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.     

Photoelectrospectrometry of bilayer lipid membranes

Three different bilayer lipid membrane systems were studied under visible and ultraviolet illumination. The first system consisted of a bilayer lipid membrane formed with a mixture of phospholipids and cholesterol, to one side of which purple membrane fragments from Halobacterium halobium were added. The second system consisted of a membrane formed from spinach chloroplast extract. When either of these membrane systems was illuminated with ultraviolet and visible radiation, photopotentials were observed and photoelectric action spectra were recorded (the technique is termed photoelectrospectrometry). Each spectrum had a definite structure which was characteristic of each of the modified membranes. The third system studied consisted of an otherwise photoinactive membrane formed with a mixture of phospholipids and cholesterol, to one side of which chymotrypsin was added. When the membrane was illuminated with visible light no photoresponse was observed. On the other hand, a photopotential which increased with incubation time was observed when the membrane was illuminated with ultraviolet light. Since, in our systems, the photoresponses have been observed to be due to certain species incorporated into the membrane, it appears that photoelectrospectrometry is a useful tool for studying lipid-protein interactions, constituent organization and energy transfer in membranes.     

Interaction of influenza virus proteins with planar bilayer lipid membranes. I. Characterization of their adsorption and incorporation into lipid bilayers

Alterations in the surface potential difference (delta U) of asolectin planar bilayer lipid membranes were measured following the adsorption of isolated matrix protein (M-protein) or neuraminidase of influenza virus. The method used was based upon measurement of the bilayer lipid membrane capacitance current second harmonic. The delta U dependence on the M-protein and neuraminidase concentration indicates different mechanisms of adsorption of these viral proteins by the lipid bilayer. The conductance (G0) dependence of the bilayer lipid membrane with different compositions on the concentration of isolated surface glycoproteins, hemagglutinin and neuraminidase, M-protein or neuraminidase was investigated. The change in G0 for M-protein was observed only after adsorption saturation had been achieved. Neuraminidase alone does not affect the membrane conductivity. The surface charge and lipid composition of the lipid bilayer influences the adsorption and incorporation of influenza virus M-protein and surface glycoproteins. The reversibility of protein incorporation into the bilayers was investigated by a perfusion technique. The results show reversibility of surface glycoprotein incorporation while M-protein binding appears to be irreversible.     

Regulation of channel function due to physical energetic coupling with a lipid bilayer

Regulation of membrane protein functions due to hydrophobic coupling with a lipid bilayer has been investigated. An energy formula describing interactions between lipid bilayer and integral ion channels with different structures, which is based on the screened Coulomb interaction approximation, has been developed. Here the interaction energy is represented as being due to charge-based interactions between channel and lipid bilayer. The hydrophobic bilayer thickness channel length mismatch is found to induce channel destabilization exponentially while negative lipid curvature linearly. Experimental parameters related to channel dynamics are consistent with theoretical predictions. To measure comparable energy parameters directly in the system and to elucidate the mechanism at an atomistic level we performed molecular dynamics (MD) simulations of the ion channel forming peptide–lipid complexes. MD simulations indicate that peptides and lipids experience electrostatic and van der Waals interactions for short period of time when found within each other’s proximity. The energies from these two interactions are found to be similar to the energies derived theoretically using the screened Coulomb and the van der Waals interactions between peptides (in ion channel) and lipids (in lipid bilayer) due to mainly their charge properties. The results of in silico MD studies taken together with experimental observable parameters and theoretical energetic predictions suggest that the peptides induce ion channels inside lipid membranes due to peptide–lipid physical interactions. This study provides a new insight helping better understand of the underlying mechanisms of membrane protein functions in cell membrane leading to important biological implications.     

A new method for membrane reconstitution: Fusion of protein-containing vesicles with planar bilayer membranes below lipid phase transition temperature

A new method of membrane reconstitution was developed by fusion of channel protein containing vesicles with planar bilayer membranes. The fusion process only occurred below and near the phase transition temperature of the lipid used. We obtained the following results: 1. Our system is solvent-free and vesicles do not come into contact with the air/water interface. This obviates a possible denaturation of hydrophobic proteins. 2. Channel forming proteins and protein complexes, respectively, are active in a frozen lipid matrix. 3. We detected an unknown channel in cilia fragments. 4. Purified acetylcholine receptors form fluctuating channels in a membrane consisting of a pure synthetic lecithin (two component system).     

