Activation of vesicular stomatitis virus fusion with cells by pretreatment at low pH

Fusion of vesicular stomatitis virus (VSV) with Vero cells was measured after exposure of the virus to low pH under a variety of experimental conditions. The method of relief of fluorescence self-quenching of the probe octadecylrhodamine was used to monitor fusion. Incubation of the virus at pH 5.5 prior to binding to cells led to significant enhancement of fusion at the plasma membrane, whereas fusion via the endocytic pathway was inhibited. Fusion of pH 5.5-pretreated VSV showed a similar pH threshold for fusion as nontreated virus, and it was blocked by antibody to VSV G protein. Activation of VSV by pretreatment at low pH was only slightly dependent on temperature. In contrast, when VSV was first bound to target cells and subsequently exposed at 4 degrees C to the low pH, activation of the fusion process did not occur. The pH 5.5-mediated activation of VSV could be reversed by returning the pH to neutral in the absence of target membranes. The low pH pretreatment also led to aggregation of virus; large aggregates could be pelleted by low speed centrifugation and only the effects of the supernatant, which consist of single virions and/or microaggregates, were considered. The data were analyzed in the framework of an allosteric model according to which viral spike glycoproteins undergo a pH-dependent conformational transition to an active (fusion-competent) state. Based on that analysis we conclude that the conformational transition to the active state is rate-limiting for fusion and that the viral spike glycoproteins are fusion-competent only in their protonated form.     

Role of viral envelope sialic acid in membrane fusion mediated by the vesicular stomatitis virus envelope glycoprotein.

Fusion of vesicular stomatitis virus (VSV) with cells and liposomes before and after treatment with neuraminidase was studied using the R18 dequenching assay. Desialylation of VSV significantly enhanced the extent of fusion with Vero cells but affected neither the pH dependence nor the binding of VSV to Vero cells. The enhanced fusion of asialo-VSV was observed both at the plasma membrane as well as via the endocytic pathway. Both VSV and asialo-VSV fused with liposomes made of neutral phospholipid, but only asialo-VSV fused with liposomes containing a 1:1 mixture of neutral and negatively charged phospholipid. To examine factors which contribute to the extent of fusion, we analyzed the various activation and inactivation reactions that take place as a result of low-pH triggering of VSV prebound to the target membrane. Lag times for the onset of fusion were similar for VSV and asialo-VSV, indicating that desialylation did not affect the activation reactions. However, exposure of VSV bound to target membranes at pH 6.5 for 400 s led to considerable inactivation, whereas little inactivation was seen after desialylation of VSV. These results are analyzed in terms of a model which allows us to determine which components of the overall fusion process are dominated by viral envelope sialic acid.     

Acid pH-induced fusion of cells by herpes simplex virus glycoproteins gB an gD

Enveloped animal viruses enter host cells either by direct fusion at neutral pH or by endocytosis. Herpes simplex virus (HSV) is believed to fuse with the plasma membrane of cells at neutral pH, and the glycoproteins gB and gD have been implicated in virus entry and cell fusion. Using cloned gB or gD genes, we show that cells expressing HSV-1 glycoproteins gB or gD can undergo fusion to form polykaryons by exposure only to acidic pH. The low pH-induced cell fusion was blocked in the presence of monoclonal antibodies specific to the glycoproteins. Infection of cells expressing gB or gD glycoproteins with HSV-1 inhibited the low pH-induced cell fusion. The results suggest that although the glycoproteins gB and gD possess fusogenic activity at acidic pH, other HSV proteins may regulate it such that in the virus-infected cell, this fusion activity is blocked.     

Early Events of Fusion Between Epstein Barr Virus and Human Lymphoblastoid Cells (Raji) Detected by R18 Fluorescence Dequenching Measurements

Relief of fluorescence self-quenching was used to monitor fusion (14) of Epstein Barr virus (EBV) with Raji cells after exposure of the virus to a variety of experimental conditions such as neutral or low pH, enzymatic modification of the viral spike glycoproteins, or inhibition of the protein kinase C (PKC) activity. Incubation of the virus at pH 5.9 prior to the binding to the cell membrane led to a significant enhancement of fusion with the plasma membrane. Treatment of Raji cells with an agent known to elevate the endosomal and lysosomal pH (lysosomotropic agent) (3, 12) partially prevented fusion at neutral pH. Desialylation of EBV significantly reduced the extent of fusion with Raji cells. Protein kinase C inhibitor reduced EBV fusion with Raji cells, while treatment with the tumor promoter and the PKC activator TPA caused an increase in the final extent of fusion. Our results suggest that EBV fuses with lymphoblastoid cells in the endocytic vescicles after being rapidly internalized and that protein kinase C is involved in the process of viral entry into cells.     

