Sialylneolacto-N-tetraose c (LSTc)-bearing Liposomal Decoys Capture Influenza A Virus

Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand binding interactions for a high avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, whereas control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains.     

Ficoll and dextran enhance adhesion of Sendai virus to liposomes containing receptor (ganglioside GD1a)

Previous work has shown that high-speed centrifugation (300,000 g) of Sendai virus and liposomes in 40% (w/v) sucrose layered under a discontinuous sucrose gradient removes Sendai virus bound to liposomes containing the ganglioside GD1a, a Sendai virus receptor. Centrifugation also removes virus bound to liposomes containing other negatively charged lipids. This work shows that centrifugation of virus through a discontinuous ficoll gradient does not remove virus bound to liposomes containing GD1a but does remove virus from liposomes containing various other negatively charged lipids including the ganglioside GM1, which is not a Sendai virus receptor. The amount of virus that adheres to liposomes increases with increasing content of GD1a in the liposomes. The adhesion of virus to receptor-containing liposomes during centrifugation through a ficoll gradient results from the presence of ficoll and increases with increasing ficoll concentration. Virus also adheres to receptor-containing liposomes during centrifugation in the presence of dextran. These data indicate that caution should be used in interpreting associations demonstrated by centrifugation through dextran and ficoll gradients. They also indicate that binding of virus by ganglioside receptors can be modulated by carbohydrate polymers, which are thought not to have any specific interaction with either viruses or gangliosides.     

Membrane fusion activity of influenza virus.

A simple assay is described to monitor fusion between fowl plague virus (FPV, an avian influenza A virus) and liposomes which allows the simultaneous quantitation of both lytic and non-lytic fusion events. As in fusion between viruses and the plasma membrane and in FPV-induced cell-cell fusion, the reaction only occurs at pH 5.5 or below, and it is fast, highly efficient, and essentially non-lytic when fresh virus and liposomes are used. The fusion occurs over a broad temperature range, and has no requirement for divalent cations. The fusion factor of influenza virus is a hemagglutinin (HA) spike which protrudes from the virus membrane and which is also responsible for virus binding to the host cell. The finding that fusion occurs as efficiently with liposomes containing or lacking virus receptor structures, further emphasizes the remarkable division of labor in the HA molecule: the receptor-binding sites are located in the globular HA1 domains and the fusion activation peptide is found at the N-terminal of HA2 in the stem region of the protein. The mechanism of fusion is discussed in terms of the three-dimensional structure of the HA and the conformational change which the protein undergoes at the fusion pH optimum.     

The role of target membrane sialic acid residues in the fusion activity of the influenza virus: the effect of two types of ganglioside on the kinetics of membrane merging

The influenza virus enters target cells via the action of hemagglutinin proteins (HA) inserted into the viral envelope. HA promotes membrane fusion between the viral envelope and endosomal membrane at low pH, following viral binding to sialic acid-containing receptors on target cells, and internalization by endocytosis. The effect of target membrane sialic acid residues on the fusion activity of the influenza virus towards model membranes was evaluated by both reduction, (i.e. treating somatic cells with neuraminidase- (NA-) prior to virus-cell interactions), and by supplementing liposomes with the gangliosides GD1a and GT1b. The harshness of the neuraminidase pretreatment of target cells required to affect virus-induced membrane merging was found to greatly depend on the assay conditions, i.e. whether a virus-cell prebinding step at neutral pH was included prior to acidification. Minor concentrations of neuraminidase were found to greatly reduce virus fusion, but only in the absence of a prebinding step; they had no effect if this step was included. Although membrane merging was greatly reduced following cell neuraminidase pretreatment, virus-cell association at low pH was not disturbed proportionately. This probably reflects unspecific virus-cell binding under these conditions, probably of inactivated or aggregated virus particles, which does not translate into membrane merging. This seems to suggest both that target membrane sialic acid can protect the virus from losing its activity before triggering membrane merging, and that the importance of this interaction is not merely to ensure virus-target proximity. With liposomes, we found that both types of ganglioside supported efficient fusion, with GD1a promoting a slightly faster initial rate. However, in this case, virus-target proximity closely mirrored fusion activity, thus pointing to differential specificity between targets routinely used to assay influenza virus fusion activity.     

