Rapid membrane fusion of individual virus particles with supported lipid bilayers

Many enveloped viruses employ low-pH-triggered membrane fusion during cell penetration. Solution-based in vitro assays in which viruses fuse with liposomes have provided much of our current biochemical understanding of low-pH-triggered viral membrane fusion. Here, we extend this in vitro approach by introducing a fluorescence assay using single particle tracking to observe lipid mixing between individual virus particles (influenza or Sindbis) and supported lipid bilayers. Our single-particle experiments reproduce many of the observations of the solution assays. The single-particle approach naturally separates the processes of membrane binding and membrane fusion and therefore allows measurement of details that are not available in the bulk assays. We find that the dynamics of lipid mixing during individual Sindbis fusion events is faster than 30 ms. Although neither virus binds membranes at neutral pH, under acidic conditions, the delay between membrane binding and lipid mixing is less than half a second for nearly all virus-membrane combinations. The delay between binding and lipid mixing lengthened only for Sindbis virus at the lowest pH in a cholesterol-dependent manner, highlighting the complex interaction between lipids, virus proteins, and buffer conditions in membrane fusion.     

Increase in lipid fluidity of cellular membranes induced by adsorption of RNA and DNA virions.

Changes in the dynamic behavior of membrane lipids of mammalian cells induced by adsorption of animal viruses were quantitatively monitored by fluorescence polarization analysis with the aid of the fluorescent probe 1,6-diphenyl 1,3,5-hexatriene embedded in the surface membrane lipid core of intact cells. Adsorption of encephalomyocarditis, West Nile, and polyoma viruses to hamster (baby hamster kidney) and mouse (3T3) cells is accompanied by a rapid and significant increase in the degree of fluidity of membrane lipids of the infected cells. These changes in membrane fluidity, which are virus dose dependent, are inhibited by low temperature and by treatment of the cells before-hand with compounds known to block viral receptors on the cell surface. It is suggested that increase in membrane lipid fluidity, induced by the adsorption of virions, is an early event in the process of cell-virus interactions.     

Fusion of Sindbis virus with model membranes containing phosphatidylethanolamine: implications for protein-induced membrane fusion

The pH-induced fusion of Sindbis virus with model lipid membranes containing phosphatidylethanolamine has been studied using a quantitative fluorescence technique. The headgroup and acyl chain domains of the lipids have been altered systematically to determine their effect on fusion. Unsaturated phosphatidylethanolamines (PE) have been found to promote fusion, either by themselves, or in combination with phosphatidylcholines (PC). Cholesterol added to a mixture of unsaturated PE and PC was also shown to increase the extent of viral fusion. The results of these studies have been interpreted in terms of a tentative model for the molecular aspects of the target membrane which are necessary for viral fusion. In this model, the target membrane must have a sufficiently-sized domain containing poorly hydrated lipids which are capable of existing in a non-bilayer arrangement.     

Microviscosity of togavirus membranes studied by fluorescence depolarization: influence of envelope proteins and the host cell.

The microviscosities of the hydrophobic regions of the membranes of intact Semliki forest and Sindbis viruses grown on BHK-21 cells, of liposomes derived from the extracted viral lipids, and of protease-treated virions were measured by fluorescence depolorization using the fluorescence probe 1, 6-diphenyl-1,3,5-hexatriene. The intact virus membranes were found to have a higher microviscosity than did virus-derived liposomes, indicating the viral envelope proteins contribute to microviscosity. However, protease-treated virus, devoid of protruding spikes but with residual lipophilic peptide tails, was found to have a microviscosity more similar to that of the intact virus than to that of protein-free liposomes. Sindbis virus grown in BHK-21 cells at 37 C had a much higher microviscosity than did Sindbis virus grown on Aedes albopicuts cells at 22 C. Sindbis virus grwon in A. albopictus and BHK-21 cells also gave higher microviscosity values than did the intact host cells. These data indicate that both the virion proteins and the cellular lipids selected during viral growth and maturation contribute to the increased microviscosity of togavirus membranes.     