Early events of fusion between Epstein Barr virus and human lymphoblastoid cells (Raji) detected by R18 fluorescence dequenching measurements

Relief of fluorescence self-quenching was used to monitor fusion (14) of Epstein Barr virus (EBV) with Raji cells after exposure of the virus to a variety of experimental conditions such as neutral or low pH, enzymatic modification of the viral spike glycoproteins, or inhibition of the protein kinase C (PKC) activity. Incubation of the virus at pH 5.9 prior to the binding to the cell membrane led to a significant enhancement of fusion with the plasma membrane. Treatment of Raji cells with an agent known to elevate the endosomal and lysosomal pH (lysosomotropic agent) (3, 12) partially prevented fusion at neutral pH. Desialylation of EBV significantly reduced the extent of fusion with Raji cells. Protein kinase C inhibitor reduced EBV fusion with Raji cells, while treatment with the tumor promotor and the PKC activator TPA caused an increase in the final extent of fusion. Our results suggest that EBV fuses with lymphoblastoid cells in the endocytic vesicles after being rapidly internalized and that protein kinase C is involved in the process of viral entry into cells.     

Infectious Cell Entry Mechanism of Influenza Virus

Interaction between influenza virus WSN strain and MDCK cells was studied by using spin-labeled phospholipids and electron microscopy. Envelope fusion was negligibly small at neutral pH but greatly activated in acidic media in a narrow pH range around 5.0. The half-time was less than 1 min at 37°C at pH 5.0. Virus binding was almost independent of the pH. Endocytosis occurred with a half-time of about 7 min at 37°C at neutral pH, and about 50% of the initially bound virus was internalized after 1 h. Electron micrographs showed binding of virus particles in coated pits in the microvillous surface of plasma membrane and endocytosis into coated vesicles. Chloroquine inhibited virus replication. The inhibition occurred when the drug was added not later than 10 min after inoculation. Chloroquine caused an increase in the lysosomal pH 4.9 to 6.1. The drug did not affect virus binding, endocytosis, or envelope fusion at pH 5.0. Electron micrographs showed many virus particles remaining trapped inside vacuoles even after 30 min at 37°C in the presence of drug, in contrast to only a few particles after 10 min in vacuoles and secondary lysosomes in its absence. Virus replication in an artificial condition, i.e., brief exposure of the inoculum to acidic medium followed by incubation in neutral pH in the presence of chloroquine, was also observed. These results are discussed to provide a strong support for the infection mechanism of influenza virus proposed previously: virus uptake by endocytosis, fusion of the endocytosed vesicles with lysosome, and fusion of the virus envelope with the surrounding vesicle membrane in the secondary lysosome because of the low pH. This allows the viral genome to enter the target cell cytoplasm.     

pH-dependent hemolysis and cell fusion of rhabdoviruses

Human erythrocytes pretreated with fungal semialkali protease or trypsin became susceptible to hemagglutination by vesicular stomatitis virus (VSV) and rabies virus. Both viruses exhibited extensive hemolytic and fusion activities against erythrocytes pretreated with these enzymes. The hemolysis and fusion were pH dependent and the activities were most apparent at pH 5.0 and decreased with increase in pH. However, VSV still exhibited slight hemolytic activity at neutral pH. Hemolysis was also dependent on the dose of virus and was inhibited by treatment of the viruses with antiviral antibody. Results of sodium dodecyl sulfate polyacrylamide gel electrophoresis of erythrocyte membranes suggested that most of the carbohydrates were removed from the membrane proteins by the treatment with proteolytic enzymes.     