Membrane fusion activity of influenza virus. Effects of gangliosides and negatively charged phospholipids in target liposomes

Fusion of influenza virus with liposomes composed of negatively charged phospholipids differs from fusion with biological membranes or zwitterionic liposomes with ganglioside receptors [Stegmann, T., Hoekstra, D., Scherphof, G., & Wilschut, J. (1986) J. Biol. Chem. 261, 10966-10969]. In this study, we investigated how the kinetics and extent of fusion of influenza virus, monitored with a fluorescence resonance energy-transfer assay, are influenced by the surface charge and the presence of receptors on liposomal membranes. The results were analyzed in terms of mass action kinetic model, providing separate rate constants for the initial virus-liposome adhesion, or aggregation, and for the actual fusion reaction. Incorporation of increasing amounts of cardiolipin (CL) or phosphatidylserine (PS) into otherwise zwitterionic phosphatidylcholine (PC)/phosphatidylethanolamine (PE) vesicles results in a gradual shift of the pH threshold of fusion to neutral, relative to the pH threshold obtained with PC/PE vesicles containing the ganglioside GD1a, while also the rate of fusion increases. This indicates the emergence of a fusion mechanism not involving the well-documented conformational change in the viral hemagglutinin (HA). However, only with pure CL liposomes this nonphysiological fusion reaction dominates the overall fusion process; with pure PS or with zwitterionic vesicles containing CL or PS, the contribution of the nonphysiological fusion reaction is small. Accordingly, preincubation of the virus alone at low pH results in a rapid inactivation of the viral fusion capacity toward all liposome compositions studied, except pure CL liposomes. The results of the kinetic analyses show that with pure CL liposomes the rates of both virus-liposome adhesion and fusion are considerably higher than with all other liposome compositions studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of N-acetylneuraminic (sialic) acid in the pH dependence of influenza virion fusion with planar phospholipid membranes

It is known that fusion of influenza virus to host cell membranes is strongly promoted by acidic pH. We have determined conditions required to obtain pH-dependent fusion of influenza virus to planar bilayer membranes. The rate of viral fusion was determined from the flash rate of R18-labeled virions delivered to the surface of the planar membrane by pressure-ejection from a pipette. For a bilayer formed only of phospholipids and cholesterol, the fusion rate was independent of pH and unaffected by the phospholipid composition. When the gangliosides GD1a + GT1b were included in the planar membrane, however, the fusion rate varied steeply with pH. The rate at pH 7.4 in the presence of the gangliosides was about an order of magnitude less than in their absence. At pH less than approximately 5.5, the rate was about an order of magnitude greater in the presence of gangliosides than in their absence. The fusion rate with planar membranes containing globoside, a ceramide-backboned glycolipid, was also independent of pH, indicating that the pH dependence required sialic acid on the carbohydrate moiety of the glycolipid. The gangliosides GM1a and GM3, both of which possess sialic acid, produced the same pH-dependent fusion rate as seen with GD1a + GT1b, indicating that the presence, but not the location, of terminal sialic acids is critical. Incubating virus with soluble sialyllactose blocked fusion to both ganglioside-free and ganglioside-containing planar membranes. These results show that the pH dependence of influenza virion fusion arises from the interaction of the sialic acid receptor with the influenza hemagglutinin. A model for sialic acid-hemagglutinin interactions accounting for pH-dependent fusion is presented.     

Kinetics of fusion and lipid transfer between virus receptor containing liposomes and influenza viruses as measured with the octadecylrhodamine B chloride assay

Octadecylrhodamine B chloride (R18) and ganglioside GD1a (virus receptor) were incorporated into small unilamellar liposomes [Hoekstra et al. (1984) Biochemistry 23, 5675-5681]. Upon interaction of these liposomes with PR8 influenza viruses without prebinding, two types of dequenching were observed at 37 degrees C, both second-order processes: a fast reaction at pH 5.3, 2k = 17.53 x 10(-3) (Q.s)-1, and a slow reaction at pH 7.4, 2k = 0.335 x 10(-3) (Q.s)-1. The maximal level of dequenching was the same for both. Upon prebinding of liposomes to PR8 viruses (30 min, 0 degrees C, pH 7.4) at high concentrations, a very fast dequenching occurred when the prebinding mixture was diluted into prewarmed (37 degrees C) 10 mM PBS, pH 5.3. For the initial phase, a first-order rate constant of 0.5 s-1 could be extrapolated. After a quick drop in velocity during the first 30 s, the reaction was kinetically indistinguishable from the one found without prebinding. A second-order process with 2k = 16.52 x 10(-3) (Q.s)-1 became rate-limiting. The fast reactions at pH 5.3 can be abolished by inactivation or removal of the virus hemagglutinin. We conclude that the reaction at pH 5.3 reflects the hemagglutinin-dependent fusion process known to occur between influenza viruses and partner membranes at low pH; however, second-order kinetics indicate that specific binding rather than fusion is the rate-limiting step. For the slow dequenching, which is not affected by prebinding, the rate constant is 20 times lower than for the fast reaction, and the process is independent of viral hemagglutinin.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Temperature-dependent kinetics of the activities of influenza virus

