Kinetics and extent of fusion between Sendai virus and erythrocyte ghosts: application of a mass action kinetic model

The kinetics and extent of fusion between Sendai virus and erythrocyte ghosts were investigated with an assay for lipid mixing based on the relief of self-quenching of fluorescence. The results were analyzed in terms of a mass action kinetic model, which views the overall fusion reaction as a sequence of a second-order process of virus-cell adhesion followed by the first-order fusion reaction itself. The fluorescence development during the course of the fusion process was calculated by numerical integration, employing separate rate constants for the adhesion step and for the subsequent fusion reaction. Dissociation of virus particles from the cells was found to be of minor importance when fusion was initiated by mixing the particles at 37 degrees C. However, besides the initiation of fusion, extensive dissociation does occur after a preincubation of a concentrated suspension of particles at 4 degrees C followed by a transfer of the sample to 37 degrees C. The conclusion drawn from the levels of fluorescence increase obtained after 20 h of incubation is that in principle most virus particles can fuse with the ghosts at 37 degrees C and pH 7.4. However, the number of Sendai virus particles that actually fuse with a single ghost is limited to 100-200, despite the fact more than 1000 particles can bind to one cell. This finding may imply that 100-200 specific fusion sites for Sendai virus exist on the erythrocyte membrane. A simple equation can yield predictions for the final levels of fluorescence for a wide range of ratios of virus particles to ghosts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sendai virus-erythrocyte membrane interaction: quantitative and kinetic analysis of viral binding, dissociation, and fusion.

A kinetic and quantitative analysis of the binding and fusion of Sendai virus with erythrocyte membranes was performed by using a membrane fusion assay based on the relief of fluorescence self-quenching. At 37 degrees C, the process of virus association displayed a half time of 2.5 min; at 4 degrees C, the half time was 3.0 min. The fraction of the viral dose which became cell associated was independent of the incubation temperature and increased with increasing target membrane concentration. On the average, one erythrocyte ghost can accommodate ca. 1,200 Sendai virus particles. The stability of viral attachment was sensitive to a shift in temperature: a fraction of the virions (ca. 30%), attached at 4 degrees C, rapidly (half time, ca. 2.5 min) eluted from the cell surface at 37 degrees C, irrespective of the presence of free virus in the medium. The elution can be attributed to a spontaneous, temperature-induced release, rather than to viral neuraminidase activity. Competition experiments with nonlabeled virus revealed that viruses destined to fuse do not exchange with free particles in the medium but rather bind in a rapid and irreversible manner. The fusion rate of Sendai virus was affected by the density of the virus particles on the cell surface and became restrained when more than 170 virus particles were attached per ghost. In principle, all virus particles added displayed fusion activity. However, at high virus-to-ghost ratios, only a fraction actually fused, indicating that a limited number of fusion sites exist on the erythrocyte membrane. We estimate that ca. 180 virus particles maximally can fuse with one erythrocyte ghost.     

Adsorption of LLCMK2 cell-grown Sendai virus onto human red blood cells and its release from the virus adsorbed cells

An early stage of virus adsorption was studied in a system of Sendai virus metabolically labeled with [3H]leucine in LLCMK2 cells and of human red blood cells (RBCs). The efficiency of viral release from the virus-bound RBCs by incubation at 37 C depended on the number of virus particles which had been used for adsorption onto the RBC at 4 C. When 7.8 x 10(2) virus particles were previously adsorbed onto the RBC at 4 C, most of the viruses were dissociated from the RBC at 37 C. In the case of adsorption of 3 to 12 virus particles per RBC, however, most of the viruses were not dissociated from the RBC by incubation at 37 C. Such RBC-bound viruses were released by incubation with various bacterial neuraminidases (Clostridium perfringens, etc.) or with a large number of LLCMK2 cell-grown Sendai virus (LLCMK2-Sendai) particles, but not released by treatment with hemagglutinin-neuraminidase protein (Sendai-gp) isolated from egg-grown Sendai virus.     