Probing the interaction between vesicular stomatitis virus and phosphatidylserine

The entry of enveloped animal viruses into their host cells always depends on membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion between the viral envelope and the endosomal membrane at the acidic environment of this compartment. In this work, we evaluated VSV interactions with membranes of different phospholipid compositions, at neutral and acidic pH, using atomic force microscopy (AFM) operating in the force spectroscopy mode, isothermal calorimetry (ITC) and molecular dynamics simulation. We found that the binding forces differed dramatically depending on the membrane phospholipid composition, revealing a high specificity of G protein binding to membranes containing phosphatidylserine (PS). In a previous work, we showed that the sequence corresponding amino acid 164 of VSV G protein was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Here, we used this sequence to explore VSV–PS interaction using ITC. We found that peptide binding to membranes was exothermic, suggesting the participation of electrostatic interactions. Peptide–membrane interaction at pH 7.5 was shown to be specific to PS and dependent on the presence of His residues in the fusion peptide. The application of the simplified continuum Gouy–Chapman theory to our system predicted a pH of 5.0 at membrane surface, suggesting that the His residues should be protonated when located close to the membrane. Molecular dynamics simulations suggested that the peptide interacts with the lipid bilayer through its N-terminal residues, especially Val¹⁴⁵ and His¹⁴⁸. Fabiana A.Carneiro and Pedro A. Lapido-Loureiro contributed equally to this work An erratum to this article can be found at     

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.     

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.     

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.     

Characterization of the fusion of enveloped viruses with the plasma membrane of Saccharomyces cerevisiae spheroplasts

Vesicular stomatitis virus (VSV) was associated at low pH with Saccharomyces cerevisiae spheroplasts. In the cold, the association was characterized as reversible binding to the spheroplast surface. At 37 degrees C, the association became irreversible due to fusion of the viral envelope with the yeast plasma membrane according to the following data. Proteinase K digestion degraded the viral envelope glycoprotein G but left the internal N and M proteins of VSV intact and associated with the spheroplasts. The plasma membrane could be stained by indirect immunofluorescent labeling using antiserum against VSV. By immunoelectron microscopy, no VSV particles could be detected at the spheroplast surface. Instead, the G protein could be visualized at the external aspect of the plasma membrane using specific antiserum and protein A-gold. Fusion of VSV with spheroplasts occurred below pH 4.75 at temperatures of 30-42 degrees C. It was strictly dependent on the prior removal of the yeast cell wall. The fusion process was fast, calcium-independent, and nonleaky, leaving the spheroplasts viable for at least 4 h. On the average, less than 100 VSV particles could be fused per one spheroplast. Similar data were obtained with Semliki Forest virus.     

Conformational changes in Sindbis virus envelope proteins accompanying exposure to low pH.

The attachment of high multiplicities of Sindbis virus to tissue-cultured cells followed by brief treatment at low pH has been shown to produce cell fusion (fusion from without). In this report, experiments to determine the effects of low pH on the physical and biological properties of Sindbis virus are described. Exposure of purified Sindbis virions to mildly acidic conditions resulted in a rapid and irreversible alteration in particle density and sedimentation characteristics, followed by a slower loss of infectivity. Infectivity was not restored by a return to neutral pH; rather, the loss of virus infectivity seemed to be initiated by exposure to low pH but continued at neutral pH. The formation of a virus-cell complex in which virions were attached to the cell surface protected the particles from low-pH inactivation, although low pH could still expose virus functions responsible for cell fusion. Low pH was found to induce a conformational change in the E2 polypeptide of the intact virion. These results are discussed with respect to the process of Sindbis virus infection of tissue-cultured cells.     

Pressure-induced fusogenic conformation of vesicular stomatitis virus glycoprotein

Vesicular stomatitis virus (VSV) is composed of a ribonucleoprotein core surrounded by a lipid envelope presenting an integral glycoprotein (G). The homotrimeric VSV G protein exhibits a membrane fusion activity that can be elicited by low pH. The fusion event is crucial to entry into the cell and disassembly followed by viral replication. To understand the conformational changes involved in this process, the effects of high hydrostatic pressure and urea on VSV particles and isolated G protein were investigated. With pressures up to 3.0 kbar VSV particles were converted into the fusogenic conformation, as measured by a fusion assay and by the binding of bis-ANS. The magnitude of the changes was similar to that promoted by lowering the pH. To further understand the relationship between stability and conversion into the fusion-active states, the stability of the G protein was tested against urea and high pressure. High urea produced a large red shift in the tryptophan fluorescence of G protein whereas pressure promoted a smaller change. Pressure induced equal fluorescence changes in isolated G protein and virions, indicating that virus inactivation induced by pressure is due to changes in the G protein. Fluorescence microscopy showed that pressurized particles were capable of fusing with the cell membrane without causing infection. We propose that pressure elicits a conformational change in the G protein, which maintains the fusion properties but suppresses the entry of the virus by endocytosis. Binding of bis-ANS indicates the presence of hydrophobic cavities in the G protein. Pressure also caused an increase in light scattering of VSV G protein, reinforcing the hypothesis that high pressure elicits the fusogenic activity of VSV G protein. This "fusion-intermediate state" induced by pressure has minor changes in secondary structure and is likely the cause of nonproductive infections.     