The temperature dependence of membrane interactions between PR8 influenza virus and virus receptor (GD1a)-containing liposomes was studied. For quantitation, the octadecylrhodamine B chloride (R18) membrane marker was incorporated into liposomes at quenched concentrations. Upon interaction with target membranes, the marker gets diluted, and dequenching can be measured in a fluorescence spectrophotometer. Rate constants were calculated from the dequenching curves under low pH conditions, which allow for fusion, and at neutral pH, where no specific fusion occurs. Activation energies were determined from Arrhenius plots. The results were compared with the temperature dependence of other viral activities like infectivity, hemolysis, and fusion with erythrocytes. For the slow reaction at pH 7.4, where only non-specific lipid transfer takes place, the activation energy was about 24 kcal/mole between 15 degrees C and 45 degrees C. For the fast, hemagglutinin (HA)-specific fusion reaction (pH 5.3), a very low activation energy (approximately 7 kcal/mole) was found between 25 degrees C and 37 degrees C, whereas below 25 degrees C it was much higher (approximately 34 kcal/mole). The temperature range with low activation energy coincides with the one for optimal infectivity, hemolysis, and fusion with erythrocytes. Furthermore, it is the same range in which the conformational change of HA takes place, which in the absence of a partner membrane leads to an irreversible inactivation of the fusion protein.     

Role of a transbilayer pH gradient in the membrane fusion activity of the influenza virus hemagglutinin: Use of the R18 assay to monitor membrane merging

It had been suggested that influenza virus-mediated membrane fusion might be dependent on a pH gradient across a target membrane. We have designed experiments in which this issue could be addressed. Two populations of liposomes were prepared, both simulating the plasma membrane of target cells, but with the pH of the internal aqueous medium buffered either at pH 7.4 (physiological cytosol pH) or at pH 5.0 (endosomal pH at which influenza virus displays maximal fusion activity). By monitoring fusion using the R18 assay, we found that the internal pH of the target liposomes did not influence membrane merging as mediated by the influenza virus hemagglutinin, thus demonstrating that a transmembrane pH gradient is not required in this fusion process.     

Influence of calcium on lipid mixing mediated by influenza hemagglutinin

We studied the influence of calcium on lipid mixing mediated by influenza hemagglutinin (HA). Lipid mixing between HA-expressing cells and liposomes containing disialoganglioside, influenza virus receptor, was studied at 37 degrees C and neutral pH after a low-pH pulse at 4 degrees C. With DSPC/cholesterol liposomes, calcium present after raising the temperature significantly promoted lipid mixing only when it was triggered by a short low-pH application. In case of DOPC/cholesterol liposomes, calcium promotion was observed regardless of the duration of the low-pH pulse. Calcium present during a short, but not long, low-pH application to HA-expressing cells with bound DSPC/cholesterol liposomes at 4 degrees C inhibited subsequent lipid mixing. We hypothesize that calcium influences lipid mixing because it binds to a vestigial esterase domain of hemagglutinin or causes expulsion of the fusion peptide from an electronegative cavity. We suggest that calcium promotes the transition from early and reversible conformation(s) of low pH-activated HA towards an irreversible conformation that underlies both HA-mediated lipid mixing and HA inactivation.     

Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rescue of vector-expressed fowl plague virus hemagglutinin in biologically active form by acidotropic agents and coexpressed M2 protein.

The hemagglutinin of the Rostock strain of fowl plague virus was expressed in CV-1 cells by a simian virus 40 vector, and its stability in the exocytotic transport process was examined by a fusion assay. A 50-fold increase in the fusion activity of the hemagglutinin was observed when expression occurred in the presence of ammonium chloride, Tris-HCl, or high doses of amantadine. When chloroquine, another acidotropic agent, was used, the hemagglutinin exposed at the cell surface had to be activated by trypsin, because intracellular cleavage was inhibited by this compound. Hemagglutinin mutants resistant to intracellular cleavage did not require acidotropic agents for full expression of fusion activity, when treated with trypsin after arrival at the cell surface. These results indicate that fowl plague virus hemagglutinin expressed by a simian virus 40 vector is denatured in the acidic milieu of the exocytotic pathway and that cleavage is a major factor responsible for the pH instability. Coexpression with the M2 protein also markedly enhanced the fusion activity of the hemagglutinin, and this effect was inhibited by low doses of amantadine. These results support the concept that M2, known to have ion channel function, protects the hemagglutinin from denaturation by raising the pH in the exocytotic transport system. The data also stress the importance of acidotropic agents or coexpressed M2 for the structural and functional integrity of vector-expressed hemagglutinin.     

