Sendai Virus Fusion Activity as Modulated by Target Membrane Components

We have studied the differences between erythrocytes and erythrocyte ghosts as target membranes for the study of Sendai virus fusion activity. Fusion was monitored continuously by fluorescence dequenching of R18-labeled virus. Experiments were carried out either with or without virus/target membrane prebinding. When Sendai virus was added directly to a erythrocyte/erythrocyte ghost suspension, fusion was always lower than that obtained when experiments were carried out with virus already bound to the erythrocyte/erythrocyte ghost in the cold, since with virus prebinding fusion can be triggered more rapidly. Although virus binding to both erythrocytes and erythrocyte ghosts was similar, fusion activity was much more pronounced when erythrocyte ghosts were used as target membranes. These observations indicate that intact erythrocytes and erythrocyte ghosts are not equivalent as target membranes for the study of Sendai virus fusion activity. Fusion of Sendai virus with both target membranes was inhibited when erythrocytes or erythrocyte ghosts were pretreated with proteinase K, suggesting a role of target membrane proteins in this process. Treatment of both target membranes with neuraminidase, which removes sialic acid residues (the biological receptors for Sendai virus) greatly reduced viral binding. Interestingly, this treatment had no significant effect on the fusion reaction itself.     

Determination of electric field threshold for electrofusion of erythrocyte ghosts. Comparison of pulse-first and contact-first protocols.

Rabbit erythrocyte ghosts were fused by means of electric pulses to determine the electrofusion thresholds for these membranes. Two protocols were used to investigate fusion events: contact-first, and pulse-first. Electrical capacitance discharge (CD) pulses were used to induce fusion. Plots of fusion yield vs peak field strength yielded curves that intersected the field strength axis at positive values (pseudothresholds) which depended on the protocol and decay half time of the pulses. It was found that plots of pseudothreshold vs reciprocal half time were linear for each protocol; when extrapolated to reciprocal half time = 0 (i.e., t----infinity), these lines intersected the ordinate at values of the field strength considered to be the true electrofusion thresholds. In this fashion, the contact-first protocol gave an electrofusion threshold of 46.5 +/- 11.5 V/mm for hemoglobin-free ghosts (white ghosts) and 40.9 +/- 8.8 V/mm for ghosts with fractional hemoglobin (pink ghosts), while the threshold for the pulse-first protocol applied to pink ghosts was determined to be 93.4 +/- 11.0 V/mm. Although the thresholds depended on the electrofusion protocol, plots of critical field strength vs reciprocal time had the same slopes, i.e., approximately 24 Vs/mm. The results suggest that the fusogenic state induced by an electric pulse in either the contact-first protocol or the pulse-first protocol (long-lived fusogenic state) may in fact share a common mechanism, if the two states are not actually identical.     

Erythrocyte-mediated microinjection of horseradish peroxidase into BHK cells

In order to establish the distribution with time of proteins microinjected into mammalian cells, horseradish peroxidase (HRP) was microinjected into baby hamster kidney (BHK) cells using chicken erythrocyte ghosts. At time intervals following initiation of fusion between ghosts and target cells, samples were fixed with aldehydes and the peroxidase visualized by reaction with diaminobenzidine and viewing by light and electron microscopy. At 10 min, the reaction product was observed within the cytoplasm of 60% of the microinjected cells, but was excluded from the nucleus and membranous organelles. In the other 40% of microinjected cells, the reaction product was also observed within the nucleus. At 30 min, the reaction product was observed to be evenly distributed throughout the cell, including the nucleus but excluded from organelles. By 6 h, the reaction product was present almost exclusively within the nucleus of 63% of microinjected cells. At all time points, 20–30% of the erythrocytes ghosts appear to have been taken up by cells by phagocytosis rather than fusion, as evidenced by the presence of peroxidase reaction product within intact and fragmented erythrocyte ghosts in the cytoplasm of target cells. Cells incubated with a lanthanum solution following fusion excluded this electron dense tracer, indicating that the cytoplasmic compartment is not opened during exposure to polyethylene glycol.     