The interaction between Sendai virus and cell membranes: A quantitative analysis of 125I-Sendai virus particles' association with human red blood cells

Intact Sendai virus particles were radiolabeled by the use of chloramine-T and Na ¹²⁵I. The method described is reproducible, efficient and appropriate for the preparation of large quantities of biologically active virus with relatively high specific activity. Gel electrophoresis analysis of the radiolabeled virus revealed that approx. 50% of the total ¹²⁵I incorporated in the virus are associated with the two viral envelope glycoproteins, while the remaining 50% are evenly distributed throughout the other viral polypeptides. The ¹²⁵I-virus particles were used to study some of the kinetic parameters of the interaction between Sendai virus particles and human erythrocytes. Binding of virus particles at 4 °C is irreversible, non-cooperative and exhibits a characteristic saturation curve. A maximum of 1–2 × 10³ virus particles bound per cell was derived from the saturation curve. Non-radioactive native virus particles as well as isolated glycophorin molecules competitively inhibit binding of the ¹²⁵I-virus particles to human erythrocytes. Incubation at 37 °C of the virus-erythrocyte complex resulted in the release of about 33% of the bound virus to the surrounding medium.     

The interaction between Sendai virus and cell membranes : A quantitative analysis of I-Sendai virus particles' association with human red blood cells

Intact Sendai virus particles were radiolabeled by the use of chloramine-T and Na ¹²⁵I. The method described is reproducible, efficient and appropriate for the preparation of large quantities of biologically active virus with relatively high specific activity. Gel electrophoresis analysis of the radiolabeled virus revealed that approx. 50% of the total ¹²⁵I incorporated in the virus are associated with the two viral envelope glycoproteins, while the remaining 50% are evenly distributed throughout the other viral polypeptides. The ¹²⁵I-virus particles were used to study some of the kinetic parameters of the interaction between Sendai virus particles and human erythrocytes. Binding of virus particles at 4 °C is irreversible, non-cooperative and exhibits a characteristic saturation curve. A maximum of 1–2 × 10³ virus particles bound per cell was derived from the saturation curve. Non-radioactive native virus particles as well as isolated glycophorin molecules competitively inhibit binding of the ¹²⁵I-virus particles to human erythrocytes. Incubation at 37 °C of the virus-erythrocyte complex resulted in the release of about 33% of the bound virus to the surrounding medium.     

Immunofluorescence and electron microscopy of the cytoplasmic surface of the human erythrocyte membrane and its interaction with Sendai virus

A method was developed for directly observing the inner surfaces of plasma membranes by light and electron microscopy. Human erythrocytes were attached to cover slips (glass or mica) treated with aminopropylsilane and glutaraldehyde, and then disrupted by direct application of a jet of buffer, which removed the distal portion of the cells, thus exposing the cytoplasmic surface (PS) of the flattened membranes. Antispectrin antibodies and Sendai virus particles were employed as sensitive markers for, respectively, the PS and the external surface (ES) of the membrane; their localization by immunofluorescence or electron microscopy demonstrated that the major asymmetrical features of the plasma membrane were preserved. The fusion of Sendai virus particles with cells was investigated using double- labeling immunofluorescence techniques. Virus adsorbed to the ES of cells at 4 degrees C was not accessible to fluorescein-labeled antibodies applied from the PS side. After incubation at 37 degrees C, viral antigens could be detected at the PS. These antigens, however, remained localized and did not diffuse from the site of attachment, as is usually seen in viral antigens accessible on the ES. They may therefore represent internal viral antigens not incorporated into the plasma membrane as a result of virus-cell fusion.     

Protein blot analysis of virus receptors: identification and characterization of the Sendai virus receptor

Receptors for Sendai virions in human erythrocyte ghost membranes were identified by virus overlay of protein blots. Among the various erythrocyte polypeptides, only glycophorin was able to bind Sendai virions effectively. The detection of Sendai virions bound to glycophorin was accomplished either by employing anti-Sendai virus antibodies or by autoradiography, when 125I-labeled Sendai virions were used. The binding activity was associated with the viral hemagglutinin/neuraminidase (HN) glycoprotein, as inferred from the observation that the binding pattern of purified HN glycoprotein to human erythrocyte membranes was identical to that of intact Sendai virions. No binding was observed when blots, containing either human erythrocyte membranes or purified glycophorin, were probed with the viral fusion factor (F glycoprotein). Active virions competed effectively with the binding of 125I-labeled Sendai virions (or purified HN glycoprotein), whereas no competition was observed with inactivated Sendai virus. The results of the present work clearly show that protein blotting can be used to identify virus receptors in cell membrane preparations.     