Effects of low pH on influenza virus. Activation and inactivation of the membrane fusion capacity of the hemagglutinin

The hemagglutinin of influenza virus undergoes a conformational change at low pH, which results in exposure of a hydrophobic segment of the molecule, crucial to expression of viral fusion activity. We have studied the effects of incubation of the virus at low pH either at 37 or 0 degrees C. Treatment of the virus alone at pH 5.0 induces the virus particles to become hydrophobic, as assessed by measuring the binding of zwitterionic liposomes to the virus. At 37 degrees C this hydrophobicity is transient, electron microscopic examination of the virus reveals a highly disorganized spike layer, and fusion activity toward ganglioside-containing zwitterionic liposomes, measured at 37 degrees C with a kinetic fluorescence assay, is irreversibly lost. By contrast, after preincubation of the virus alone at pH 5.0 and 0 degrees C fusion activity remains unaffected. Yet, the preincubation at 0 degrees C does result in exposure of the hydrophobic segment of hemagglutinin, but now hydrophobicity is sustained and viral spike morphology unaltered. Hydrophobicity also remains to a significant extent upon pH neutralization, but fusion activity is negligible under these conditions. It is concluded that for optimal expression of fusion activity the virus must be bound to the target membrane before exposure to low pH. Furthermore, even after exposure of the hydrophobic segment of hemagglutinin, fusion occurs only at low pH. Finally, fusion occurs only at elevated temperature, possibly reflecting the unfolding of hemagglutinin trimers or the cooperative action of several hemagglutinin trimers in the reaction.     

Stability of yeast iso-1-ferricytochrome c as a function of pH and temperature.

Absorbance-detected thermal denaturation studies of the C102T variant of Saccharomyces cerevisiae iso-1-ferricytochrome c were performed between pH 3 and 5. Thermal denaturation in this pH range is reversible, shows no concentration dependence, and is consistent with a 2-state model. Values for free energy (delta GD), enthalpy (delta HD), and entropy (delta SD) of denaturation were determined as functions of pH and temperature. The value of delta GD at 300 K, pH 4.6, is 5.1 +/- 0.3 kcal mol-1. The change in molar heat capacity upon denaturation (delta Cp), determined by the temperature dependence of delta HD as a function of pH (1.37 +/- 0.06 kcal mol-1 K-1), agrees with the value determined by differential scanning calorimetry. pH-dependent changes in the Soret region indicate that a group or groups in the heme environment of the denatured protein, probably 1 or both heme propionates, ionize with a pK near 4. The C102T variant exhibits both enthalpy and entropy convergence with a delta HD of 1.30 kcal mol-1 residue-1 at 373.6 K and a delta SD of 4.24 cal mol-1 K-1 residue-1 at 385.2 K. These values agree with those for other single-domain, globular proteins.     

A reassortment-incompetent live attenuated influenza virus vaccine for protection against pandemic virus strains

Although live-attenuated influenza vaccines (LAIV) are safe for use in protection against seasonal influenza strains, concerns regarding their potential to reassort with wild-type virus strains have been voiced. LAIVs have been demonstrated to induce enhanced mucosal and cell-mediated immunity better than inactivated vaccines while also requiring a smaller dose to achieve a protective immune response. To address the need for a reassortment-incompetent live influenza A virus vaccine, we have designed a chimeric virus that takes advantage of the fact that influenza A and B viruses do not reassort. Our novel vaccine prototype uses an attenuated influenza B virus that has been manipulated to express the ectodomain of the influenza A hemagglutinin protein, the major target for eliciting neutralizing antibodies. The hemagglutinin RNA segment is modified such that it contains influenza B packaging signals, and therefore it cannot be incorporated into a wild-type influenza A virus. We have applied our strategy to different influenza A virus subtypes and generated chimeric B/PR8 HA (H1), HK68 (H3), and VN (H5) viruses. All recombinant viruses were attenuated both in vitro and in vivo, and immunization with these recombinant viruses protected mice against lethal influenza A virus infection. Overall, our data indicate that the chimeric live-attenuated influenza B viruses expressing the modified influenza A hemagglutinin are effective LAIVs.     

