The plasma membrane lipid composition affects fusion between cells and model membranes

Investigations were carried out on the effect of plasma membrane lipid modifications on the fusogenic capacity of control and ras-transformed fibroblasts. The plasma membrane lipid composition was modified by treatment of cells with exogenous phospholipases C and D, sphingomyelinase and cyclodextrin. The used enzymes hydrolyzed definite membrane lipids thus inducing specific modifications of the lipid composition while cyclodextrin treatment reduced significantly the level of cholesterol. The cells with modified membranes were used for assessment of their fusogenic capacity with model membranes with a constant lipid composition. Treatment with phospholipases C and D stimulated the fusogenic potential of both cell lines whereas the specific reduction of either sphingomyelin or cholesterol induced the opposite effect. The results showed that all modifications of the plasma membrane lipid composition affected the fusogenic capacity irrespective of the initial differences in the membrane lipid composition of the two cell lines. These results support the notion that the lipid composition plays a significant role in the processes of membrane-membrane fusion. This role could be either direct or through modulation of the activity of specific proteins which regulate membrane fusion.     

Insertion of lipid domains into plasma membranes by fusion with erythrocytes

After prelabeling the plasma membrane with several lipid-specific fluorescent probes, erythrocytes with symmetric lipid bilayers were fused with culture cells using either poly(ethylene glycol) or Sendai virus as fusogen. Several nonspecific probes were transferred to, and became uniformly distributed within, the culture cell membrane upon fusion. In contrast, when merocyanine 540, which displays preferential binding to bilayers in which the lipids are loosely packed, was used to prelabel erythrocytes, fluorescence remained localized within a small confined area of the membrane, even 24 h after fusion. These results suggest that insertion of the lipids of the erythrocyte membrane into the plasma membrane of the culture cell can produce discrete domains which persist as such for long periods following fusion. Because the inserted proteins of the erythrocyte membrane similarly do not freely diffuse throughout the culture cell membrane, interactions between membrane proteins and lipids may be involved in this singular compartmentalization.     

Interaction of influenza virus proteins with planar bilayer lipid membranes II. Effects of rimantadine and amantadine

The dependence of the surface potential difference (ΔU), transversal elasticity module (E₁) and membrane conductivity (G₀) on the concentrations of the antiviral drugs, rimantadine and amantadine was studied in the planar bilayer lipid membrane system. The method used was based on independent measurements of the second and third harmonics of the membrane capacitance current. The binding constants of bilayer lipid membranes obtained from the drug adsorption isotherms were 2.1 · 10⁵ M^?1 and 1.3 · 10⁴ M^?1 for rimantadine and amantadine, respectively. The changes in G₀ took place only after drug adsorption saturation had been achieved. The influence of rimantadine and amantadine on the interaction of bilayer lipid membranes with matrix protein from influenza virus was also investigated. The presence of 70 μg/ml rimantadine in the bathing solution resulted in an increase in the concentration of M-protein at which the adsorption and conductance changes were observed. The effects of amantadine were similar to those of rimantadine but required a higher critical concentration of amantadine. The results obtained suggest that the antiviral properties of rimantadine and amantadine may be related to the interaction of these drugs with the cell membrane, which can affect virus penetration into the cell as well as maturation of the viral particle at the cell membrane.     

Parameters affecting low-pH-mediated fusion of liposomes with the plasma membrane of cells infected with influenza virus

