Erythrocyte membrane structural features that are critical for the lytic reaction of Spirographis spallanzani coelomic fluid hemolysin

1. Hemolytic activity of Spirographis spallanzani coelomic fluid depends on factor(s) strongly influenced by calcium but not by sulfhydril or disulfide reagents.2. The lytic reaction was suppressed by low zinc ion concentrations but it was not influenced by the presence of proteinase inhibitors.3. These data indicate that S. spallanzani hemolysin is a non-enzymatic, calcium-dependent, zincinhibitable factor that occurs naturally in the coelomic fluid.4. In the absence of calcium, enzymatic desialization converted sheep erythrocytes into susceptible targets, suggesting the involvement of erythrocyte surface sialic acid.5. However, the inhibitory effect of the sugar on anti-rabbit lysis was partially removed by addition of calcium.6. Attempts to characterize membrane components that are critical for hemolysis were performed by inhibition experiments.7. We found that saccharides, glycoproteins, mucosubstances as well as rabbit erythrocyte soluble tryptic fragments were ineffective in inhibiting hemolysis.8. Sonicated dispersion of phosphatidyl choline, phosphatidyl glycerol, phosphatidyl ethanol, sphingomyelin and cholesterol did not influence the hemolytic reaction.9. Rabbit erythrocyte extracted from membrane lipids (chloroform phase) did not modify the lytic activity against rabbit red blood cells.10. Conversely, the methanol phase consistently reduced the lytic capacity of the fluid.11. The heat-stable, trypsin-resistant inhibitory factor was most probably a small molecule, since dialysis removed the inhibitory effect.     

Mechanism of hemolysis and erythrocyte transformation caused by lipogrammistin-A, a lipophilic and acylated cyclic polyamine from the skin secretion of soapfishes (Grammistidae).

The mechanism of hemolysis and erythrocyte transformation caused by lipogrammistin-A (LGA), a lipophilic and acylated cyclic polyamine from the skin secretion of soapfishes (Grammistidae), was investigated. The dependency of hemolysis on the erythrocyte concentration indicated that the amount of membrane-bound LGA required for 50% hemolysis is about 13% of the total phospholipids in erythrocytes on a molar basis. A synthetic analogue which lacked a long alkyl chain exhibited much less activity, suggesting that the alkyl chain is important for membrane-binding. In addition, microscopic observations showed that LGA elicited the invagination of erythrocytes at sublytic concentrations, which makes LGA one of the most potent agents with this transforming activity known to date. Its protonated secondary amino group is responsible for the unequal distribution of LGA in the inner leaflet of the lipid bilayer, which leads to invagination, since acetylation at the amino group markedly reduced the invagination activity. Furthermore, the size of LGA-induced lesions on erythrocyte membrane was estimated to be 7-29 A based on osmotic protection experiments, where the external addition of isotonic molecules in this size range gradually increased the effective dose of LGA. Based on these lines of evidence, the mode of LGA action on erythrocytes is deduced to be as follows. First, LGA molecules bind to erythrocyte membrane by lipophilicity. Second, the molecules accumulate in the inner leaflet of the lipid bilayer by interaction of their cationic ammonium groups with acidic residues of membrane lipid in the inner surface. This uneven distribution of LGA distorts the bilayer structure, and results in a change in cell shape and consequent small lesions. Third, small solutes permeate through the lesions, which induces an osmotic change across the membrane, which leads to colloid-osmotic rupture. This mode of action of LGA on erythrocytes accompanied by cell invagination is the first reported example for natural defense substances.     

The activity of membranes reconstituted from HVJ envelope proteins and lipids to induce hemolysis and fusion between liposomes and erythrocytes

A simple method for preparation of lipid-free envelope proteins (HN protein and F protein) of HVJ (Sendai virus) was developed. Reconstituted 'envelopes' were then prepared from envelope proteins and various lipids by the detergent dialysis method, and the activity to induce hemolysis and fusion between liposome and erythrocyte was studied. Lipid-free envelope protein aggregates could induce hemolysis and liposome-erythrocyte fusion. The activity was however greatly augmented by incorporation of envelope proteins into membrane of viral total lipids. Hemolytic and fusogenic activity was somewhat augmented by incorporation of envelope proteins into dipalmitoylphosphatidylcholine/cholesterol (1:1, molar ratio) and dimyristoylphosphatidylcholine/cholesterol (1:1), though the augmentation was lower than that observed with viral total lipids. When 'envelopes' were reconstituted with the proteins and viral total lipids supplemented with phosphatidylethanolamine, two kinds of 'envelopes' were prepared; one was permeable to Dextran (Mr 75000) and hemolytic, and the other was impermeable to Dextran and nonhemolytic. The latter acquired hemolytic activity after subjection to freezing and thawing, and its barrier function was lost concomitantly. The study suggests that envelope proteins (HN protein and F protein) could function without lipids but their activity was greatly influenced by not only the composition of additional lipids but also mode of arrangement of components on the reconstituted membranes.     