Effect of Temperature on Radiosensitivity of Newcastle Disease Virus

Newcastle disease virus was irradiated at temperatures ranging from 2.2 to 60 C. An interaction between the thermal and ionizing energy was observed in the temperature region of 49 to 60 C. At 2.2 C, the hemagglutinin was considerably more radioresistant than the infectivity property. It is believed that radiation inactivation of Newcastle disease virus infectivity at low temperatures was due to nucleic acid degradation and at higher temperatures was due to protein denaturation.     

Hepatitis C virus glycoproteins mediate low pH-dependent membrane fusion with liposomes

It has been suggested that the hepatitis C virus (HCV) infects host cells through a pH-dependent internalization mechanism, but the steps leading from virus attachment to the fusion of viral and cellular membranes remain uncharacterized. Here we studied the mechanism underlying the HCV fusion process in vitro using liposomes and our recently described HCV pseudoparticles (pp) bearing functional E1E2 envelope glycoproteins. The fusion of HCVpp with liposomes was monitored with fluorescent probes incorporated into either the HCVpp or the liposomes. To validate these assays, pseudoparticles bearing either the hemagglutinin of the influenza virus or the amphotropic glycoprotein of murine leukemia virus were used as models for pH-dependent and pH-independent entry, respectively. The use of assays based either on fusion-induced dequenching of fluorescent probes or on reporter systems, which produce fluorescence when the virus and liposome contents are mixed, allowed us to demonstrate that HCVpp mediated a complete fusion process, leading to the merging of both membrane leaflets and to the mixing of the internal contents of pseudoparticle and liposome. This HCVpp-mediated fusion was dependent on low pH, with a threshold of 6.3 and an optimum at about 5.5. Fusion was temperature-dependent and did not require any protein or receptor at the surface of the target liposomes. Most interestingly, fusion was facilitated by the presence of cholesterol in the target membrane. These findings clearly indicate that HCV infection is mediated by a pH-dependent membrane fusion process. This paves the way for future studies of the mechanisms underlying HCV membrane fusion.     

Liposomes That Provide T-Dependent Help to Weak Antigens (T-Independent Antigens)

The design of an adjuvant for eliciting a thymus-dependent response to LPS, a well-defined thymus-independent antigen, is presented. Hybrid liposomes containing LPS and HA2 peptide from the hemagglutinin protein of influenza virus within the liposome bilayer were prepared (LPS/HA2 liposomes). The HA2 polypeptide contains epitopes recognized by T-helper lymphocytes and T-cytotoxic lymphocytes. Outbred mice immunized with LPS/HA2 liposomes produced anti-LPS-specific IgG responses. IgG subclass analysis indicated that IgG1, IgG2, and IgG3 antibodies were produced by these animals. LPS liposomes (liposomes without HA2) stimulated a T-independent response only. This was demonstrated by the detection of IgG3 but not IgG1 or IgG2 in serum of mice immunized with LPS liposomes. These results support the concept that the simultaneous incorporation into liposomes of a polypeptide with T-cell recognition sites along with a T-independent antigen can lead to the generation of cognate T-cell help for the T-independent antigen. The synthesis and characterization of a neo-lipopolysaccharide T-independent antigen for incorporation in hybrid HA2 liposomes are also presented. Findings are discussed relative to the liposome model used and implications for development of vaccines for use in humans.     

Fusion activity of influenza virus. A comparison between biological and artificial target membrane vesicles

We have investigated the pH-dependent fusion activity of influenza virus toward human erythrocyte ghosts, utilizing a recently developed fluorescence assay, which permits continuous monitoring of the fusion reaction. The rate of fusion is negligible at neutral pH but shows a sharp increase at pH values just below 5.5. This pH dependence profile closely corresponds to that of virus-induced hemolysis. Fusion is rapidly inactivated by a low-pH preincubation of the virus alone either at 37 or at 0 degrees C. The presence of ghosts during this low-pH preincubation, carried out at 0 degree C under which condition there is hardly any fusion, causes a significant protection of the viral fusion activity against inactivation. Fusion initiated at low pH can be arrested instantaneously by readjustment of the pH to neutral. The characteristics of fusion of influenza virus with ghosts deviate from those of fusion with cardiolipin liposomes (Stegmann, T., Hoekstra, D., Scherphof, G., and Wilschut, J. (1985) Biochemistry 24, 3107-3113). Fusion with ghosts is consistent with a requirement of the well-documented pH-dependent conformational change in the viral hemagglutinin, whereas fusion with cardiolipin liposomes does not exhibit a strict dependence on the conformational change. Rather, the negative surface charge on the liposomes plays a critical role, as zwitterionic liposomes containing gangliosides show fusion behavior similar to that of erythrocyte ghosts.