Unilamellar liposomes can be fused at low pH with the plasma membrane of cells that express the hemagglutinin glycoprotein of influenza virus on their surface [van Meer, G., & Simons, K. (1983) J. Cell Biol. 97, 1365-1374]. Here, we have resolved this fusion process into two kinetically distinct steps. The first and more rapid step converts the bound liposome to a form that can no longer be released by neuraminidase. The second step is the actual membrane fusion as measured by the loss of resonance energy transfer between two liposomal fluorescent phospholipids, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dioleoylphosphatidylethanolami ne (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine (N-Rh-PE). In contrast to the first step, the rate of the second one was highly dependent on the liposomal lipid composition and the cell type used. The replacement of 50% of the phosphatidylcholine (PC) in egg PC-cholesterol liposomes by unsaturated phosphatidylethanolamine (PE) species increased the rate of fusion at least 2-fold. Of the PE-containing liposomes that were associated with Madin-Darby canine kidney (MDCK) cells after 30 s of fusion, 80% had actually fused with the plasma membrane. Fringe pattern fluorescence photobleaching experiments showed that after fusion a fraction of the cell-associated N-Rh-PE diffused laterally in the plasma membrane. Without fusion, the N-Rh-PE was completely immobile. Under optimal conditions, the mobile fractions were 65% on MDCK cells and 78% on baby hamster kidney cells. The mobility was acquired simultaneously with the dilution of the fluorescent phospholipids as measured from the loss of resonance energy transfer. The mobile fraction of N-Rh-PE on the cell surface can therefore be used as a second independent measure of actual membrane fusion. Finally, we observed that upon fusion up to 80% of the nonexchangeable liposome markers cholesterol [14C]oleate and glycerol tri[14C]oleate became accessible to cellular hydrolases. The results showed that this hydrolysis assay can also be used to monitor the second step of the fusion process.     

Interaction of the antivirus agents remantadine and amantadine with lipid membranes and the influence on the curvature of human red cells

Surface potential difference, conductance, and elasticity changes of bilayer lipid membranes induced by the antivirus drugs amantadine and remantadine were measured. An influence on the human erythrocyte shape was shown. Both drugs are stomatocytogenic. The adsorption at the cytoplasmatic membrane was electrophoretically proved. The heat-induced vesiculation is partly inhibited. No microvesicles were observed. Instead, large tails which did not detach from the cell body were seen. The general conclusion is that these amphiphilic adamantane derivatives are membrane agents which modify membrane interaction processes, possibly by influencing the bending properties.     

Reconstitution of functional influenza virus envelopes and fusion with membranes and liposomes lacking virus receptors.

Reconstituted influenza virus envelopes were obtained following solubilization of intact virions with Triton X-100. Quantitative determination revealed that the hemolytic and fusogenic activities of the envelopes prepared by the present method were close or identical to those expressed by intact virions. Hemolysis as well as virus-membrane fusion occurred only at low pH values, while both activities were negligible at neutral pH values. Fusion of intact virions as well as reconstituted envelopes with erythrocyte membranes--and also with liposomes--was determined by the use of fluorescently labeled viral envelopes and fluorescence dequenching measurements. Fusion with liposomes did not require the presence of specific virus receptors, namely sialoglycolipids. Under hypotonic conditions, influenza virions or their reconstituted envelopes were able to fuse with erythrocyte membranes from which virus receptors had been removed by treatment with neuraminidase and pronase. Inactivated intact virions or reconstituted envelopes, namely, envelopes treated with hydroxylamine or glutaraldehyde or incubated at low pH or 85 degrees C, neither caused hemolysis nor possessed fusogenic activity. Fluorescence dequenching measurements showed that only fusion with liposomes composed of neutral phospholipids and containing cholesterol reflected the viral fusogenic activity needed for infection.     

Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of influenza virus proteins with planar bilayer lipid membranes. II. Effects of rimantadine and amantadine

The dependence of the surface potential difference (delta U), transversal elasticity module (E1) and membrane conductivity (G0) on the concentrations of the antiviral drugs, rimantadine and amantadine was studied in the planar bilayer lipid membrane system. The method used was based on independent measurements of the second and third harmonics of the membrane capacitance current. The binding constants of bilayer lipid membranes obtained from the drug adsorption isotherms were 2.1 X 10(5) M-1 and 1.3 X 10(4) M-1 for rimantadine and amantadine, respectively. The changes in G0 took place only after drug adsorption saturation had been achieved. The influence of rimantadine and amantadine on the interaction of bilayer lipid membranes with matrix protein from influenza virus was also investigated. The presence of 70 micrograms/ml rimantadine in the bathing solution resulted in an increase in the concentration of M-protein at which the adsorption and conductance changes were observed. The effects of amantadine were similar to those of rimantadine but required a higher critical concentration of amantadine. The results obtained suggest that the antiviral properties of rimantadine and amantadine may be related to the interaction of these drugs with the cell membrane, which can affect virus penetration into the cell as well as maturation of the viral particle at the cell membrane.     