Virus-induced fusion of human erythrocyte ghosts I. Effects of macromolecules on the final stages of the fusion reaction

Human erythrocyte ghosts prepared by hypotonic hemolysis can be fused by Sendai virus, provided that certain macromolecules (bovine serum albumin, dextran and others) are sequestered in the ghosts. Since fusion of the ghosts is dependent on intactness of the F(fusion)-glycoprotein of the virion, and since the other requirements for this reaction are also similar to those for the Sendai virus-induced fusion of intact erythrocytes, this system can be used as a model for the Sendai virus-induced cell fusion reaction. Sequestered macromolecules seem to be required for rounding of locally fused ghosts. Under low osmotic swelling conditions, such as use of ghosts sealed without macromolecules or using bovine serum albumin-loaded ghosts sealed in the presence of external macromolecules, no apparently complete cell fusion (large spherical polyghost formation) could be observed. Even under these conditions, however, occurence of local cell fusion could be demonstrated either by transfer of fluorescent-labeled albumin from one ghost to an other, or by observation of polyghost formation after osmotic swelling in the cold. Thus, final stages of the fusion reaction can be divided into local cell-cell fusion which could not be observed by phase-contrast microscopy, and rounding (i.e. formation of spherical polyghost). For the observation of fusion of ghosts, the last step seems to be important.     

Quantitative introduction of a given macromolecule into cells by fusion with erythrocyte ghosts using a fluorescence activated cell sorter.

FITC-conjugated bovine serum albumin (FITC-BSA) molecules were quantitatively introduced into human erythrocyte ghosts by gradual hemolysis. When the ghosts and L cells were fused with UV-inactivated HVJ (Sendai virus), FITC-BSA was introduced into the cytoplasm of the L cells and fluorescence could be observed inthe cells with a fluorescence microscope. A mixture of L cells and ghosts was introduced into a fluorescence activated cell sorter (FACS), which could separate the mononuclear cells on the basis of their light-scattering profile. Four distinct populations of mononuclear cells were found by fluorescence analysis. These populations were separated from the cell mixture and found to correspond to cells fused with one, two and three ghosts and unfused cells. After separation, the cells from each population could form colonies in culture. As a given macromolecule can be quantitatively introduced into erythrocyte ghosts with the FITC-BSA, after fusion of these ghosts with cells, this sorting method is useful for separating cells containing a definite number of macromolecules.     

Extended recombinant bacterial ghost system.

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri-plasma and internal lumen of the ghosts can be fully utilized. This extended recombinant ghost system represents a new strategy for adjuvant free combination vaccines.     

A quantitative study of ultramicroinjection of macromolecules into animal cells.

Improvements in the technique of ultramicroinjection of macromolecules into animal cells are described. The method is based on the Sendai virus-induced fusion of animal cells with erythrocyte ghosts containing trapped macromolecules. Fusion of hepatoma tissue culture (HTC) cells with ghosts prepared by hemolysis of erythrocytes in the presence of cytochrome C is much more efficient than fusion with ghosts prepared in the presence of bovine serum albumin (BSA) as in previous investigations. La+++ is more fficient in promoting fusion and less toxic to cells than Mn++, which was used previously. Thus in all subsequent experiments, erythrocytes were hemolyzed in the presence of cytochrome C plus other macromolecules to be trapped, and the resultant ghosts fused in the presence of La+++. The percentage of HTC cells which fused with ghosts reached 80% in many experiments. Ghosts containing 125I-BSA were used to measure the number of BSA molecules injected into HTC cells. About 10(6) BSA molecules were injected per fused cell. The overall efficiency of injection was low (about 0.02% of the starting material).     

Fusion of cell ghosts with intact cells in Dictyostelium discoideum: Differential response of opposite mating-type cells

The sexual cycle of Dictyostelium discoideum is initiated by the fusion of cells that are of opposite mating types (e.g. NC4- and HM1-type cells). Cells grown in light on agar plates are not capable of sexual cell fusion, but become capable when cultured in the dark in a liquid medium. Cells in the incapable state are called fusion-incompetent cells, and cells in the latter state, fusion-competent cells. To gain some understanding of the mechanism of cell fusion, cell ghosts prepared by freeze-thawing intact cells were incubated with intact cells. The cell ghosts killed the intact cells by directly fusing with them, the extent of fusion depending on the particular strains employed and the fusion-competency of the intact cells and of the cells from which the cell ghosts had been prepared. A detailed examination revealed that fusion-competent NC4 cells were always more easily killed by cell ghosts than fusion-incompetent NC4 cells. It also became apparent that cell ghosts prepared from fusion-competent NC4 cells killed all cell types far more efficiently than did those prepared from fusion-incompetent NC4 cells. However, fusion-competent and fusion-incompetent HM1 cells were equally sensitive to cell ghosts, and cell ghosts prepared from fusion-competent HM1 cells had the same ability to kill as those prepared from fusion-incompetent HM1 cells. From these findings, it thus appears that opposite mating-type cells have distinct membrane properties related to sexual cell fusion.     

