Peptide inhibitors of enveloped virus infection inhibit phospholipid vesicle fusion and Sendai virus fusion with phospholipid vesicles

Small hydrophobic peptides that are capable of inhibiting Sendai virus infection of cells (Richardson, C. D., Scheid, A., and Choppin, P. W. (1980) Virology 105, 205-222) are also capable of inhibiting membrane fusion in a pure lipid vesicle system. Large unilamellar vesicles of N-methyl dioleoylphosphatidylethanolamine containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid and/or p-xylene bis (pyridinium bromide) were formed by extrusion. Vesicle fusion (contents mixing) and leakage were then monitored with the 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylene bis(pyridinium bromide) fluorescence assay. Sendai virus fusion with lipid vesicles was measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride, a lipid mixing assay for fusion. The efficiency with which the peptides carbobenzoxy-D-Phe-L-PheGly, carbobenzoxy-L-Phe-L-Tyr, and carbobenz-oxy-Gly-L-Phe inhibit fusion of N-methyl dioleoyl-phosphatidylethanolamine large unilamellar vesicles directly paralleled their previously known effectiveness in blocking virus infectivity of cultured cells. In addition, above a certain concentration threshold, the inhibitory peptides decreased the initial rate of leakage from lipid vesicles. The inhibition by these peptides of virus-vesicle fusion followed the same order of potency as for vesicle-vesicle fusion. The observation of the same relative potency of these peptides toward inhibition of virus-cell infection, and virus-vesicle and vesicle-vesicle membrane fusion suggested that these peptides inhibited virus-cell infection by inhibiting the ability of the virus to fuse with the cell. Furthermore, these results suggest that the mechanism of inhibition of all three fusion events may have steps in common.     

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)     

Energy transfer measurements of fusion between Sendai virus and vesicles corrected for decreased absorption of acceptor probe

The fusion of Sendai virus at pH 4-7 with artificial lipid vesicles composed of phosphatidylserine or phosphatidylcholine was quantified by measuring fluorescence energy transfer from N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phosphatidylethanolamine to N-(lissamine-rhodamine-B-sulfonyl)-phosphatidylethanolamine in the target membranes. About 60% of the phosphatidylserine vesicles and virus appeared to fuse at pH 4 and about 100% at pH 5. Fusion was much less under all other conditions. The apparent fusion at pH 4, however, was due to a decrease in absorption of the acceptor probe, instead of dilution of acceptor as a result of fusion of labeled vesicles with unlabeled virus. After correction for this fusion-independent effect of Sendai virus, the extent of fusion was only 4-20% at pH 4 but still 80-100% at pH 5. These findings paralleled the loss of hemagglutinating and hemolytic activities of the virus induced by incubation at pH 4 but not at pH 5. Vesicle-virus hybrids were observed with the electron microscope after incubation at pH 5 but not at pH 7. The assay of membrane fusion by fluorescence energy transfer can be misleading unless correction is made for changes in energy transfer due to fusion-independent effects.     

Fusion of Sendai virus with vesicles of oligomerizable lipids: a microcalorimetric analysis of membrane fusion

Sendai virus fuses efficiently with small and large unilamellar vesicles of the lipid 1,2-di-n-hexadecyloxypropyl-4- (beta-nitrostyryl) phosphate (DHPBNS) at pH 7.4 and 37 degrees C, as shown by lipid mixing assays and electron microscopy. However, fusion is strongly inhibited by oligomerization of the head groups of DHPBNS in the bilayer vesicles. The enthalpy associated with fusion of Sendai virus with DHPBNS vesicles was measured by isothermal titration microcalorimetry, comparing titrations of Sendai virus into (i) solutions of DHPBNS vesicles (which fuse with the virus) and (ii) oligomerized DHPBNS vesicles (which do not fuse with the virus), respectively. The observed heat effect of fusion of Sendai virus with DHPBNS vesicles is strongly dependent on the buffer medium, reflecting a partial charge neutralization of the Sendai F and HN proteins upon insertion into the negatively-charged vesicle membrane. No buffer effect was observed for the titration of Sendai virus into oligomerized DHPBNS vesicles, indicating that inhibition of fusion is a result of inhibition of insertion of the fusion protein into the target membrane. Fusion of Sendai virus with DHPBNS vesicles is endothermic and entropy-driven. The positive enthalpy term is dominated by heat effects resulting from merging of the protein-rich viral envelope with the lipid vesicle bilayers rather than by the fusion of the viral with the vesicle bilayers per se.     