Fusion of Madin-Darby canine kidney cells by HVJ (Sendai virus): absence of direct association of virus particles with the site of membrane fusion

The process of cell fusion of Madin-Darby canine kidney (MDCK) cells by HVJ (Sendai virus) was investigated to determine whether the HVJ particles were directly associated with the site of membrane fusion. Confluent monolayer cultures of MDCK cells are sealed together by tight junctions on the apices of their lateral membranes, so added virus particles can be adsorbed only to the apical surfaces of the cells. After incubation with HVJ at 37 degrees C for 30 min, the cells still appeared mononucleate and unfused by light microscopy, but electron microscopic examination showed that fusion at the lateral membranes had occurred below the tight junctions. Furthermore, when fluorescein isothiocyanate (FITC)-labeled macromolecules, which cannot pass across the gap junctions, were injected into the cells at this stage, labeled macromolecules were found to diffuse into the adjacent cells. These findings strongly suggest that cell fusion was initiated in the lateral membrane, a region distinct from the site of adsorbed HVJ particles. Thus, the virus particles were not directly associated with the fusion site, but induced fusion of the lateral membranes indirectly.     

Construction of fusogenic vesicles bearing specific antibodies. Targeting of reconstituted Sendai virus envelopes towards neuraminidase-treated human erythrocytes

The cross-linking reagents succinimidyl-4-(p-maleimidophenyl)-butyrate and N-succinimidyl-3-(2-pyridyldithio)-propionate were used to covalently attach antibodies against human erythrocytes to the thiol-containing paraffin, dodecanethiol. The complex formed, dodecanethiol-maleimidophenylbutyrate (or pyridyldithiopropionate)-antibody was inserted into the membranes of reconstituted Sendai virus envelopes. This was achieved by addition of the dodecanethiol-maleimidophenylbutyrate-antibody to a detergent solution (Triton X-100) containing the viral envelope phospholipids and glycoproteins. Removal of the detergent led to the formation of vesicles containing the viral glycoprotein and the dodecanethiol-maleimidophenylbutyrate (or pyridyldithiopropionate)-antibody complexes within the same membrane. Reconstituted Sendai virus envelope-bearing antibodies against human erythrocytes were able to fuse with human erythrocytes (as was reflected by reconstituted Sendai virus envelope-induced hemolysis) from which the natural virus receptors were removed by treatment with neuraminidase. Thus, it appears that anti-human erythrocyte antibodies could substitute for the viral binding protein (hemagglutinin/neuraminidase glycoprotein) in mediating functional binding of the virus particles to the cell plasma membranes. Furthermore, from the results of the present work, it may be inferred that in addition to being the viral-binding protein, hemagglutinin/neuraminidase glycoprotein actively participates in the process of virus-cell fusion.     

Dynamic association of band 3 with triton shells in human erythrocyte ghosts

To test a possibility that free band 3 and ankyrin-linked band 3 are exchanged in situ, band 3 was labeled with 125I, using intact red blood cells and lactoperoxidase. The cytoplasmic surface of this labeled band 3 was considered to be intact. When Triton shells were incubated with Triton supernatants prepared from 125I-labeled intact erythrocytes at 37 degrees C in the presence of Mg-ATP under isotonic conditions, the incorporation of free 125I-labeled band 3 to shells was observed. This incorporation was affected by the presence of Triton X-100 in the incubation mixture, and significantly decreased when the content of Triton X-100 was less than 0.04% (v/v). On the other hand, ankyrin-linked 125I-labeled band 3 was released when shells prepared from 125I-labeled intact erythrocytes were incubated with the Triton supernatants at 37 degrees C under the same condition as when free 125I-labeled band 3 incorporation was observed. These results strongly suggest that free and ankyrin-linked band 3 exchanged with each other in the presence of Triton X-100. A water-soluble 43 kDa fragment of band 3 inhibited the incorporation of free 125I-labeled band 3 to the shells and also inhibited the Mg-ATP-dependent shape change of ghosts in the absence of Triton X-100. Both of these inhibitory effects remained, even after 10 min of heat treatment at 100 degrees C, but drastically decreased by treatment with trypsin. Our results strongly suggest that a dynamic exchange of the free band 3 for ankyrin-linked band 3 may occur in intact erythrocytes, and it may even contribute to the shape change of erythrocytes.     