Low-pH association of proteins with the membranes of intact red blood cells. II. Studies of the mechanism

The low-pH interaction of proteins with erythrocyte membranes has been found to be correlated with pH-induced changes in the erythrocyte membrane. Using a 90 degree lightscattering method it was shown that red blood cell hemolysis was slow between pH 5.8 and 5 (t1/2 above 1 h) but became fast at and below pH 4.7 (t1/2 less than 20 min). At pH 4.7, the presence of glycophorin in the incubation medium inhibited the hemolysis of erythrocytes and this protective effect was found to be dependent on the glycophorin concentration. Electron microscope experiments showed the presence of membrane defects after 10 s incubation at pH 4.6 in the absence of glycophorin in the incubation medium. These defects could further develop into openings with average widths of 14 nm after 1.5 min incubation under the acidic conditions. Fluorescence and flow cytometry studies showed that at pH 4.7, but not at pH 7.4, glycophorin tightly associates with phosphatidylcholine liposomes, and that liposome associated glycophorin molecules are recognized by anti-glycophorin monoclonal antibodies.     

Susceptibility of erythrocytes from several animal species to Vibrio vulnificus hemolysin

The hemolytic activity of Vibrio vulnificus hemolysin (VVH) against erythrocytes from several animal species (sheep, horse, cow, rabbit, chicken) was investigated. VVH was active against erythrocytes from all species, but the amount of VVH causing 50% hemolysis under identical conditions (hemolytic susceptibility to VVH) differed. The degree of 125I-labeled VVH (125I-VVH) binding to each erythrocyte species correlated with the susceptibility of the cells to hemolysis. However, marked differences in the binding ability of 125I-VVH were not observed against liposomes constructed with lipids from each erythrocyte membrane. On the other hand, release of hemoglobin (Hb) differed for each of the erythrocyte species despite administration of approximately the same hemolytic VVH concentration to each species. Furthermore, under hypotonic conditions, the stability of each erythrocyte species varied markedly; the more susceptible the erythrocyte to VVH, the more unstable it was under such conditions. These results, therefore, suggest that the susceptibility of erythrocytes to VVH may be closely associated with the binding ability of VVH and erythrocyte membrane stability.     

Mobilization and aggregation of integral membrane proteins in erythrocytes induced by interaction with influenza virus at acidic pH

Effect of influenza virus on erythrocyte membranes was investigated by electron microscopy and fluorescence photobleaching recovery measurements. The virus induced mobilization of integral proteins in erythrocyte membrane at acidic pH, where it fused with the cell membrane to cause hemolysis and also cell fusions but not at neutral pH. At lower temperatures (e.g., 4 degrees C), the proteins aggregated in the membrane and, consequently, large protein-free lipid bilayer area was produced. At higher temperatures (e.g., 37 degrees C) the protein distribution became randomized. Spectrin meshwork underneath the erythrocyte membrane was also markedly modified by the virus at acidic pH. Diffuse fibril structure was converted into dense spots and the membrane area lacking the fibril structure was produced. Isolated hemagglutinin rosettes also caused mobilization and aggregation of the integral proteins at acidic pH but to smaller extent than that induced by virus. The membrane perturbation detected as the protein mobilization by the action of hemagglutinin was assigned to be the cause for envelope fusion.     