Mass action kinetics of virus-cell aggregation and fusion.

A simple approximate solution for the mass action kinetics of small particles (viruses or vesicles) binding to large particles (cells) and their subsequent fusion has been derived. The solution is evaluated in terms of the measurable fluorescence changes expected when the virus or vesicles are labeled with fluorescent probes, which are diluted into the cellular membrane by fusion. Comparison with numerical integrations shows that the approximate solution is extremely accurate. Analytic simplifications for a variety of special cases of this general problem are also shown.     

Influence of phospholipid asymmetry on fusion between large unilamellar vesicles.

The ability of lipid asymmetry to regulate Ca(2+)-stimulated fusion between large unilamellar vesicles has been investigated. It is shown that for 100-nm-diameter LUVs composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, phosphatidylinositol, and dioleoylphosphatidic acid (DOPC/DOPE/PI/DOPA; 25:60:5:10) rapid and essentially complete fusion is observed by fluorescent resonance energy transfer techniques when Ca2+ (8 mM) is added. Alternatively, for LUVs with the same lipid composition but when DOPA was sequestered to the inner monolayer by incubation in the presence of a pH gradient (interior basic), little or no fusion is observed on addition of Ca2+. It is shown that the extent of Ca(2+)-induced fusion correlates with the amount of exterior DOPA. Further, it is shown that LUVs containing only 2.5 mol % DOPA, but where all the DOPA is in the outer monolayer, can be induced to fuse to the same extent and with the same rate as LUVs containing 5 mol % DOPA. These results strongly support a regulatory role for lipid asymmetry in membrane fusion and indicate that the fusogenic tendencies of lipid bilayers are largely determined by the properties of the monolayers proximate to the fusion interface.     

Asymmetric fusion between phospholipid vesicles and vesicles formed from synthetic di(n-alkyl)phosphates

We have investigated the fusion behavior of a mixed vesicle system consisting of vesicles prepared from the simple synthetic surfactants di(n-dodecyl)phosphate (DDP) or di(n-tetradecyl)phosphate (DTP) and vesicles prepared from the phospholipids phosphatidylserine (PS) or dioleoylphosphatidylcholine (DOPC). Fusion between the vesicles, induced by Ca2+, was determined by a resonance energy transfer assay for lipid mixing, sucrose density gradient analysis, and electron microscopy. We demonstrate that synthetic surfactant vesicles can specifically engage in asymmetric fusion events, provided that the incubation temperature is kept below the gel-liquid crystalline phase-transition temperature (Tc) of the synthetic amphiphile (29 and 48 degrees C for DDP and DTP, respectively) and that the physical state of the target membrane is fluid. Asymmetric fusion of DDP or DTP vesicles was most efficient with PS vesicles, but it also occurred with zwitterionic PC vesicles. In the latter case, fusion proceeded spontaneously, but the process was markedly accelerated upon addition of Ca2+. Furthermore, in contrast to a massive transformation of bilayer into nonbilayer hexagonal HII tubular structures, as occurs upon symmetric Ca(2+)-induced fusion of DDP vesicles, asymmetric fusion with phospholipid bilayers predominantly leads to the formation of larger vesicles. This indicates that both PS and DOPC stabilize the DDP bilayer structure in the fusion product.     

pH-dependent fusion induced by vesicular stomatitis virus glycoprotein reconstituted into phospholipid vesicles

Purified G-protein from vesicular stomatitis virus was reconstituted into egg phosphatidylcholine vesicles by detergent dialysis of octyl glucoside. A homogeneous population of reconstituted vesicles could be obtained, provided the protein to lipid ratio was high (about 0.3 mol % protein) and the detergent removal was slow. The reconstituted vesicles were assayed for fusion activity using electron microscopy and fluorescence energy transfer. The fusion activity mediated by the viral envelope protein was dependent upon pH, temperature, and target membrane lipid composition. Incubation of reconstituted vesicles at low pH with small unilamellar vesicles containing negatively charged lipids resulted in the appearance of large cochleate structures, as shown by electron microscopy using negative stain. This process did not cause leakage of a vesicle-encapsulated aqueous marker. The rate of fusion was pH-dependent with a pK of about 4 and the apparent energy of activation for the fusion was 16 +/- 1 kcal/mol. G-protein-mediated fusion showed a large preference for target membranes which contain phosphatidylserine or phosphatidic acid. Inclusion of 36% cholesterol in any of the lipid compositions had no effect on the rate of fusion. These reconstituted vesicles provide a system to study the mechanism of pH-dependent fusion induced by a viral spike protein.     