Efficient introduction of contents of liposomes into cells using HVJ (Sendai virus)

The conditions for efficient introduction of the contents of liposomes into cells were examined using fragment A of diphtheria toxin (DA) as a marker; one molecule of DA can kill a cell when introduced into the cytoplasm. Liposomes containing DA (DA liposomes) were toxic to cells treated with HVJ (Sendai virus) at 4 degrees C just before exposure to DA liposomes at 37 degrees C, but were not toxic to untreated cells. This toxicity was temperature-dependent. DA outside of liposomes was not toxic to HVJ-treated cells. Results also showed that liposomes could fuse with HVJ at 37 degrees but not at 4 degrees C and that liposomes preincubated with HVJ at 37 degrees C could associate with cells. DA liposomes preincubated with HVJ at 37 degrees C were highly toxic to cells. This toxicity was dependent on the duration of preincubation with HVJ and the dose of HVJ. When plasmid DNA coded herpes simplex virus thymidine kinase was trapped in liposomes and fused with Ltk- cells with HVJ, the thymidine kinase activity was expressed in about 10% of the cells. These data show that naked liposomes fuse efficiently with cells with HVJ and that the contents of the liposomes can be introduced into the cytoplasm 100-10 000 times more efficiently by treatment of the cells or liposomes with HVJ.     

Active function of membrane receptors for enveloped viruses : I. Specific requirement for liposome-associated sialoglycolipids, but not sialoglycoproteins, to allow lysis of phospholipid vesicles by reconstituted Sendai viral envelopes

Phospholipid liposomes composed of phosphatidylcholine (PC) and cholesterol (chol), bearing the sialoglycoprotein glycophorin (GP), are able to effectively bind Sendai virus particles, but not to be lysed by them. Incorporation of gangliosides (gangl) into the above phospholipid vesicles (yielding liposomes composed of PC/chol/gangl/GP), although not increasing their ability to interact with Sendai virions, rendered them susceptible to the viral lytic activity. This was inferred from the ability of the virus to induce release of carboxyfluorescein (CF) upon interaction at 37 degrees C with liposomes composed of PC/chol/gangl/GP. Lysis of liposomes required the presence of the two viral envelope glycoproteins, namely the hemagglutinin/neuraminidase (HN) and the fusion (F) polypeptides, and was inhibited by phenylmethyl sulfonylfluoride (PMSF), dithiothreitol (DTT) and trypsin, showing that virus-induced lysis of PC/chol/gangl/GP liposomes reflects the fusogenic activity of the virus. Incubation of Sendai virus particles with liposomes containing the acidic phospholipid dicetylphosphate (DCP) but lacking sialic acid containing receptors, also resulted in release of the liposome content. Lysis of these liposomes was due to the activity of the viral HN glycoprotein, therefore not reflecting the natural viral fusogenic activity. Fluorescence dequenching studies, using fluorescently labeled reconstituted Sendai virus envelopes (RSVE), have shown that the viral envelopes are able to fuse with neutral, almost to the same extent, as with negatively charged liposomes. However, fusion with negatively charged liposomes, as opposed to fusion with neutral liposomes, was mediated by the viral HN glycoprotein and not by the viral fusion polypeptide.     

Use of virus-attached antibodies or insulin molecules to mediate fusion between Sendai virus envelopes and neuraminidase-treated cells

Anti-human erythrocyte antibodies or insulin molecules were covalently coupled to the glycoproteins (the hemagglutinin/neuraminidase and the fusion polypeptides) of Sendai virus envelopes with N-succinimidyl 3-(2-pyridyldithio)propionate and succinimidyl 4-(p-maleimidophenyl)butyrate as cross-linking reagents. Reconstituted Sendai virus envelopes, bearing covalently attached anti-human erythrocyte antibodies or insulin molecules, were able to bind to but not fuse with virus receptor depleted human erythrocytes (neuraminidase-treated human erythrocytes). Only coreconstitution of Sendai virus glycoproteins, bearing attached anti-human erythrocyte antibodies or insulin molecules with intact, untreated viral glycoproteins, led to the formation of fusogenic, targeted reconstituted Sendai virus envelopes. Binding and fusion of reconstituted Sendai virus envelopes, bearing anti-human erythrocyte antibodies or insulin molecules, with neuraminidase-treated human erythrocytes were blocked by the monovalent fraction, obtained after papain digestion of immunoglobulins, made of anti-human erythrocyte antibodies or free insulin molecules, respectively. The results of this work demonstrate an active role of the viral binding protein (hemagglutinin/neuraminidase polypeptide) in the virus membrane fusion process and show a novel and efficient method for the construction of targeted, fusogenic Sendai virus envelopes.     