Rabbit platelet calcium ATPase differs from the human erythrocyte (Ca2+ + Mg2+)-ATPase in its response to three purified phospholipases A2, exogenous phospholipids and calmodulin

Human erythrocyte (Ca2+ + Mg2+)-ATPase and calcium ATPase of rabbit platelets were compared by their responses to a variety of treatments. These included three purified phospholipases A2 (acidic, neutral and basic) from Agkistrodon halys blomhoffii, as well as several phospholipids and lysophospholipids. The erythrocyte enzyme was stimulated 2-3-fold by all three phospholipases with maximal stimulation occurring at different concentrations of the three enzymes. The basic phospholipase was the most potent, followed by the neutral and acidic enzymes in that order. The calcium ATPase activity of the platelet was also stimulated by phospholipase treatment, but only by 10-20%. The stimulatory activity was attributable to hydrolysis of a very small portion of the total membrane phospholipid. Inactivation of the phospholipases by heating or chemical modification with p-bromophenacyl bromide abolished their ability to stimulate. Addition of polyphosphoinositides stimulated both ATPases. However, another acidic phospholipid, lysophosphatidic acid, stimulated only the erythrocyte enzyme and failed to affect the platelet calcium ATPase. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) had no effect on either enzyme, while the platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), its lyso compound and lysoPC inhibited both ATPases. Calmodulin stimulated the erythrocyte enzyme, but did not affect the platelet calcium ATPase. These results demonstrate that the protein-lipid interactions operative in the erythrocyte and platelet calcium ATPases are quite different.     

Effect of ethanol on the hemolytic stability of erythrocytes

The stability of rabbit erythrocytes to hemolysis induced by different compounds in the presence or absence of ethanol or acetaldehyde has been analyzed. Ethanol slightly reduced erythrocyte stability against acidic hemolysis only after long-term preincubation, but the effect of ethanol on stability to oxidative hemolysis manifested itself immediately after its addition to the cells. Ethanol decreased both stability of cells to oxidative damage and dispersion of the hemolytic curve. Comparison of the effects of ethanol and acetaldehyde showed that the destabilizing effect of ethanol might be caused by either its direct action or the effect of its metabolites formed during preincubation of ethanol with erythrocytes. Possible mechanisms of ethanol and acetaldehyde effects on erythrocyte stability are discussed.     

Calmodulin-independent inhibition of platelet phospholipase A2 by calmodulin antagonists

We tested the effects of calmodulin, two types of calmodulin antagonists, and various phospholipids on the phospholipase A2 activities of intact platelets, platelet membranes, and partially purified enzyme preparations. Trifluoperazine, chlorpromazine (phenothiazines) and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7), at concentrations which antagonize the effects of calmodulin, significantly inhibited thrombin- and Ca2+ ionophore-induced production of arachidonic acid metabolites by suspensions of rabbit platelets and Ca2+-induced arachidonic acid release from phospholipids of membrane fractions, but not phospholipase A2 activity in purified enzyme preparations. The addition of acidic phospholipids, but not calmodulin, stimulated phospholipase A2 activity in purified enzyme preparations while decreasing its Km for Ca2+. The dose-response and kinetics of inhibition by calmodulin antagonists of acidic phospholipid-activated phospholipase A2 activity in purified preparations were similar to those of Ca2+-induced arachidonic acid release from membrane fractions. Calmodulin antagonists were also found to inhibit Ca2+ binding to acidic phospholipids in a similar dose-dependent manner. Our results suggest that the platelet phospholipase A2 is the key enzyme involved in arachidonic acid mobilization in platelets and is regulated by acidic phospholipids in a Ca2+-dependent manner and that calmodulin antagonists inhibit phospholipase A2 activity via an action on acidic phospholipids.     

Cholesterol inhibits the lytic activity of melittin in erythrocytes

Although cell lysis by the hemolytic peptide, melittin, has been extensively studied, the role of specific lipids of the erythrocyte membrane on melittin-induced hemolysis remains unexplored. In this report, we have explored the modulatory role of cholesterol on the hemolytic activity of melittin by specifically depleting cholesterol from rat erythrocytes using methyl-beta-cyclodextrin (MbetaCD). Our results show that the hemolytic activity of melittin is increased by approximately 3-fold upon depletion of erythrocyte membrane cholesterol by approximately 55% without any appreciable loss of phospholipids. This result constitutes the first report demonstrating that the presence of cholesterol inhibits the lytic activity of melittin in its natural target membrane, i.e., the erythrocyte membrane. These results are relevant in understanding the role of cholesterol in the mechanism of action of melittin in the erythrocyte membrane.     