Penetration and fusion of phospholipid vesicles by lysozyme

The lysozyme-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles as studied at a wide range of pH is found to correlate well with the binding of this protein to the vesicles. An identical 6000 molecular weight segment of lysozyme at the N-terminal region is found to be protected from tryptic digestion when initially incubated with vesicles at several pH values. Only this segment is labeled by dansyl chloride, which is partitioned into the bilayer. These results suggest the penetration of one segment of lysozyme into the bilayer. Photoactivated labeling of the membrane-penetrating segment of lysozyme with 3-(trifluoromethyl)-3-([125I]iodophenyl)diazirine ([125I]TID) and subsequent identification of the labeled residues by Edman degradation and gamma-ray counting indicate that four amino acids from the N-terminal are located outside the hydrophobic core of the bilayer. Although treatment of the membrane-embedded segment with aminopeptidase failed to cleave any amino acids from the N-terminal, it appears that a loop of lysozyme segment near the N-terminal penetrates into the bilayer at acidic pH. A helical wheel diagram shows that the labeling is done mainly on one surface of the alpha-helix. The penetration kinetics as studied by time-dependent [125I]TID labeling coincide with the fusion kinetics, strongly suggesting that the penetration of the lysozyme segment into the vesicles is the cause of the fusion.     

Melittin induces fusion of unilamellar phospholipid vesicles

Melittin, the soluble lipophilic peptide of bee venom, causes fusion of phospholipid vesicles when vesicle suspensions are heated or cooled through their thermal phase transition. Fusion was detected using a new photochemical method (Morgan, C.G., Hudson, B. and Wolber, P. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 26–30) which monitors lipid mixing. Electron microscopy and gel filtration confirmed that most of the lipid formed large vesicular structures. Fluorescence experiments with a water-soluble, membrane-impermeable complex of terbium (Wilschut, J. and Papahadjopoulos, D. (1979) Nature 281, 690–692) demonstrate that these ionic contents are released during fusion. The large structures formed by melittin-induced fusion are impermeable to these ions and are resistant to further fusion. This is in contrast to the behavior observed for the cationic detergent cetyltrimethylammonium bromide (CETAB). The large size of the vesicles formed, the extreme speed of the fusion event and the appearance of electron microscope images of the vesicles prior to fusion suggest that the mechanism of the fusion process includes a preaggregation step.     

Monovalent cation-induced fusion of acidic phospholipid vesicles

Fusion of small unilamellar vesicles (SUV) consisting of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and phosphatidylglycerol (PG) from egg yolk, dipalmitoylphosphatidylserine (DPPS) and phosphatidylserine (PS) from bovine brain was studied as a function of monovalent cation concentration. Fusion was detected by measuring the changes in the excimer to monomer fluorescence intensity ratio (IE/M) of pyrene-labeled phospholipid analogues upon fusion of the pyrene-labeled and unlabeled vesicles. No fusion was observed from vesicles consisting of DMPC, PS from bovine brain or PG from egg yolk upon addition of NaCl (up to 1 M). However, considerable fusion was evident for vesicles consisting of DMPG or DPPS upon addition of monovalent cations (300 mM to 1 M). Fusion kinetics were fast reaching a plateau after 5 min of addition of cations. The order of efficiency of different monovalent cations to induce the fusion of DMPG vesicles as judged by the changes of the IE/M ratio was Li+ greater than Na+ greater than K+ greater than Cs+. DSC-scan of sonicated DMPG vesicles showed, in the absence of salt, a phase transition at 19.2 degrees C with enthalpy of 1.1 kcal.mol-1. After incubation in the presence of 600 mM NaCl the DSC scan showed a narrow phase transition at 24.1 degrees C with enthalpy of 6.9 kcal.mol-1 and a pronounced pretransition, both supporting that the fusion of the vesicles had occurred in the presence of NaCl. The results indicate that sonicated vesicles consisting of acidic phospholipids with fully saturated fatty acids fuse in the presence of monovalent cations, whereas those containing unsaturated fatty acids do not.     