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells

Receptor sites for insulin on GH3 cells were characterized. Uptake of 125I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37 degrees C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with 125I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for 125I-labeled insulin binding sites. 125I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. 125I-labeled insulin was degraded at both 4 and 37 degrees C by GH3 cells, but not by medium conditioned by these cells. After a 5 min incubation at 37 degrees C, products of 125I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of 125I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of 125I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of 125I-labeled insulin, as well as intact hormone, were recovered from GH3 cells. After 30 min incubation at 37 degrees C, 80% of the cell-bound radioactivity was not extractable from GH3, cells with acetic acid.     

Immunoelectron microscopic study on interactions of noninfectious sendai virus and murine cells.

The early interactions of LLC-MK2 cell-grown noninfectious Sendai virus and a murine cell line, P815 mastocytoma ascitic cells, were studied by electron microscopy, using the ferritin-conjugated antibody technique with anti-virus glycoprotein serum. For comparison, the interactions of egg-grown infectious Sendai virus with the same cells were also examined. When noninfectious virus was adsorbed to the cells in the cold, the cell membranes become partially invaginated at the site of contact of adsorbed virions, but ferritin-conjugated antibodies did not penetrate into the areas of envelope-cell membrane association. This pattern of virus attachment was similar to that of infectious virus attachment. Upon subsequent incubation at 37 degrees C, most of the adsorbed noninfectious virions were taken into cytoplasmic vesicles and then degraded, although a few virions remained attached to the cell membrane. No evidence of fusion of envelopes of noninfectious virions was obtained. On the other hand, envelopes of infectious virions fused with the cell membrane, and the transferred viral antigens diffused on the cell surfaces and then decreased in number.     

Degradation and internalization of bound insulin in human erythrocytes.

Internalization and degradation of insulin by human erythrocytes were studied. Erythrocytes were incubated with 125I-insulin at 4 degrees C, 15 degrees C, and 37 degrees C for varying time intervals. These erythrocytes were then subjected to a low pH wash to release bound insulin followed by TCA precipitation. After 4, 22, and 24 hours of insulin binding at 4 degrees C, 92 to 95% of the bound 125I-insulin was dissociable and 92 to 98% of the extractable insulin was undegraded. After 3.5 hours of incubation at 15 degrees, 82% of the bound insulin was dissociable and 60% of this was intact. However, after 60, 90, 120, and 180 minutes of incubation at 37 degrees C, only 42, 34, 24, and 37%, respectively, of the bound insulin was dissociable. The undissociated insulin in the 37 degrees C studies was considered to be intracellular. With increasing time of incubation at 37 degrees C, the extractability of cell bound insulin and the proportion of undegraded dissociable insulin were decreased. When 125I-insulin binding was 95% blocked by preincubating the erythrocytes with anti-insulin receptor antibody, 95% of the degradation of 125I-insulin was also blocked. These studies indicate that mature human erythrocytes degrade internalized insulin and this process is time, temperature, and insulin receptor dependent.     

Tetanus toxin interaction with human erythrocytes. I. Properties of polysialoganglioside association with the cell surface

Human erythrocytes in suspension acquire gangliosides containing di- and trisialosyl residues added to the maintenance medium. This is reflected in the increased cell-associated sialic acid content and ability to bind 125I-labeled tetanus toxin. A salt-sensitive and a salt-insensitive ganglioside-mediated toxin-cell surface association is detected which is reduced after sialidase treatment of ganglioside-supplemented cells. The salt-insensitive ganglioside-cell association is saturable after 2 h incubation in 0.3 M mannitol buffer and has an optimum at pH 5. The association process is higher at 37 degrees C than at 4 degrees C, depends on cell density, and is considerably higher in metabolically active cells compared to lysed cells. Pretreatment of cells with trypsin decreases the salt-resistant toxin association with ganglioside-supplemented cells. In contrast, glutaraldehyde-fixed cells treated with trypsin and supplemented with gangliosides bind more toxin which is insensitive to salt. Ganglioside-mediated tetanus toxin binding to the intact erythrocyte membrane can be utilized as a model system for studying the role of glycolipids in membrane function.     