Membrane lipid fluidity and its effect on the activation energy of membrane-associated enzymes.

1. The fatty acid composition of mitochondrial membranes from sheep and rats was altered by feeding these animals diets which were rich in unsaturated fatty acids. Changes in membrane lipid fluidity resulting from the altered membrane lipid composition were assessed by determining the upper temperature limit of the disorder-order transition (Tf) and the Arrhenius activation energy (Ea) of succinate oxidase. 2. After feeding the unsaturated fatty acid-rich diet to sheep the Ea, in the temperature range above Tf, increased from 8 to 63 kJ . mol-1 while Tf decreased from 32 to 15 degrees C. Rats fed an unsaturated fatty acid-rich diet exhibited an increase in Ea from 17 to 63 kJ . mol-1 and a decrease in Tf from 23 to 4 degrees C. 3. This decrease in Tf was related to an increase in the ratio of linoleic acid to stearic acid in the membrane lipid. Tf was not related to the proportion of unsaturated fatty acids in the membrane lipids, although an increase in unsaturation usually led to a decrease in Tf. 4. The results show that membrane lipid fluidity has a direct influence on the conformation of the active site of some membrane-associated enzymes, with the result that such enzymes display a higher Ea when the membrane lipids are comparatively more fluid. The increase in Ea of membrane-associated enzymes which accompanies changes in the physical state of membrane suggests that some proteins may phase separate with the more fluid lipids at low temperatures.     

Effect of membrane protein on lipid bilayer structure: a spin-label electron spin resonance study of vesicular stomatitis virus.

Spin-label electron spin resonance (ESR) methods have been used to study the structure of the envelope of vesicular stomatitis virus (VSV). The data indicate that the lipid is organized in a bilayer structure. Proteolytic digestion of the glycoproteins which are the spike-like projections on the outer surface of the virus particle increases the fluidity of the lipid bilayer. Since the lipid composition of the virion reflects the composition of the host plasma membrane and the protein composition is determined by the viral genome, VSV was grown in both MDBK and BHK21-F cells to determine the effect of a change in lipid composition on the structure of the lipid bilayer of VSV. The lipid bilayer of the virion was found to be more rigid when derived from MDBK cells than from BHK21-F cells. Studies comparing spin-labeled intact cells and cell membrane fractions suggest that upon labeling the whole cell the spin label probes the plasma membrane. Comparison of spin-labeled VSV particles and their host cells indicates that the lipid bilayer of the plasma membrane is considerably more fluid than that of the virion. These results are discussed in terms of the effect of membrane-associated protein on the structure of the lipid bilayer.     

血红蛋白对人红细胞膜流动性的影响

本文报道了pH7.5时血红蛋白和红细胞膜的结合效应.在10—45℃温度范围内观察到血红蛋白对膜脂质流动性的限制作用.看来这种限制作用不是脂质过氧化所致,而是血红蛋白和红细胞膜直接作用的结果.对流动性大的膜,血红蛋白的效应也随之增大.高铁血红蛋白及红细胞膜去胆固醇皆能修饰血红蛋白和膜的相互作用.     

Aging of the erythrocyte IV. Spin-label studies of membrane lipids,proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Complete exchange of viral cholesterol.

The exchange of the cholesterol in the membranes of two enveloped viruses, Sindbis virus and vesicular stomatitis virus, with cholesterol present in lipid vesicles and in serum was measured. Biosynthetically labeled viral cholesterol underwent spontaneous and complete transfer to both lipid vesicles and to serum. The rate with which and the extent to which this process occurred were very similar for these two viruses. During incubation with lipid vesicles in excess, half of the viral cholesterol underwent transfer in approximately 4 h and more than 90% underwent transfer in 24h at 37 degrees C. Similar rates and extents of movement of viral cholesterol were observed when incubations were carried out with vesicles which contained cholesterol and phospholipid in the same molar ratio as in the virus or with egg lecithin vesicles which contained no cholesterol. When labeled cholesterol was present initially in the lipid vesicles, movement of cholesterol from the vesicles to the virus was observed. One implication of the fact that viral cholesterol undergoes extensive exchange with serum cholesterol is that cellular cholesterol is in equilibrium with that in the extracellular fluid.     