Abscisic acid enhances aggregation and fusion of phospholipid vesicles

The plant hormone abscisic acid (ABA) is shown to enhance the aggregation and fusion of small unilamellar lipid vesicles composed of 80 mol% dimyristoylphosphatidylcholine (DMPC) and 20 mol% dimyristoylphosphatidylcholine (DMPE). Aggregation and fusion did not occur with single component (100 mol%) DMPC vesicles. Fusion was followed by two fundamentally different techniques, fluorescence resonance energy transfer which monitors intermixing of bilayers and ANTS-DPX which monitors intermixing of the sequestered aqueous interiors. It is suggested that a previously unreported role of ABA may be as a membrane fusagen.     

Lysozyme induced fusion of negatively charged phospholipid vesicles

Lysozyme promotes fusion of negatively charged phospholipid vesicles prepared by ethanolic injection. Vesicle fusion was a leaky process as revealed by the release of encapsulated carboxyfluorescein or Tb-DPA complex. Extensive proteolysis of lysozyme inhibited the fusion process. The fusion process was critically dependent on the medium ionic strength; 100 mM of any salt was sufficient to inhibit totally the fusion activity of the protein. The high efficiency of lysozyme (80% RET) was almost constant in the pH range from 4.0 to 9.0, but it was sharply diminished when the pH of the medium was at the isoelectric point of the protein (pI 11.0). Fusion induced by chemically modified lysozyme, showed that the pH profile changed according to the isoelectric point of the protein derivative. These observations stress the importance of electrostatic interactions in the process of fusion induced by lysozyme.     

Conformation and stability of Sendai virus fusion protein

The conformation and stability of Sendai virus fusion (F) protein were studied by circular dichroism spectroscopy, and the protein predictive models of Chou and Fasman and Robson and Suzuki were used to elucidate the secondary structure of Sendai virus F protein. The F protein conformation is predicted to contain 33% alpha-helix, 53% beta-sheet and 15% beta-turn by the Chou and Fasman model, and 30% alpha-helix, 55% beta-sheet, 9% beta-turn and 7% random coil by the Robson and Suzuki model. C.d. studies of F protein purified in the presence of the non-ionic detergent, n-octylglucoside, indicated the presence of 49% alpha-helix and 31% beta-sheet at pH 7.0, 54% alpha-helix and 28% beta-sheet at pH 9.0 and 50% alpha-helix and 23% beta-sheet at pH 5.4. A small change in conformation of the protein occurred when the pH was titrated from 7.0 to 5.4 and from 7.0 to 9.0 and a more pronounced conformational change occurred when the pH was changed from 9.0 to 5.4. The F protein in 0.2% n-octylglucoside was resistant to denaturation by 4 M guanidine hydrochloride, the reducing agent 20 mM mercaptoethanol, and to increases in temperature from 5 to 80 degrees C. Monoclonal anti-F protein antibody showed an increased binding to whole virus when the pH was changed from 7.0 to 9.0. The antibody binding was decreased when the pH was shifted from 9.0 to 5.4 Maximum haemolytic activity was observed with virus that was preincubated at pH 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the fusogenic properties of Sendai virus: kinetics of fusion with erythrocyte membranes