Fusion of Sendai virus with human HL-60 and CEM cells: different kinetics of fusion for two isolates.

The kinetics of fusion of Sendai virus (Z strain) with the human promyelocytic leukemia cell line HL-60, and the human T lymphocytic leukemia cell line CEM was investigated. Fusion was monitored by fluorescence dequenching of octadecylrhodamine (R-18) incorporated in the viral membrane. For one virus isolate (Z/G), the overall rate of fusion (at 37 degrees C) increased as the pH was lowered, reaching a maximum at about pH 5, the lowest pH tested. For another isolate (Z/SF) the rate and extent of fusion were lower at pH 5 than at neutral pH. Lowering the pH from neutral to 5 after several minutes of incubation of either isolate with HL-60 cells resulted in an enhanced rate of fluorescence dequenching. Nevertheless, experiments utilizing NH4Cl indicated that fusion of the virus with cells was not enhanced by the mildly acidic pH of the endosome lumen. Analysis of the kinetics of fusion by means of a mass action model resulted in good simulation and predictions for the time-course of fusion. For the isolate which showed maximal fusogenic activity at pH 5, the rate constant of fusion (approx. 0.1 s-1) at neutral pH was in the range found previously for virus-liposome fusion, whereas the rate constant of adhesion was close to the upper limit for diffusion-controlled processes (1.4.10(10) M-1 s-1). However, for the other isolate (Z/SF) the rate constant of fusion at neutral pH was very small (less than 0.01 s-1), whereas the rate constant of adhesion was larger (greater than or equal to 2.10(10) M-1 s-1). Lowering the temperature decreased the fusion rate. Experiments involving competition with excess unlabeled virions indicated that not all binding sites for Sendai virus on HL-60 cells are fusion sites. The virus fusion activity towards HL-60 cells at neutral pH was not altered significantly by pre-incubation of the virus at pH 5 or 9, in contrast to earlier observations with liposomes and erythrocyte ghosts, or results based on erythrocyte hemolysis or cell-cell fusion.     

Influenza virus-model membrane interaction. A morphological approach using modern cryotechniques

The membrane fusion activity of influenza virus was characterized morphologically using a model system composed of a highly purified influenza B virus suspension and ganglioside-containing zwitterionic liposomes. Electron microscopical analysis was performed after a combination of fast-freezing with either freeze-fracture or freeze-substitution-thin sectioning, ensuring maximal time resolution and avoiding preparation artifacts. In a parallel fluorescence 'lipid mixing' fusion assay, influenza virus-membrane fusion was characterized biochemically. Biochemical and morphological data are in full agreement, indicating negligible membrane fusion activity at neutral pH and high fusion activity at low pH. The freeze-fracture morphology strongly suggests a local point contact between viral and liposomal membrane at neutral pH, and a local point fusion mechanism for influenza virus-membrane fusion upon lowering of the pH. Fusion is followed by lipid mixing, lateral diffusion of viral spike proteins and exposure of viral contents at the inner liposomal surface.     

Adaptation of alphaviruses to heparan sulfate: interaction of Sindbis and Semliki forest viruses with liposomes containing lipid-conjugated heparin