Effect of erythrocyte transbilayer phospholipid distribution on fusion with vesicular stomatitis virus

To identify the specific component(s) in the target membrane involved in fusion of vesicular stomatitis virus (VSV), we examined the interaction of the virus with human erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion was monitored spectrofluorometrically by the octadecylrhodamine dequenching assay. Fusion of VSV with lipid-symmetric erythrocyte ghosts was rapid at 37 degrees C and low pH, whereas little or no fusion was observed with lipid-asymmetric ghosts. Conversion of phosphatidylserine in the lipid-symmetric ghost membrane to phosphatidylethanolamine by means of the enzyme phosphatidylserine decarboxylase did not alter the target membrane's susceptibility to VSV fusion. Spin-labeled phospholipid analogues with phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine headgroups incorporated into the outer leaflet of lipid-asymmetric erythrocytes did not render those membranes fusogenic. Electron spin resonance spectra showed an increased mobility of a phosphatidylcholine spin-label incorporated into the outer leaflet of lipid-symmetric erythrocyte ghosts as compared to that of lipid-asymmetric ghosts. These results indicate that the susceptibility to VSV fusion is not dependent on any particular phospholipid but rather is related to packing characteristics of the target membrane.     

Interaction of Sendai virions with resealed human erythrocyte ghosts. Lateral mobility of the viral glycoproteins in the cell membrane following fusion

Two independent methods demonstrated that resealed human erythrocyte ghosts undergo Sendai virus-mediated cell-cell fusion to a much lower degree (about 4%) than intact erythrocytes, in spite of similar levels of viral envelope-cell fusion in the two preparations. Fluorescence photobleaching recovery (FPR) showed similar lateral mobilities of the viral glycoproteins following fusion with either ghosts or whole erythrocytes. It is suggested that although viral glycoprotein mobilization in the cell membrane is essential for cell-cell fusion, the target cell properties are also important; in the absence of the required cellular parameters, the mobilization may not be a sufficient condition.     

Fusion of cell ghosts with sexually opposite type cells in Dictyostelium discoideum

In the sexual cycle of Dictyostelium discoideum, haploid cells of two opposite mating types, strains HM1 and NC4, acquire fusion-competence under certain conditions, such as suspension culture in the dark, and fuse specifically to form giant zygote cells. Each giant cell engulfs the surrounding cells, gradually increases in size, and finally develops into a macrocyst that is a sexual structure in D. discoideum. Fusion-competent HM1 cells suspended in a solution were frozen and thawed to make cell ghosts. When cell ghosts were introduced into fusion-competent and -incompetent intact NC4 cells, the cell ghosts killed them in a short time, but the fusion-competent cells were killed in preference to the fusion-incompetent cells. This killing occurred through the fusion of the cell ghosts directly to intact cell membranes. Since the fusion was specific, the fusion between ghosts and cells appears to be essentially the same as that between intact cells during the sexual cycle in molecular mechanisms.     

A fluorescence assay for monitoring and analyzing fusion biological membrane vesicles in vitro

A new technique has been developed to study fusion of biological membrane vesicles. Bovine chromaffin granule ghosts (CGG) were loaded with fluorescein isothiocyanate-dextran (FITC-dextran) at self-quenching concentrations. Loaded ghosts were then made to fuse with empty CGG. Fusion was induced by synexin, a protein previously proposed to be involved in exocytosis. The fusion process was monitored by measuring the dequenching of the fluorescence. Dequenching occurred as FITC-dextran was diluted into the increased volume due to fusion with empty ghosts. Spurious signals from leakage or breakage of vesicles were removed by including a specific anti-fluorescein antibody in the reaction medium. This new technique may prove to be of more general use for studying membrane fusion processes in other systems.     

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)     

Gating Movements of Colicin A and Colicin Ia Are Different

The effect of temperature on fusion of Sendai virus with target membranes and mobility of the viral glycoproteins was studied with fluorescence methods. When intact virus was used, the fusion threshold temperature (20–22°C) was not altered regardless of the different types of target membranes. Viral glycoprotein mobility in the intact virus increased with temperature, particularly sharply at the fusion threshold temperature. This effect was suppressed by the presence of erythrocyte ghosts and/or dextran sulfate in the virus suspension. In these cases also, no change in the fusion threshold temperature was observed. On the other hand, reconstituted viral envelopes (virosomes) bearing viral glycoproteins but lacking matrix proteins were capable of fusing with erythrocyte ghosts even at temperatures lower than the fusion threshold temperature and no fusion threshold temperature was observed over the range of 10–40°C. The mobility of viral glycoproteins on virosomes was much greater and virtually temperature-independent. The intact virus treated with an actin-affector, jasplakinolide, reduced the extent of fusion with erythrocyte ghosts and the mobility of viral glycoproteins, while the treatment of virosomes with the same drug did not affect the extent of fusion of virosomes with erythrocyte ghosts and the mobility of the glycoproteins. These results suggest that viral matrix proteins including actins affect viral glycoprotein mobility and may be responsible for the temperature threshold phenomenon observed in Sendai virus fusion.This revised version was published online in August 2005 with a corrected cover date.     

