular tissue. Additionally, fusogenic liposomes also effectively delivered the Ags into epithelial cells and macrophages in nasopharyngeal-associated lymphoreticular tissue and nasal passages. In vitro Ag presentation assays clearly showed that fusogenic liposomes effectively presented encapsulated Ags via the MHC class II-dependent pathway of epithelial cells as well as macrophages. Fusogenic liposomes also have an adjuvant activity against mucosal epithelial cells to enhance MHC class II expression. According to these high delivery and adjuvant activities of fusogenic liposomes, nasal immunization with OVA-encapsulated fusogenic liposomes induced high levels of OVA-specific CD4(+) Th1 and Th2 cell responses. Furthermore, Ag-specific CTL responses and Ab productions were also elicited at both mucosal and systemic sites by nasal immunization with Ag-encapsulated fusogenic liposomes. These results indicate that fusogenic liposome is a versatile and effective system for the stimulation of Ag-specific immune responses at both mucosal and systemic compartments.     

Elastase activated liposomal delivery to nucleated cells.

The specific activation of liposomes for delivery has been explored by enzyme mediated cleavage of a peptide substrate covalently conjugated to a fusogenic lipid. We have previously shown an elastase sensitive peptide conjugated to 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine [corrected] (DOPE) could be activated by enzymatic cleavage, triggering liposome-liposome lipid mixing and fusion with erythrocyte ghosts (Pak et al., Biochim. Biophys. Acta, 1372 (1998) 13-27). Further optimization of this system has been aimed at obtaining substrate cleavage at or below physiological elastase levels and to demonstrate triggered delivery to living cells. Therefore a new peptide-lipid, MeO-suc-AAPV-DOPE (N-methoxy-succinyl-Ala-Ala-Pro-Val-DOPE), has been developed that exhibits greater sensitivity and selectivity for elastase cleavage and subsequent conversion to DOPE. This peptide-lipid was used with DODAP (dioleoyl dimethylammonium propane, a pH dependent cationic lipid) in a 1:1 mol ratio with the expectation that endocytosis would lead to a liposome with an overall positive charge if enzymatic cleavage had occurred. Elastase treated liposomes displayed pH dependent enhancement of binding, lipid mixing, and delivery of 10000 MW dextrans, relative to untreated liposomes, when incubated with HL60 human leukemic cells. Heat denatured elastase did not activate DODAP/MeO-suc-AAPV-DOPE liposomes, indicating enzymatic activity of elastase is necessary. Liposomes bound to ECV304 endothelial cells at physiological pH could be activated by elastase to deliver an encapsulated fluorescent probe, calcein, into the cell cytoplasm. These results suggest enzyme substrate peptides linked to a fusogenic lipid may be used to elicit specific delivery from liposomes to cells.     

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.