Aging of the erythrocyte. IV. Spin-label studies of membrane lipids, proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Effect of cholesterol on the interaction of the HIV GP41 fusion peptide with model membranes. Importance of the membrane dipole potential

Fusion of viral and cell membranes is a key event in the process by which the human immunodeficiency virus (HIV) enters the target cell. Membrane fusion is facilitated by the interaction of the viral gp41 fusion peptide with the cell membrane. Using synthetic peptides and model membrane systems, it has been established that the sequence of events implies the binding of the peptide to the membrane, followed by a conformational change (transformation of unordered and helical structures into beta-aggregates) which precedes lipid mixing. It is known that this process can be influenced by the membrane lipid composition. In the present work we have undertaken a systematic study in order to determine the influence of cholesterol (abundant in the viral membrane) in the sequence of events leading to lipid mixing. Besides its effect on membrane fluidity, cholesterol can affect a less known physical parameter, the membrane dipole potential. Using the dipole potential fluorescent sensor di-8-ANEPPS together with other biophysical techniques, we show that cholesterol increases the affinity of the fusion peptide for the model membranes, and although it lowers the extent of lipid mixing, it increases the mixing rate. The influence of cholesterol on the peptide affinity and the lipid mixing rate are shown to be mainly due to its influence of the membrane dipole potential, whereas the lipid mixing extent and peptide conformational changes seem to be more dependent on other membrane parameters such as membrane fluidity and hydration.     

Seasonal changes in the structure and function of mitochondrial membranes of artichoke tubers: acyl Fatty Acid composition and the effect of growth conditions

Changes in the temperature response, fluidity, function and the acyl fatty acid composition, were determined for a mitochondria-rich membrane fraction from Jerusalem artichoke (Helianthus tuberosus L.) tubers during dormancy for a crop which matured in midsummer. The temperature of both the upper and lower limits of the membrane lipid transition decreased during dormancy from 26 C and 1 C to 4 C and −5 C, respectively. This was similar to the changes observed with crops maturing in late autumn. The order parameter of a spin label intercalated into the membrane lipids decreased from about 0.6 to 0.5 during dormancy and returned to the original value before sprouting, showing that membrane fluidity increased during dormancy. The activation energy of succinate oxidase of tuber mitochondria was generally high at middormancy when membrane lipids were more fluid and decreased as the membranes became more rigid at the end of dormancy. The fatty acid composition of the membrane lipids did not alter significantly during dormancy. The results indicate that neither decreasing day length nor low soil temperature during tuber maturation is essential for the initiation of the membrane changes necessary for tubers to avoid low temperature injury during dormancy. The increase in membrane fluidity during dormancy could not be accounted for by an increase in the proportion of unsaturated fatty acids in the membrane lipids.     

Fluid lipid fraction in rod outer segment membrane.

Rod outer segment membrane is analyzed using the spin resonance label technique by means of two probes. The solubility of the first label,2,2,6,6-tetramethylpiperidin-1-oxyl, is correlated with the membrane fluidity which is measured using a stearic acid spin probe. The two values are compared to the solubility-fluidity relationship which characterizes a model system in which all lipids are in a fluid state. The analysis leads to the conclusion that only two thirds of the membrane lipids are fluid. This conclusion is reinforced by the observation that partial lipid removal leaves rigid lipids associated with the rhodopsin molecules.     

