PubMed Health⌕ Search

Biomedical subjects

J Wilschut

Publications and source records attributed to J Wilschut.

At least 73 records · Page 4Linked to original sources
PubMed1991

Interaction of non-enveloped plant viruses and their viral coat proteins with phospholipid vesicles.

The interaction of the non-enveloped plant viruses TMV (rod-shaped) and CCMV (spherical) and of their coat proteins in several well-defined aggregation states, with artificial membranes was investigated to study the early stages of the cellular infection process. Information about the separate steps in the interaction mechanisms was obtained by employing three assays, performed as a function of vesicle size, net membrane charge, pH and ionic strength. The assays allow to discriminate between aggregation of vesicles (turbidity assay) and membrane destabilization (vesicle leakage assay and lipid mixing assay). The aggregation of the vesicles is a result of electrostatic interactions between the viral material and vesicles surface (cross-linking), while the destabilization of the membrane is a result of penetration or bilayer disruption by hydrophobic protein domains. TMV virions and its coat protein, and CCMV virions, due to their net negative charge, predominantly interact with positively charged membranes. The coat protein of CCMV was found to interact with negatively charged membranes, an interaction that can be assigned to its basical N-terminal sequence. Changing the aggregational state of the viral coat proteins yielded most significant interactions in case of TMV coat protein aggregated in the disk form and CCMV coat protein aggregated in empty capsids with oppositely charged membranes. These protein aggregates are found to be the best compromise between efficiency (capacity of the protein to bridge vesicles and destabilize their membranes) and concentration of protein aggregates. The results are discussed with respect to previously proposed biological models of the early stages of plant virus infection.

Capsid↗
PubMed1989

Intervesicular exchange of lipids with weak acid and weak base characteristics: influence of transmembrane pH gradients.

Transmembrane pH gradients have previously been shown to induce an asymmetric transmembrane distribution of simple lipids that exhibit weak acid or basic characteristics (Hope, M.J. and Cullis, P.R. (1987) J. Biol. Chem. 262, 4360-4366). In the present study we have examined the influence of proton gradients on the inter-vesicular exchange of stearylamine and oleic acid. We show that vesicles containing stearylamine immediately aggregate with vesicles containing phosphatidylserine and that disaggregation occurs subsequently as stearylamine equilibrates between the two vesicle populations. Despite visible flocculation during the aggregation phase, vesicle integrity is maintained. Stearylamine is the only lipid to exchange, fusion does not occur and vesicles are able to maintain a proton gradient. When stearylamine is sequestered to the inner monolayer in response to a transmembrane pH gradient (inside acidic) aggregation is not observed and diffusion of stearylamine to acceptor vesicles is greatly reduced. The ability of delta pH-dependent lipid asymmetry to modulate lipid exchange is also demonstrated for fatty acids. Oleic acid can be induced to transfer from one population of vesicles to another by maintaining a basic interior pH in the acceptor vesicles. Moreover, it is shown that the same acceptor vesicles are capable of depleting serum albumin of bound fatty acid. These results are discussed with respect to the mechanism and modulation of lipid flow between membranes both in vitro and in vivo.

Acids↗
PubMed1989

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)

Animals↗
PubMed1988

A synthetic peptide corresponding to the C-terminal 25 residues of phage MS2 coded lysis protein dissipates the protonmotive force in Escherichia coli membrane vesicles by generating hydrophilic pores.

The RNA phage MS2 encodes a protein, 75 amino acids long, that is necessary and sufficient for lysis of the host cell. DNA deletion analysis has shown that the lytic activity is confined to the C-terminal half of the protein. We have examined the effects of a synthetic peptide, covering the C-terminal 25 amino acids of the lysis protein, on the electrochemical potential, generated in Escherichia coli membrane vesicles and in liposomes reconstituted with cytochrome c oxidase. In all cases the peptide dissipates the electrochemical potential. The peptide also induces the release of carboxyfluorescein (376 daltons), but not of inuline (5500 daltons), from protein-free liposomes. The phenomena are observed at a lipid to peptide molar ratio of approximately 100:1. The possible connection between the dissipation of the proton-motive force and bacteriolysis is discussed.

Amino Acid Sequence↗
PubMed1987

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.

Cell Membrane↗
PubMed1987

Fusion of influenza virus in an intracellular acidic compartment measured by fluorescence dequenching.