二氧化硅对红细胞膜损伤的冷冻断裂研究

应用冷冻断裂技术观察二氧化硅粉尘对红细胞膜的损伤效应,结果表明,二氧化硅可明显地引起膜内形态结构的改变.     

Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane

The zwitterionic detergent CHAPS, a derivative of the bile salts, is widely used in membrane protein solubilization. It is a “facial” detergent, having a hydrophilic side and a hydrophobic back. The objective of this work is to characterize the interaction of CHAPS with a cell membrane. To this aim, erythrocytes were incubated with a wide range of detergent concentrations in order to determine CHAPS partition behavior, and its effects on membrane lipid order, hemolytic effects, and the solubilization of membrane phospholipids and cholesterol. The results were compared with those obtained with the nonionic detergent Triton X-100. It was found that CHAPS has a low affinity for the erythrocyte membrane (partition coefficient K = 0.06 mM^− 1), and at sub-hemolytic concentrations it causes little effect on membrane lipid order. CHAPS hemolysis and phospholipid solubilization are closely correlated. On the other side, binding of Triton X-100 disorders the membrane at all levels, and has independent mechanisms for hemolysis and solubilization. Differential behavior was observed in the solubilization of phospholipids and cholesterol. Thus, the detergent resistant membranes (DRM) obtained with the two detergents will have different composition. The behaviors of the two detergents are related to the differences in their molecular structures, suggesting that CHAPS does not penetrate the lipid bilayer but binds in a flat position on the erythrocyte surface, both in intact and cholesterol depleted erythrocytes. A relevant result for Triton X-100 is that hemolysis is not directly correlated with the solubilization of membrane lipids, as it is usually assumed.     

Differences in the pattern of attack of acidic,neutral, and basic phospholipases A2 of A. halys blomhofii on human erythrocyte membranes: Problems in interpretation of phospholipid location

Purified acidic (pI 4.9), neutral (pI 6.9), and basic (pI 8.7) phospholipase A₂ from Agkistrodon halys blomhofii showed characteristically different patterns of hemolysis and phospholipid hydrolysis of intact human erthyrocytes. Acidic and neutral enzymes were nonlytic in the early periods of incubations with intact erythrocytes whereas the basic enzyme caused immediate hemolysis (5–8%). Under nonlytic conditions acidic and neutral enzymes hydrolyzed only phosphatidyl choline (PC) (20 and 50%, respectively), whereas basic enzyme hydrolyzed not only PC (60%) but nearly 15% of the phosphatidylethanolamine (PE). Both PC and PE were hydrolyzed significantly when the three phospholipases A₂ were incubated individually with erythrocyte lysate or hypotonic ghosts (sealed or unsealed). The order of substrate preference for acidic and neutral enzymes was always PC > PE. On the contrary basic enzyme exhibited the property of substrate specificity reversal. It hydrolyzed PC faster than PE when the membranes were sealed whereas PE hydrolysis was faster than PC hydrolysis in unsealed membranes. Interestingly only the basic enzyme showed activity in the absence of Ca²⁺ and in the presence of 0.5 mm EDTA. Phospholipase C (Bacillus cereus or Clostridium perfringens) did not show the property of substrate specificity reversal although their ability to hydrolyze PC and PE was different. In general this study demonstrates the unique activity patterns of three physically different pure phospholipases A₂ on human erythrocyte membranes which could be of value in selectively modifying membrane phospholipids. In addition it also throws an important light on the fact that results obtained with phospholipases should be interpreted with caution particularly as regards the localization of phospholipids in membranes.     

Estimation by radiation inactivation of the size of functional units governing Sendai and influenza virus fusion

The target sizes associated with fusion and hemolysis carried out by Sendai virus envelope glycoproteins were determined by radiation inactivation analysis. The target size for influenza virus mediated fusion with erythrocyte ghosts at pH 5.0 was also determined for comparison; a value of 57 +/- 15 kDa was found, indistinguishable from that reported previously for influenza-mediated fusion of cardiolipin liposomes [Gibson, S., Jung, C. Y., Takahashi, M., & Lenard, J. (1986) Biochemistry 25, 6264-6268]. Sendai-mediated fusion with erythrocyte ghosts at pH 7.0 was likewise inactivated exponentially with increasing radiation dose, yielding a target size of 60 +/- 6 kDa, a value consistent with the molecular weight of a single F-protein molecule. The inactivation curve for Sendai-mediated fusion with cardiolipin liposomes at pH 7.0, however, was more complex. Assuming a "multiple target-single hit" model, the target consisted of 2-3 units of ca. 60 kDa each. A similar target was seen if the liposomes contained 10% gangliosides or if the reaction was measured at pH 5.0, suggesting that fusion occurred by the same mechanism at high and low pH. A target size of 261 +/- 48 kDa was found for Sendai-induced hemolysis, in contrast with influenza, which had a more complex target size for this activity (Gibson et al., 1986). Sendai virus fusion thus occurs by different mechanisms depending upon the nature of the target membrane, since it is mediated by different functional units. Hemolysis is mediated by a functional unit different from that associated with erythrocyte ghost fusion or with cardiolipin liposome fusion.     