Fluid lipid fraction in rod outer segment membrane

Rod outer segment membrane is analyzed using the spin label technique by means of two probes. The solubility of the first label, 2,2,6,6-tetramethylpiperidin-1-oxyl, is correlated with the membrane fluidity which is measured using a stearic acid spin probe. The two values are compared to the solubility-fluidity relationship which characterizes a model system in which all lipids are in a fluid state. The analysis leads to the conclusion that only two thirds of the membrane lipids are fluid. This conclusion is reinforced by the observation that partial lipid removal leaves rigid lipids associated with the rhodopsin molecules.     

Lipid-protein interactions in thylakoid membranes of chilling-resistant and -sensitive plants studied by spin label electron spin resonance spectroscopy

Lipid-protein interactions in thylakoid membranes from lettuce, pea, tomato, and cucumber have been studied using spin-labeled analogues of the thylakoid membrane lipid components, monogalactosyl diglyceride and phosphatidylglycerol. The electron spin resonance spectra of the spin-labeled lipids all consist of two components, one corresponding to the fluid lipid environment in the membranes and the other to the motionally restricted lipids interacting with the integral membrane proteins. Comparison of the spectra from the same spin label in thylakoid membranes from different plants shows that the overall lipid fluidity in the membranes decreases with chilling sensitivity. Spectral subtraction has been used to quantitate the fraction of the membrane lipids in contact with integral membrane proteins. Thylakoid membranes of cucumber, a typical chilling-sensitive plant, have been found to have a higher proportion of motionally restricted lipids and a different lipid selectivity for lipid-protein interaction, as compared with those of pea, a typical chilling-resistant plant. This correlation with chilling sensitivity holds generally for the different plants studied. It seems likely that the chilling sensitivity in thylakoid membranes is not determined by lipid fluidity alone, but also by the lipid-protein interactions which could affect protein function in a more direct manner.     

Fluorescence-polarization measurements on normal and mutant human skin fibroblasts

Measurements of fluorescence polarization in intact diploid skin fibroblasts after exposure to 1,6-diphenyl-1,3,5-hexatriene were used to estimate the fluidity of the lipid phase(s) of cellular membranes. The membrane lipids of cells derived from four patients with homozygous familial hypercholesterolemia were in a more fluid state than those of cells obtained from 13 other individuals of normal and nonrelated mutant genotypes when all cultures were grown on medium with native serum. The only other cell type having membrane lipids of increased fluidity under these conditions was one fibroblast line derived from a patient with the Lesch-Nyhan syndrome. Examination of two additional nonconsanguinous lines of Lesch-Nyhan fibroblasts, however, revealed that an abnormally high level of lipid fluidity was not a common property of the membranes of cells of this genotype. Incubation of cultures in medium containing lipid-depleted serum (virtually devoid of lipoprotein-bound sterol) caused a reversible increase in the fluidity of the membranes of normal cells to values similar to those of the hypercholesterolemic cells, but had no effect on the membranelipid fluidity of the latter. By contrast, exposure of cultures to cholesterol not bound to lipoprotein in serum-free medium resulted in a decrease in the lipid fluidity of the membranes of both normo- and hypercholesterolemic fibroblasts.     

Effect of nicotinic acid on microviscosity in mixed liposomal system of lecithin and sphingomyelin

In rat liver plasma membrane, the molar ratio of sphingomyelin and phospholipid is approximately 1:4, whereas, the molar ratio of phospholipid and cholesterol is 3:1. Considering this ratio to be typical for a real biological membrane, we have studied the effect of anticholesterol and the vasodialatory drug nicotinic acid (NA) on the fluidity profile of a liposomal system of lipids mixed in this ratio using the fluorescence polarization probe 1,6-diphenyl-1-1,3,5-hexatriene. The study reveals that when NA is added to the aqueous dispersion of the mixed lipid system (molar ratio of lipid:NA, 1:1) it creates a more fluid environment for the probe molecule and modifies the fluidity profile of the cholesterol-incorporated liposomal system by eliminating the effect of cholesterol to some extent. The drug also affects the activation energy of diffusion of this system. These results on fluidity have been compared with those in cases of liposomes of individual lipids. The effect of NA on fluidity may be attributed to a mechanical interaction of the drug molecules with the lipid molecules.     