The fusion of influenza virus with cultured cells has been investigated. The virus was labelled with the fluorescent probe octadecyl rhodamine B and fusion was monitored as fluorescence dequenching due to dilution of the probe from the viral into a cellular target membrane. Fusion with the plasma membrane does not occur, unless the extracellular pH is temporarily lowered. At neutral pH fusion occurs only after a lag phase of 10-15 min, the time required for virus internalization, and the reaction is inhibited by NH4Cl, indicating that it takes place in an intracellular acidic compartment, most likely the endosome. This suggests that influenza virus infects cells via the endocytic pathway.

Ammonium Chloride↗
PubMed1987

Ca2+-induced fusion of large unilamellar phosphatidylserine/cholesterol vesicles.

The effect of cholesterol on the Ca2+-induced aggregation and fusion of large unilamellar phosphatidylserine (PS) vesicles has been investigated. Mixing of aqueous vesicle contents was followed continuously with the Tb/dipicolinate assay, while the dissociation of pre-encapsulated Tb/dipicolinate complex was taken as a measure of the release of vesicle contents. Vesicles consisting of pure PS or PS/cholesterol mixtures at molar ratios of 4:1, 2:1 and 1:1 were employed at three different lipid concentrations, each at four different Ca2+ concentrations. The results could be well simulated in terms of a mass-action kinetic model, providing separately the rate constants of vesicle aggregation, c11, and of the fusion reaction itself, f11. In the analyses the possibility of deaggregation of aggregated vesicles was considered explicitly. Values of both c11 and f11 increase steeply with the Ca2+ concentration increasing from 2 to 5 mM. With increasing cholesterol content of the vesicles the value of c11 decreases, while the rate of the actual fusion reaction, f11, increases. Remarkably, the effect of cholesterol on both aggregation and fusion is quite moderate. The presence of cholesterol in the vesicle bilayer does not affect the leakage of vesicle contents during fusion.

Calcium↗
PubMed1987

Functional reconstitution of influenza virus envelopes.

We have examined several procedures for the reconstitution of influenza virus envelopes, based on detergent removal from solubilized viral membranes. With octylglucoside, no functionally active virosomes are formed, irrespective of the rate of detergent removal: in the final preparation the viral spike proteins appear predominantly as rosettes. Protein incorporation in reconstituted vesicles is improved when a method based on reverse-phase evaporation of octylglucoside-solubilized viral membranes in an ether/water system is employed. However, the resulting vesicles do not fuse with biological membranes, but exhibit only a non-physiological fusion reaction with negatively charged liposomes. Functional reconstitution of viral envelopes is achieved after solubilization with octaethyleneglycol mono(n-dodecyl)ether (C12E8), and subsequent detergent removal with Bio-Beads SM-2. The spike protein molecules are quantitatively incorporated in a single population of virosomes of uniform buoyant density and appear on both sides of the membrane. The virosomes display hemagglutination activity and a strictly pH-dependent hemolytic activity. The virosomes fuse with erythrocyte ghosts, as revealed by a fluorescence resonance energy transfer assay. The rate and the pH dependence of fusion are essentially the same as those of the intact virus. The virosomes also fuse with cultured cells, either at the level of the endosomal membrane or directly with the cellular plasma membrane upon a brief exposure to low pH.

Animals↗
PubMed1986

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

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

Animals↗
PubMed1986

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.

Cold Temperature↗
PubMed1986

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.

Animals↗
PubMed1985

Ca2+-induced fusion of phosphatidylserine vesicles: mass action kinetic analysis of membrane lipid mixing and aqueous contents mixing.