A novel fluorescence assay [Hoekstra, D., De Boer, T., Klappe, K., & Wilschut, J. (1984) Biochemistry 23, 5675-5681] has been used to characterize the fusogenic properties of Sendai virus, using erythrocyte ghosts and liposomes as target membranes. This assay involves the incorporation of the "fusion-reporting" probe in the viral membrane, allowing continuous monitoring of the fusion process in a very sensitive manner. Fusion was inhibited upon pretreatment of Sendai virus with trypsin. Low concentrations of the reducing agent dithiothreitol (1 mM) almost completely abolished viral fusion activity, whereas virus binding was reduced by ca. 50%, indicating that the fusogenic properties of Sendai virus are strongly dependent on the integrity of intramolecular disulfide bonds in the fusion (F) protein. Pretreatment of erythrocyte ghosts with nonlabeled Sendai virus inhibited subsequent fusion of fluorophore-labeled virus irrespective of the removal of nonbound virus, thus suggesting that the initial binding of the virus to the target membrane is largely irreversible. As a function of pH, Sendai virus displayed optimal fusion activity around pH 7.5-8.0. Preincubation of the virus at suboptimal pH values resulted in an irreversible diminishment of its fusion capacity. Since virus binding was not affected by the pH, the results are consistent with a pH-induced irreversible conformational change in the molecular structure of the F protein, occurring under mild acidic and alkaline conditions. In contrast to virus binding, fusion appeared to be strongly dependent on temperature, increasing ca. 25-fold when the temperature was raised from 23 to 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fusion of influenza virus with cardiolipin liposomes at low pH: mass action analysis of kinetics and extent

The kinetics and extent of low pH induced fusion between influenza virus and large unilamellar cardiolipin liposomes were investigated with an assay for lipid mixing based on fluorescence resonance energy transfer. 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-liposome adhesion or aggregation 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 initial aggregation step and for the subsequent fusion reaction. Analytical solutions were found for several limiting cases. Deaggregation of virus--liposome aggregates was explicitly taken into account but was found to be a minor effect under the conditions studied. The calculations gave good simulations and predictions for the kinetics and extent of fusion at different virus/liposome concentrations and ratios. At pH 5.0 and 37 degrees C, very high rate constants for aggregation and fusion were obtained, and essentially all of the virus particles were involved in the fusion process. Experiments at different virus/liposome ratios showed that fusion products may consist of a single virus particle and several liposomes but not of a single liposome and several virus particles. At pH 6.0, the rate constant for aggregation was the same as at pH 5.0, but the rate constant of fusion was about 5-fold lower, and only 25-40% of the virus particles were capable of fusing with the liposomes. The analytical procedure presented enables elucidation of the crucial role of the composition of target membrane vesicles in the initial adhesion and subsequent fusion of the virus at various pH values.     

Effects of apolipoprotein structure on the kinetics of apolipoprotein transfer between phospholipid vesicles

The kinetics and mechanism of transfer of 14C-labeled human apolipoproteins A-I, A-II and C-III1 between small unilamellar vesicles (SUV) have been investigated. Ion exchange chromatography was used for rapid separation of negatively charged egg phosphatidylcholine (PC)/dicetyl phosphate donor SUV containing bound 14C-labeled apoprotein from neutral egg PC acceptor SUV present in 10-fold molar excess. The transfer kinetics of these apolipoproteins at 37 degrees C are consistent with the existence of fast, slow and apparently 'nontransferrable' pools of SUV-associated lipoprotein: the transfers from these pools occur on timescales of seconds (or less), minutes/hours and days/weeks, respectively. For donor SUV containing about 15 apoprotein molecules per vesicle and at a donor SUV concentration of 0.15 mg phospholipid/ml incubation mixture, the sizes of the fast kinetic pools for apolipoproteins A-I, A-II and C-III1 associated with donor SUV are 2, 10 and 11%, respectively. The sizes of the slow kinetic pools for these apolipoproteins are 16, 71 and 50%, respectively. The transfer of the various apolipoproteins from the slow kinetic pool follows first order kinetics and the half-time (t1/2) values are in the order: apo C-III1 less than apo A-I. Increasing the number of apoprotein molecules per donor SUV enlarges the size of the fast pool and increases the t1/2 of slow transfer. The differences in the kinetics of apolipoprotein transfer between SUV are consequences of the variations in the primary and secondary structures of the apolipoprotein molecules. The slow transfer of apoprotein molecules is mediated by collisions between donor and acceptor SUV; the rate is dependent on the apoprotein molecular weight with larger molecules transferring more slowly from donor SUV containing the same lipid/protein molar ratio. The hydrophobicity of the apoprotein molecule is also significant with less hydrophobic molecules transferring more rapidly. Further understanding of the differences in the kinetics of transfer of these apolipoproteins will require more knowledge of their secondary and tertiary structures.