Lipid Composition of Purified Vesicular Stomatitis Viruses

Methods are described for the production of vesicular stomatitis (VS) virus of sufficient purity for reliable chemical analysis. VS virions released from infected cells were concentrated and purified at least 150-fold by sequential steps of precipitation with polyethylene glycol, column chromatography, rate zonal centrifugation, and equilibrium centrifugation. The Indiana serotype (VS(Ind) virus) propagated in L-cells was found to contain 3% ribonucleic acid, 64% protein, 13% carbohydrate, and 20% lipid; the molar ratio of cholesterol to phospholipid was 0.6 or greater. Thin-layer chromatography revealed no unusual neutral lipids or phospholipids and gas-liquid chromatography revealed no unusual fatty acids incorporated into VS virions. The antigenically distinct New Jersey serotype (VS(NJ) virus) grown in L-cells showed a similar lipid profile except that the proportion of neutral lipids was larger than in VS(Ind) virus also grown in L-cells. This differences was less pronounced when the lipid composition of VS(Ind) and VS(NJ) viruses grown in chick embryo cells was compared, but VS(NJ) virus grown in either cell type always contained larger amounts of neutral lipids other than cholesterol than did VS(Ind) virus. The lipid composition of both VS(Ind) and VS(NJ) viruses grown in L-cells or chick embryo cells more closely resembled that of plasma membrane than of whole cells. A consistent finding was the relatively large amounts of phosphatidylethanolamine and sphingomyelin and the relatively small amounts of phosphatidylcholine in both VS viruses compared with uninfected whole L-cells and chick embryo cells or their plasma membranes. The methods available for isolation of plasma membranes were inadequate for conclusive comparison of the lipids of VS virions with the lipids of the plasma membranes of their host cells. Nevertheless, the data obtained are consistent with two hypotheses: (i) the lipid composition of VS viruses primarily reflects their membrane site of maturation, and (ii) the newly synthesized viral proteins inserted into cell membranes influence the proportions of phospholipids and neutral lipids selected for incorporation into the viral membrane.     

Target Membrane Cholesterol Modulates Single Influenza Virus Membrane Fusion Efficiency but Not Rate

Host lipid composition influences many stages of the influenza A virus (IAV) entry process, including initial binding of IAV to sialylated glycans, fusion between the viral envelope and the host membrane, and the formation of a fusion pore through which the viral genome is transferred into a target cell. In particular, target membrane cholesterol has been shown to preferentially associate with virus receptors and alter physical properties of the membrane like fluidity and curvature. These properties affect both IAV binding and fusion, which makes it difficult to isolate the role of cholesterol in IAV fusion from receptor binding effects. Here, we develop a fusion assay that uses synthetic DNA-lipid conjugates as surrogate viral receptors to tether virions to target vesicles. To avoid the possibly perturbative effect of adding a self-quenched concentration of dye-labeled lipids to the viral membrane, we tether virions to lipid-labeled target vesicles and use fluorescence microscopy to detect individual, pH-triggered IAV membrane fusion events. Through this approach, we find that cholesterol in the target membrane enhances the efficiency of single-particle IAV lipid mixing, whereas the rate of lipid mixing is independent of cholesterol composition. We also find that the single-particle kinetics of influenza lipid mixing to target membranes with different cholesterol compositions is independent of receptor binding, suggesting that cholesterol-mediated spatial clustering of viral receptors within the target membrane does not significantly affect IAV hemifusion. These results are consistent with the hypothesis that target membrane cholesterol increases lipid mixing efficiency by altering host membrane curvature.