The role of the target membrane structure in fusion with Sendai virus

Fusion between membranes of Sendai virus and liposomes or human erythrocytes ghosts was studied using an assay for lipid mixing based on the relief of self-quenching of octadecylrhodamine (R18) fluorescence. We considered only viral fusion that reflects the biological activity of the viral spike glycoproteins. The liposomes were made of phosphatidylcholine, and the effects of including cholesterol, the sialoglycolipid GD1a, and/or the sialoglycoprotein glycophorin as receptors were tested. Binding of Sendai virus to those liposomes at 37 degrees C was very weak. Fusion with the erythrocyte membranes occurred at a 30-fold faster rate than with the liposomes. Experiments with biological and liposomal targets of different size indicated that size did not account for differences in fusion efficiency.     

Binding and degradation of human high-density lipoproteins by human hepatoma cell line HepG2

The catabolism of human HDL was studied in human hepatoma cell line HepG2. The binding of 125I-labeled HDL at 4 degrees C was time-dependent and reached completion within 2 h. The observed rates of binding of 125I-labeled HDL at 4 degrees C and uptake and degradation at 37 degrees C indicated the presence of both high-affinity and low-affinity binding sites for this lipoprotein density class. The specific binding of 125I-labeled HDL accounted for 55% of the total binding capacity. The lysosomal degradation of 125I-labeled HDL was inhibited 25 and 60% by chloroquine at 50 and 100 microM, respectively. Depolymerization of microtubules by colchicine (1 microM) inhibited the degradation of 125I-labeled HDL by 36%. Incubation of cells with HDL caused no significant change in the cellular cholesterol content or in the de novo sterol synthesis and cholesterol esterification. Binding and degradation of 125I-labeled HDL was not affected by prior incubation of cells with HDL. When added at the same protein concentration, unlabeled VLDL, LDL and HDL had similar inhibitory effects on the degradation of 125I-labeled HDL, irrespective of a short or prolonged incubation time. Reductive methylation of unlabeled HDL had no significant effect on its capacity to inhibit the 125I-labeled HDL degradation. The competition study indicated no correlation between the concentrations of apolipoproteins A-I, A-II, B, C-II, C-III, E and F in VLDL, LDL and HDL and the inhibitory effect of these lipoprotein density classes on the degradation of 125I-labeled HDL. There was, however, some association between the inhibitory effect and the levels of apolipoprotein D and C-I.     

The avian retrovirus avian sarcoma/leukosis virus subtype A reaches the lipid mixing stage of fusion at neutral pH

We previously showed that the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus subtype A (ASLV-A) binds to liposomes at neutral pH following incubation with its receptor, Tva, at >or=22 degrees C. We also provided evidence that ASLV-C fuses with cells at neutral pH. These findings suggested that receptor binding at neutral pH and >or=22 degrees C is sufficient to activate Env for fusion. A recent study suggested that two steps are necessary to activate avian retroviral Envs: receptor binding at neutral pH, followed by exposure to low pH (W. Mothes et al., Cell 103:679-689, 2000). Therefore, we evaluated the requirements for intact ASLV-A particles to bind to target bilayers and fuse with cells. We found that ASLV-A particles bind stably to liposomes in a receptor- and temperature-dependent manner at neutral pH. Using ASLV-A particles biosynthetically labeled with pyrene, we found that ASLV-A mixes its lipid envelope with cells within 5 to 10 min at 37 degrees C. Lipid mixing was neither inhibited nor enhanced by incubation at low pH. Lipid mixing of ASLV-A was inhibited by a peptide designed to prevent six-helix bundle formation in EnvA; the same peptide inhibits virus infection and EnvA-mediated cell-cell fusion (at both neutral and low pHs). Bafilomycin and dominant-negative dynamin inhibited lipid mixing of Sindbis virus (which requires low pH for fusion), but not of ASLV-A, with host cells. Finally, we found that, although EnvA-induced cell-cell fusion is enhanced at low pH, a mutant EnvA that is severely compromised in its ability to support infection still induced massive syncytia at low pH. Our results indicate that receptor binding at neutral pH is sufficient to activate EnvA, such that ASLV-A particles bind hydrophobically to and merge their membranes with target cells. Possible roles for low pH at subsequent stages of viral entry are discussed.