Passage of Sindbis virus (SIN) in BHK-21 cells has been shown to select for virus mutants with high affinity for the glycosaminoglycan heparan sulfate (HS). Three loci in the viral spike protein E2 (E2:1, E2:70, and E2:114) have been identified that mutate during adaptation and independently confer on the virus the ability to bind to cell surface HS (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). In this study, we used HS-adapted SIN mutants to evaluate a new model system involving target liposomes containing lipid-conjugated heparin (HepPE) as an HS receptor analog for the virus. HS-adapted SIN, but not nonadapted wild-type SIN TR339, interacted efficiently with HepPE-containing liposomes at neutral pH. Binding was competitively inhibited by soluble heparin. Despite the efficient binding of HS-adapted SIN to HepPE-containing liposomes at neutral pH, there was no fusion under these conditions. Fusion did occur, however, at low pH, consistent with cellular entry of the virus via acidic endosomes. At low pH, wild-type or HS-adapted SIN underwent fusion with liposomes with or without HepPE with similar kinetics, suggesting that interaction with the HS receptor analog at neutral pH has little influence on subsequent fusion of SIN at low pH. Finally, Semliki Forest virus (SFV), passaged frequently on BHK-21 cells, also interacted efficiently with HepPE-containing liposomes, indicating that SFV, like other alphaviruses, readily adapts to cell surface HS. In conclusion, the liposomal model system presented in this paper may serve as a novel tool for the study of receptor interactions and membrane fusion properties of HS-interacting enveloped viruses.     

pH-dependent membrane fusion activity of a synthetic twenty amino acid peptide with the same sequence as that of the hydrophobic segment of influenza virus hemagglutinin

A twenty amino acid hydrophobic peptide with the same sequence as that of the HA2 N-terminal segment of influenza virus hemagglutinin was synthesized and studied as to its fusion activity. The peptide caused rapid and efficient fusion of egg yolk phosphatidylcholine sonicated vesicles at acidic pH but not at neutral pH. The threshold pH was ca. 6.2 and the maximum fusion occurred at pH 4.8, the half-maximal pH for fusion being 5.6. The pH dependence was similar to that of the parent virus. The fusion efficiency was dependent on the ration of lipid to peptide, increasing with decreasing ratio. The fusion can be rapidly switched on and off by adjusting the pH, to the acidic side and neutral, respectively. The peptide with an acetylated or succinylated N-terminus also showed low pH-induced fusion activity but the pH range was shifted by ca. 1 unit to the acidic side. The results indicate that the HA2 hydrophobic segment in the virus fusion protein is directly involved in the fusion reaction and protonation of the acidic residues in the segment is required for the activity.     

Cell surface expression of fusogenic vesicular stomatitis virus G protein from cloned cDNA.

Vesicular stomatitis virus (VSV) enters the host cell by the receptor-mediated endocytotic pathway. This brings the virus particle into acidic vesicles inside the cell where infection occurs through a fusion event between the viral and the host vesicle membrane. In this work we have shown that the VSV glycoprotein (G) carries the fusion activity of this virus. The G protein was expressed on the surface of baby hamster kidney 21 cells from cloned cDNA which had been engineered into an expression vector and introduced into cell nuclei with the aid of a glass microneedle. A short (60 s) treatment with acid (pH less than or equal to 6.0) medium induced fusion of cells having G protein on their surface. For efficient G protein expression and cell-cell fusion we had to trim the 5' end of the G cDNA and to use as promoter the long terminal repeat of the mouse Moloney sarcoma virus.     

Pyrene phospholipid as a biological fluorescent probe for studying fusion of virus membrane with liposomes

We are using fluorescent endogenous phospholipids in virus membranes to study the factors that promote fusion on interaction with receptor membranes. To this end, vesicular stomatitis virus (VSV) grown in baby hamster kidney (BHK-21) cells was biologically labeled with fluorescent lipids, primarily phosphatidylcholine and phosphatidylethanolamine, derived from pyrene fatty acids. The pyrene lipids present in the virions showed a fluorescence spectrum typical of pyrene with an intense monomer and a broad excimer. Interaction of pyrene lipid labeled VSV with serum lipoproteins led to a spontaneous fast transfer of the small amount of pyrene fatty acids present in the envelope (t1/2 less than or equal to 7 min), followed by a considerably slower transfer of pyrene phospholipids from the membrane of the virions (t1/2 greater than or equal to 12 h). Incubation of pyrene phospholipid labeled VSV with phosphatidylserine small unilamellar vesicles resulted in fusion at low pH (pH 5.0) as measured by the change in the excimer/monomer fluorescence intensity ratio. Fusion kinetics was rapid, reaching a plateau after 4 min at pH 5.0 and 37 degrees C. Only negligible fusion was noted at neutral pH or at 4 degrees C. Fully infectious virions labeled biologically with fluorescent lipids provide a useful tool for studying mechanisms of cell-virus interactions and neutralization of viral infectivity by specific monoclonal antibodies reactive with viral membrane glycoprotein.