Quantitative determination of virus-membrane fusion events. Fusion of influenza virions with plasma membranes and membranes of endocytic vesicles in living cultured cells

Incubation of fluorescently labeled influenza virus particles with living cultured cells such as lymphoma S-49 cells or hepatoma tissue culture cells resulted in a relatively high degree of fluorescence dequenching. Increase in the degree of fluorescence (35-40% fluorescence dequenching) was observed following incubation at pH 5.0 as well as at pH 7.4. On the other hand, incubation of fluorescently labeled influenza virions with erythrocyte ghosts resulted in fluorescence dequenching only upon incubation at pH 5.0. Only a low degree of fluorescence dequenching was observed upon incubation with inactivated unfusogenic influenza or with hemagglutinino-influenza virions. The results of the present work clearly suggest that the fluorescence dequenching observed at pH 5.0 resulted from fusion with the cells' plasma membranes, while that at pH 7.4 was with the membranes of endocytic vacuoles following endocytosis of the virus particles. Our results show that only the fluorescence dequenching observed at pH 7.4--but not that obtained at pH 5.0--was inhibited by lysosomotropic agents such as methylamine and ammonium chloride, or inhibitors of endocytosis such as EDTA and NaN3.     

Dextran sulfate inhibits fusion of influenza virus and cells expressing influenza hemagglutinin with red blood cells.

The influence of dextran sulfate with molecular weights of 500,000 and 8000 on binding and fusion of influenza virus (X31 strain) and of cells expressing influenza hemagglutinin (GP4F) with red blood cells (RBC) was investigated by spectrofluorimetry using virus and RBC labeled with the fluorescent dye octadecyl rhodamine B (R18). There was no significant inhibition of binding of virus and GP4F cells to red blood cells by dextran sulfate, but the polymer strongly inhibited the low pH induced fusion. Virus-RBC fusion was completely blocked by the high molecular weight dextran sulfate at concentrations as low as 0.5 mg/ml. Inhibition of RBC-GP4F cell fusion by dextran sulfate in the same concentration range was not as pronounced but the effect was potentiated by Ca2+. The polymer was only inhibitory when added at early steps of the fusion reaction, but the pH-induced conformational change of the hemagglutinin was not affected by dextran sulfate as measured by its susceptibility to proteolytic digestion. Removal of dextran sulfate after low pH-requiring steps allowed the system to fuse at neutral pH indicating that the inhibitory effect requires the continuous presence of dextran sulfate during the fusion reaction.     

Analysis of delay times of hemagglutinin-mediated fusion between influenza virus and cell membranes

We have studied the kinetics of low pH-induced fusion between influenza virus A/PR 8/34 and human erythrocyte membranes in suspension by using an assay based on fluorescence dequenching (FDQ) of the lipophilic dye octadecylrhodamine B chloride (R 18). As shown previously (Clague et al. 1991) the onset of FDQ is preceded by a characteristic lag time (t _lag) following pH reduction. Whereas t _lag represents only a subpopulation of fusing viruses with the shortest delay time we suggest here that a representative mean lag time µ_1ag of virus-cell fusion can be deduced from the R 18-assay. Kinetics of FDQ reflects the cumulative distribution function of lag times _lag of single fusion events with the mean value µ_lag. We show that t _lag obtained from the onset of FDQ does not always reflect the fusion behaviour of the whole population of fusing viruses. While both lag times, t _lag and µ_lag exhibit a similar temperature dependence we found a significantly different dependence of both delay times on virus inactivation by low pH-pretreatment. We conclude that the mean lag time µ_lag appears to be a more appropriate parameter describing the kinetics of virus-cell fusion. The analysis of delay times offers a new approach to test the validity of different kinetic models of HA-mediated fusion and to gain valuable information about HA-mediated fusion. The analysis confirms that the inactivation process proceeds via steps of the formation of the fusion pore. Although the increase of lag times can be explained by a depletion of fusion competent HA's, our data suggest that intermediate structures of HA along the inactivation pathway can still transform into a fusion site.Abbreviations FDQ fluorescence dequenching - HA hemagglutinin - PBS phosphate buffered saline - R18 octadecylrhodamine B chloride - t _lag lag time - µ_lag mean lag time - _lag individual delay time Correspondence to: A. Herrmann     

Properties and structures of the influenza and HIV fusion peptides on lipid membranes: implications for a role in fusion