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.     

Fusion and infection of influenza and Sendai viruses as modulated by dextran sulfate: a comparative study

We have directly compared the effect of two types of dextran sulfate with distinct molecular weights (500 kDa and 5 kDa) on the fusion activity and infectivity of both Sendai and influenza viruses, two lipid-enveloped viruses that differ in their routes of entry into target cells. To correlate membrane merging and infectivity MDCK cells were used as targets for the viruses in both approaches. In either case pronounced inhibition of virus–cell interactions by dextran sulfate was only observed at low pH, even though Sendai virus fuses maximally at pH 7.4. Although membrane merging could not be fully abolished, the inhibitory effect was always greater when the higher molecular weight dextran sulfate was used. The presence of this residual fusion activity, that could not be reduced even with high concentrations of agent, suggests that a limited number of binding sites for dextran sulfate may exist on the viral envelopes. The compounds also inhibited fusion of bound virions, and all results could be reproduced using erythrocyte ghosts as target membranes in the fusion assay, instead of MDCK cells. In agreement with these observations only the infectivity of influenza virus (which requires a low pH-dependent step to enter target cells) was affected by dextran sulfate, again the higher molecular weight compound showing a more pronounced inhibitory effect.     

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.     

"Ultramicroinjection" of macromolecules or small particles into animal cells. A new technique based on virus-induced cell fusion.

A new method is described for the introduction of macromolecules and small particles into animal cells. The first step in this procedure is the trapping of particles in ghosts of human erythrocytes. This is achieved by the gradual hemolysis of erythrocytes in the presence of the particles to be trapped. The second step is the Sendai virus-induced fusion of the ghosts containing the particles with cells. By this method, ferritin and latex spheres (diameter 0.1 mum) have been "injected" into cells.     

Kinetics and mechanism of cell membrane electrofusion.

A new quantitative approach to study cell membrane electrofusion has been developed. Erythrocyte ghosts were brought into close contact using dielectrophoresis and then treated with one square or even exponentially decaying fusogenic pulse. Individual fusion events were followed by lateral diffusion of the fluorescent lipid analogue 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) from originally labeled to unlabeled adjacent ghosts. It was found that ghost fusion can be described as a first-order rate process with corresponding rate constants; a true fusion rate constant, k(f), for the square waveform pulse and an effective fusion rate constant, k(ef), for the exponential pulse. Compared with the fusion yield, the fusion rate constants are more fundamental characteristics of the fusion process and have implications for its mechanisms. Values of k(f) for rabbit and human erythrocyte ghosts were obtained at different electric field strength and temperatures. Arrhenius k(f) plots revealed that the activation energy of ghost electrofusion is in the range of 6-10 kT. Measurements were also made with the rabbit erythrocyte ghosts exposed to 42 degrees C for 10 min (to disrupt the spectrin network) or 0.1-1.0 mM uranyl acetate (to stabilize the bilayer lipid matrix of membranes). A correlation between the dependence of the fusion and previously published pore-formation rate constants for all experimental conditions suggests that the cell membrane electrofusion process involve pores formed during reversible electrical breakdown. A statistical analysis of fusion products (a) further supports the idea that electrofusion is a stochastic process and (b) shows that the probability of ghost electrofusion is independent of the presence of Dil as a label as well as the number of fused ghosts.     

Erythrocyte ghost-mediated microinjection into cells in monolayer cultures: a highly efficient and low toxic technique

We describe a technique by which macromolecules can be microinjected into mammalian cells in monolayer cultures. This technique employs erythrocyte ghosts as the vehicle for microinjection, calcium as attachment agent and polyethylene glycol as fusogen. The use of calcium allows a reduction of the time of exposure to polyethylene glycol, and results in a high injection efficiency and a high cell viability when followed by incubation in a buffer free of divalent cations. Injecting over 90% of the cells, a reduction of cell viability is not observed and the mitotic index is never lower than 2.3%. Light and electron microscopy suggest that erythrocyte ghost-cell fusion is only a short event.