We have investigated the initial kinetics of Ca2+-induced aggregation and fusion of phosphatidylserine large unilamellar vesicles at 3, 5 and 10 mM Ca2+ and 15, 25 and 35 degrees C, utilizing the Tb/dipicolinate (Tb/DPA) assay for mixing of aqueous vesicle contents and a resonance energy transfer (RET) assay for mixing of bilayer lipids. Separate rate constants for vesicle aggregation as well as deaggregation and for the fusion reaction itself were determined by analysis of the data in terms of a mass action kinetic model. At 15 degrees C the aggregation rate constants for either assay are the same, indicating that at this temperature all vesicle aggregation events that result in lipid mixing lead to mixing of aqueous contents as well. By contrast, at 35 degrees C the RET aggregation rate constants are higher than the Tb/DPA aggregation rate constants, indicating a significant frequency of reversible vesicle aggregation events that do result in mixing of bilayer lipids, but not in mixing of aqueous vesicle contents. In any conditions, the RET fusion rate constants are considerably higher than the Tb/DPA fusion rate constants, demonstrating the higher tendency of the vesicles, once aggregated, to mix lipids than to mix aqueous contents. This possibly reflects the formation of an intermediate fusion structure. With increasing Ca2+ concentrations the RET and the Tb/DPA fusion rate constants increase in parallel with the respective aggregation rate constants. This suggests that fusion susceptibility is conferred on the vesicles during the process of vesicle aggregation and not solely as a result of the interaction of Ca2+ with isolated vesicles. Aggregation of the vesicles in the presence of Mg2+ produces neither mixing of aqueous vesicle contents nor mixing of bilayer lipids.

Animals↗
PubMed1985

Low pH-induced fusion of liposomes with membrane vesicles derived from Bacillus subtilis.

We have investigated the pH-dependent interaction between large unilamellar phospholipid vesicles (liposomes) and membrane vesicles derived from Bacillus subtilis, utilizing a fluorescent assay based on resonance energy transfer (RET) (Struck, D. K., Hoekstra, D., and Pagano, R. E. (1981) Biochemistry 20, 4093-4099). Efficient interaction occurs only with negatively charged liposomes, containing cardiolipin or phosphatidylserine, as revealed by the dilution of the RET probes from the liposomal bilayer into the bacterial membrane. The initial rate of fluorophore dilution increases steeply with decreasing pH. The interaction involves a process of membrane fusion, as indicated by the proportional transfer of cholesteryl-[1-14C]oleate, 14C-labeled egg PC, and the RET probes from the liposomes to the bacterial vesicles, the formation of interaction products with an intermediate buoyant density, and the appearance of colloidal gold, initially encapsulated in the liposomes, in the internal volume of fused structures as revealed by thin-section electron microscopy. Treatment of B. subtilis vesicles with trypsin strongly inhibits the fusion reaction, indicating the protein dependence of the process. Vesicles derived from Streptococcus cremoris or from the inner membrane of Escherichia coli also show low pH-dependent fusion with liposomes. The fusion process described in this paper may well be of considerable importance to studies on the mechanisms of membrane fusion and to studies on the structure and function of bacterial membranes. In addition, the fusion reaction could be utilized to deliver foreign substances into bacterial protoplasts.

Bacillus subtilis↗
PubMed1985

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)

Erythrocyte Membrane↗
PubMed1985

Kinetics of Ca2+-induced fusion of cardiolipin-phosphatidylcholine vesicles: correlation between vesicle aggregation, bilayer destabilization, and fusion.

We have investigated the kinetics of Ca2+-induced aggregation and fusion of large unilamellar vesicles composed of an equimolar mixture of bovine heart cardiolipin and dioleoylphosphatidylcholine. Mixing of bilayer lipids was monitored with an assay based on resonance energy transfer (RET) and mixing of aqueous vesicle contents with the Tb/dipicolinate assay. The results obtained with either assay were analyzed in terms of a mass action kinetic model, providing separate rate constants for vesicle aggregation and for the fusion reaction proper. At different Ca2+ concentrations, either at 25 degrees C or at 37 degrees C, aggregation rate constants derived from the data obtained with the RET assay were the same as those derived from the Tb/dipicolinate data, indicating that mixing of bilayer lipids occurred only during vesicle aggregation events that resulted in mixing of aqueous contents as well. At 25 degrees C, identical fusion rate constants were obtained with either assay, indicating that at this temperature the probability of lipid mixing and that of aqueous contents mixing, occurring after vesicle aggregation, were the same. The fusion rate constants for the RET assay increased more steeply with increasing temperature than the fusion rate constants derived from the Tb/dipicolinate data. As a result, at 37 degrees C the tendency of the vesicles, after aggregation, to mix lipids was slightly higher than their tendency to mix aqueous contents. The aggregation rate constants increased steeply with Ca2+ concentrations increasing in a narrow range (9.5-11 mM), indicating that, in addition to a Ca2+-dependent charge neutralization on the vesicle surface, structural changes in the lipid bilayer are involved in the aggregation process.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