The fusion peptides of HIV and influenza virus are crucial for viral entry into a host cell. We report the membrane-perturbing and structural properties of fusion peptides from the HA fusion protein of influenza virus and the gp41 fusion protein of HIV. Our goals were to determine: 1), how fusion peptides alter structure within the bilayers of fusogenic and nonfusogenic lipid vesicles and 2), how fusion peptide structure is related to the ability to promote fusion. Fluorescent probes revealed that neither peptide had a significant effect on bilayer packing at the water-membrane interface, but both increased acyl chain order in both fusogenic and nonfusogenic vesicles. Both also reduced free volume within the bilayer as indicated by partitioning of a lipophilic fluorophore into membranes. These membrane ordering effects were smaller for the gp41 peptide than for the HA peptide at low peptide/lipid ratio, suggesting that the two peptides assume different structures on membranes. The influenza peptide was predominantly helical, and the gp41 peptide was predominantly antiparallel beta-sheet when membrane bound, however, the depths of penetration of Trps of both peptides into neutral membranes were similar and independent of membrane composition. We previously demonstrated: 1), the abilities of both peptides to promote fusion but not initial intermediate formation during PEG-mediated fusion and 2), the ability of hexadecane to compete with this effect of the fusion peptides. Taken together, our current and past results suggest a hypothesis for a common mechanism by which these two viral fusion peptides promote fusion.     

Tension of membranes expressing the hemagglutinin of influenza virus inhibits fusion.

The effects of membrane tension on fusion between cells expressing the hemagglutinin (HA) of influenza virus and red blood cells were studied by capacitance measurements. Inflation of an HA-expressing cell was achieved by applying a positive hydrostatic pressure to its interior through a patch-clamp pipette in the whole-cell configuration. Inflating cells to the maximum extent possible without lysis created a membrane tension and completely inhibited low-pH-induced fusion at room temperature. Fully inflated cells that were subsequently deflated to normal size resumed the ability to fuse in response to low pH. At the higher temperature of 32 degrees C, fusion conditions were sufficiently optimal that full inflation did not hinder fusion, and once formed, pores enlarged more rapidly than those of never inflated cells. It is suggested that under fusogenic conditions HA causes the formation of a dimple within the membrane in which it resides, and that membrane tension hinders fusion by preventing the formation of dimples. Because dimpling bends the bilayer portion of bound membranes so that they come into intimate contact, the damping of dimpling would suppress this initial step in the fusion process.     

Structural determinants of the interaction between influenza A virus matrix protein M1 and lipid membranes

Influenza A virus is a pathogen responsible for severe seasonal epidemics threatening human and animal populations every year. One of the ten major proteins encoded by the viral genome, the matrix protein M1, is abundantly produced in infected cells and plays a structural role in determining the morphology of the virus. During assembly of new viral particles, M1 is recruited to the host cell membrane where it associates with lipids and other viral proteins. The structure of M1 is only partially known. In particular, structural details of M1 interactions with the cellular plasma membrane as well as M1–protein interactions and multimerization have not been clarified, yet.In this work, we employed a set of complementary experimental and theoretical tools to tackle these issues. Using raster image correlation, surface plasmon resonance and circular dichroism spectroscopies, we quantified membrane association and oligomerization of full-length M1 and of different genetically engineered M1 constructs (i.e., N- and C-terminally truncated constructs and a mutant of the polybasic region, residues 95–105). Furthermore, we report novel information on structural changes in M1 occurring upon binding to membranes. Our experimental results are corroborated by an all-atom model of the full-length M1 protein bound to a negatively charged lipid bilayer.     

Influence of the spectrin network on fusion of influenza virus with red blood cells

We examined the influence of the physical state of the membrane skeleton on low pH fusion of influenza virus A/PR 8/34 with intact human red blood cells. Spectrin, the major component of the skeleton, is known to become denaturated at 50°C. After heat treatment of erythrocytes at 50°C we observed an enhanced kinetics of fusion monitored spectrofluorometrically by the octadecylrhodamine fluorescence dequenching assay, while the extent of fusion was not affected. The accelerated fusion of influenza virus after preincubation of red blood cells at 50°C is not mediated by alterations of the lipid phase of the target. From ESR measurements using spin-labelled phospholipids we conclude that heat-induced alterations of the spectrin network did not affect either the phospholipid asymmetry or the fluidity of the exoplasmic and the cytoplasmic leaflets of the erythrocyte membrane. Moreover, as deduced from our previous investigations, the swelling behaviour of red blood cells could not be responsible for the observed effect. Possible mechanisms for the spectrin effect include a change in the ability of the target membrane to bend locally, and a change in the rate of formation and development of the fusion pore.     