The relationship of membrane lipids to species specific hemolysis by hemolytic factors from Stomoxys calcitrans (L.) (Diptera: Muscidae)

Posterior-midgut homogenate from female stable flies prepared at 12 h after feeding hemolyzed erythrocytes from 6 different mammalian species more readily than homogenate prepared at 22 h. A significant correlation was obtained between the per cent sphingomyelin content of the erythrocyte membrane and the time required for lysis by the 12 h homogenate. Erythrocytes with low sphingomyelin content were more sensitive to lysis than cells with high sphingomyelin. No such correlation exists for hemolysis by 22 h homogenate. Mean corpuscular volume and osmotic fragilities of erythrocytes were not related to hemolysis either by 12 or 22 h homogenate. Determination of phospholipase C and sphingomyelinase activities showed that the hydrolysis rate of phospholipase C in homogenates prepared at 12–14 h was almost twice as much as sphingomyelinase activity. Whereas hydrolysis rates in 22–24 h homogenate were not different and markedly reduced compared to the 12–14 h homogenate. The times required for erythrocyte hemolysis related to the phospholipase C and sphingomyelinase activity profiles suggests that these enzyme activities participate in the in vitro hemolysis of red blood cells. Bovine and human erythrocytes change their biconcave contour into a spiculated spherical shape when they are exposed to midgut homogenate. This shape change is interpreted as a detergent induced modification of the red cell membrane which renders the erythrocytes more vulnerable to hemolysis.     

Binding properties of Paracentrotus lividus (Echinoidea) hemolysin.

1. Paracentrotus lividus hemolysin binds erythrocytes, zymosan particles, lipopolysaccharide and laminarin surfaces but not auto and allogeneic cell membranes. 2. The binding could, at least for erythrocytes, involve phospholipids and cholesterol. 3. The protease activity of the coelomic fluid is not related to hemolysis. 4. The finding that very low concentrations of Zn2+ inactivate the hemolysin suggests a possible regulative function of the ion in the hemolytic reaction. 5. Ultrastructural observations on rabbit erythrocyte membranes indicate that most likely the transmembrane pores are induced by the lytic molecules.     

Effects of added dietary taurine on erythrocyte lipids and oxidative stress in rabbits fed a high cholesterol diet

Lipid peroxidation leads to damage of polyunsaturated fatty acids of membrane phospholipids. The contribution of oxidative stress to hypercholesterolemia-induced hemolytic anemia and the effects of addition of taurine on erythrocyte lipid composition, oxidative stress, and hematological data were studied in rabbits fed on a high cholesterol (HC) diet (1%, w/w) for 2 months. The effects of taurine on erythrocyte hemolysis and H2O2-induced lipid peroxidation were investigated in normal rabbit erythrocytes in vitro. The HC diet resulted in increases in plasma lipids and lipid peroxide levels as well as increases in cholesterol levels and the cholesterol:phospholipid ratio in the erythrocytes. This diet caused a hemolytic anemia, but lipid peroxide levels remained unchanged in the erythrocytes of the rabbits. Taurine (2.5%, w/w) added to the food has an ameliorating effect on plasma lipids and lipid peroxide levels in rabbits fed on a HC diet. This treatment also caused decreases in elevated erythrocyte cholesterol levels and cholesterol:phospholipid ratio due to the HC diet, but it did not prevent the hemolytic anemia and did not change erythrocyte lipid peroxide levels. In addition, in an in vitro study, taurine did not protect erythrocytes against H2O2-induced hemolysis or lipid peroxidation. These results show that the HC diet causes hemolytic anemia without any changes in erythrocyte lipid peroxidation, and taurine treatment was not effective against hemolytic anemia caused by the HC diet.     