Interaction of smooth muscle plasma membranes with flat lipid membranes

The method of implantation of smooth muscle cells from plasma membranes (PM) of the rabbit intestine into flat lipid membranes (FLM) is described. The method is based on the pretreatment of PM vesicles with asolectin liposomes in the ratio that provides the activation of membrane ATPases. Thus modified FLM possesses channel conductivity.     

Interaction of human immunodeficiency virus (HIV-1) fusion peptides with artificial lipid membranes

The interaction of 11 overlapping synthetic peptides corresponding to N-terminal segment of HIV transmembrane glycoprotein gp41 (fusion domain) with artificial lipid membranes has been studied. For this purpose the increase of a bilayer lipid membrane (BLM) conductivity and the changes in ESR spectra of spin-labelled liposomes were registrated. Peptide fragment 523-532 gp160 (BRU strain) had the critical length with regard to channel-forming activity on BLM. The degree of such membranotropic action increased simultaneously with the growth of peptide length and the temperature in the cell. Peptides 518-532 and 517-532 lysed TEMPOcholine-containing liposomes at 37 degrees C. The significance of observed effects for explanation of the mechanism of HIV-induced membrane fusion is discussed.     

The interaction of viral fusion peptides with lipid membranes

In this paper, we studied fusogenic peptides of class I-III fusion proteins, which are relevant to membrane fusion for certain enveloped viruses, in contact with model lipid membranes. We resolved the vertical structure and examined the adsorption or penetration behavior of the fusogenic peptides at phospholipid Langmuir monolayers with different initial surface pressures with x-ray reflectometry. We show that the fusion loops of tick-borne encephalitis virus (TBEV) glycoprotein E and vesicular stomatitis virus (VSV) G-protein are not able to insert deeply into model lipid membranes, as they adsorbed mainly underneath the headgroups with only limited penetration depths into the lipid films. In contrast, we observed that the hemagglutinin 2 fusion peptide (HA2-FP) and the VSV-transmembrane domain (VSV-TMD) can penetrate deeply into the membranes. However, in the case of VSV-TMD, the penetration was suppressed already at low surface pressures, whereas HA2-FP was able to insert even into highly compressed films. Membrane fusion is accompanied by drastic changes of the membrane curvature. To investigate how the peptides affect the curvature of model lipid membranes, we examined the effect of the fusogenic peptides on the equilibration of cubic monoolein structures after a phase transition from a lamellar state induced by an abrupt hydrostatic pressure reduction. We monitored this process in presence and absence of the peptides with small-angle x-ray scattering and found that HA2-FP and VSV-TMD drastically accelerate the equilibration, while the fusion loops of TBEV and VSV stabilize the swollen state of the lipid structures. In this work, we show that the class I fusion peptide of HA2 penetrates deeply into the hydrophobic region of membranes and is able to promote and accelerate the formation of negative curvature. In contrast, we found that the class II and III fusion loops of TBEV and VSV tend to counteract negative membrane curvature.     

Effects of cyclosporin A on model lipid membranes

Cyclosporin A (CSA) is a widely used immunosuppressant drug for transplant therapy, however its limitation is its toxicity. The effect of CSA on model membranes such as dimyristoyl phosphatidylcholine (DMPC) bilayers was studied using small-angle X-ray diffraction and differential scanning calorimetry (DSC). CSA abolishes the pretransition and affects the transition of DMPC model membranes in a concentration-related manner as is shown by DSC. CSA induces a second peak at the high temperature side of the main transition, which is interpreted as a phase separation between areas rich and poor in CSA concentration. Small angle X-ray diffraction shows that the repeat distance of the DMPC bilayers in the lamellar Lalpha state increases as a function of concentration up to 10 mol% and remains constant thereafter. Furthermore, CSA affects the fatty acyl chains of the bilayer, especially the part of the chain proximal to the head group. In conclusion, CSA, as both small-angle X-ray diffraction and DSC show, affects in a concentration-wise manner the DMPC model membranes and perturbs the bilayer, in particular the acyl chain region.