Roles of integral protein in membrane permeabilization by amphidinols

Amphidinols (AMs) are a group of dinoflagellate metabolites with potent antifungal activity. As is the case with polyene macrolide antibiotics, the mode of action of AMs is accounted for by direct interaction with lipid bilayers, which leads to formation of pores or lesions in biomembranes. However, it was revealed that AMs induce hemolysis with significantly lower concentrations than those necessary to permeabilize artificial liposomes, suggesting that a certain factor(s) in erythrocyte membrane potentiates AM activity. Glycophorin A (GpA), a major erythrocyte protein, was chosen as a model protein to investigate interaction between peptides and AMs such as AM2, AM3 and AM6 by using SDS-PAGE, surface plasmon resonance, and fluorescent-dye leakages from GpA-reconstituted liposomes. The results unambiguously demonstrated that AMs have an affinity to the transmembrane domain of GpA, and their membrane-permeabilizing activity is significantly potentiated by GpA. Surface plasmon resonance experiments revealed that their interaction has a dissociation constant of the order of 10 μM, which is significantly larger than efficacious concentrations of hemolysis by AMs. These results imply that the potentiation action by GpA or membrane integral peptides may be due to a higher affinity of AMs to protein-containing membranes than that to pure lipid bilayers.     

Roles of integral protein in membrane permeabilization by amphidinols

Amphidinols (AMs) are a group of dinoflagellate metabolites with potent antifungal activity. As is the case with polyene macrolide antibiotics, the mode of action of AMs is accounted for by direct interaction with lipid bilayers, which leads to formation of pores or lesions in biomembranes. However, it was revealed that AMs induce hemolysis with significantly lower concentrations than those necessary to permeabilize artificial liposomes, suggesting that a certain factor(s) in erythrocyte membrane potentiates AM activity. Glycophorin A (GpA), a major erythrocyte protein, was chosen as a model protein to investigate interaction between peptides and AMs such as AM2, AM3 and AM6 by using SDS-PAGE, surface plasmon resonance, and fluorescent-dye leakages from GpA-reconstituted liposomes. The results unambiguously demonstrated that AMs have an affinity to the transmembrane domain of GpA, and their membrane-permeabilizing activity is significantly potentiated by GpA. Surface plasmon resonance experiments revealed that their interaction has a dissociation constant of the order of 10 microM, which is significantly larger than efficacious concentrations of hemolysis by AMs. These results imply that the potentiation action by GpA or membrane integral peptides may be due to a higher affinity of AMs to protein-containing membranes than that to pure lipid bilayers.     

Parameters affecting fusion between Sendai virus and liposomes. Role of viral proteins, liposome composition, and pH

A kinetic and quantitative characterization of the fusion process between Sendai virus and phospholipid vesicles is presented. Membrane fusion was monitored in a direct and continuous manner by employing an assay which relies on the relief of fluorescence self-quenching of the probe octadecylrhodamine B chloride which was located in the viral membrane. Viral fusion activity was strongly dependent on the vesicle lipid composition and was most efficient with vesicles solely consisting of acidic phospholipids, particularly cardiolipin (CL). This result implies that the fusion of viruses with liposomes does not display an absolute requirement for specific membrane receptors. Incorporation of phosphatidylcholine (PC), rather than phosphatidylethanolamine (PE), into CL bilayers strongly inhibited fusion, suggesting that repulsive hydration forces interfere with the close approach of viral and target membrane. Virus-liposome fusion products were capable of fusing with liposomes, but not with virus. In contrast to fusion with erythrocyte membranes, fusion between virus and acidic phospholipid vesicles was triggered immediately, did not strictly depend on viral protein conformation, and did not display a pH optimum around pH 7.5. On the other hand, with vesicles consisting of PC, PE, cholesterol, and the ganglioside GD1a, the virus resembled more closely the fusogenic properties that were seen with erythrocyte target membranes. Upon decreasing the pH below 5.0, the viral fusion activity increased dramatically. With acidic phospholipid vesicles, maximal activity was observed around pH 4.0, while with GD1a-containing zwitterionic vesicles the fusion activity continued to increase with decreasing pH down to values as low as 3.0.(ABSTRACT TRUNCATED AT 250 WORDS)