Effects of membrane composition and lipid structure on the photopolymerization of lipid diacetylenes in bilayer membranes

Molecules analogous to biological and synthetic lipids have been prepared with conjugated diacetylene moieties in the long alkyl chain. These lipid diacetylenes form bilayer structures when suspended in aqueous buffers. Ultraviolet light (254 nm) exposure initiates the polymerization of the diacetylenes in the lipid bilayer to give a fully conjugated, highly colored product. The reaction is topotactic, and its efficiency depends on the correct alignment of the monomeric units. Thus, the lipid diacetylenes are photopolymerizable if the hydrocarbon chains are in a regular lattice found at temperatures below the lipid transition temperature; polymerization is inhibited above this transition. The photopolymerization of a diacetylenic glycerophosphocholine in lipid bilayer membranes was observed in two-component mixtures with a nonpolymerizable lipid, either dioleoylphosphatidylcholine or distearoylphosphatidylcholine. The photochemical and thermochemical characteristics suggest that the diacetylenic glycerophosphocholine exists largely in separate domains in the mixed bilayers. Lipid diacetylenes analogous to a dialkyldimethylammonium salt and to a dialkyl phosphate have a plane of symmetry, which suggests that both chains penetrate equally into the bilayer. The photopolymerization of these symmetrical synthetic species is more than 10³-times more efficient than that of the diacetylenic glycerophosphocholine. These differences are interpretable in terms of the expected conformational preference of the lipid molecules.     

Interaction of charged lipid vesicles with planar bilayer lipid membranes: Detection by antibiotic membrane probes

A technique has been developed for monitoring the interaction of charged phospholipid vesicles with planar bilayer lipid membranes (BLM) by use of the antibiotics Valinomycin, Nonactin, and Monazomycin as surface-charge probes. Anionic phosphatidylserine vesicles, when added to one aqueous compartment of a BLM, are shown to impart negative surface charge to zwitterionic phosphatidylocholine and phosphatidylethanolamine bilayers. The surface charge is distributed asymmertically, mainly on the vesicular side of the BLM, and is not removed by exchange of the vesicular aqueous solution. Possible mechanisms for the vesicle-BLM interactions are discussed.     

Influence of an additional amino group on the potency of aminoadamantanes against influenza virus A. II - Synthesis of spiropiperazines and in vitro activity against influenza A H3N2 virus

Spiro[piperidine-2,2′-adamantane] 4 is one of the most potent synthetic anti-influenza A aminoadamantanes or other cage structure amines tested so far. Based on previous results Tataridis et al. (2007) [5h] which demonstrate the boost of in vitro potency by the presence of an additional amino group, we examined whether the incorporation of a second amino group into this heterocycle would increase the anti-influenza A virus activity. The new synthetic molecules 5–7 are capable of forming two hydrogen bonds within the receptor. We identified the diamino derivatives 5 and 6, which are active against influenza A H3N2 virus although less potent than amantadine and its equipotent spiropiperidine 4.     

Effects of pH and diethylpyrocarbonate on the conductance states of planar lipid bilayers containing haemocyanin

The addition of haemocyanin from Megathura crenulata to the aqueous phase bathing a bilayer lipid membrane resulted in the formation of ionic channels. With an applied voltage biased negative with respect to the haemocyanin-containing side, a single conductance state was observed above pH 7.0. Below pH 7.0 several conductance states were manifested, and the maximum conductance observed for a single channel decreased with decreasing pH. Extensive treatment of the haemocyanin with diethylpyrocarbonate, which reacts primarily with histidine residues, completely prevented the formation of ionic channels; however, milder treatment produced a chemically modified haemocyanin that was capable of forming ionic channels with modified conductance properties. Each channel conductance was typically much lower than that of the channels formed from unmodified haemocyanin, and there was now substantial variation in conductance from channel to channel. Following the use of hydroxylamine to remove the carbethoxy groups from the modified haemocyanin, it formed ionic channels that were restored to the original unit channel conductance.     

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca²⁺ concentration was increased to a threshold of 3–5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 μm diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca²⁺-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca²⁺ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca²⁺ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca²⁺ concentration.     

Phase transition and lateral phase separation in planar lipid membranes as sensed by the lipophilic ion dipicrylamine

The permeation of the lipophilic ion dipicrylamine through planar lipid membranes formed from dipalmitoylphosphatidylcholine in n-decane shows an anomaly near the main phase transition of this system. Both the rate constant, k_i, of ion translocation across the membrane interior and the interfacial concentration, N, of this ion have a maximum at about 36°C. Analogous experiments were performed with tetraphenylborate. A considerably lesser effect of the phase transition was found. The addition of cholesterol leads to a broadening of the maxima for k_i and N. The time course of the current following a voltage jump shows a characteristic change below a temperature of about 45°C, if the molar ratio cholesterol/ phosphatidylcholine in the membrane forming solution exceeds 1. While the current transient decays exponentially above 45°C, a sum of two exponential terms yields an adequate fit below that temperature. This is regarded as evidence for a lateral phase separation below 45°C into structurally different domains, which provide two different pathways for dipicrylamine.     

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     

Electrogenic and selective transport of biogenic amines across lipid bilayer membranes mediated by a neutral ionophore (AS701)

The neutral noncyclic imide and ether containing ionophore (AS701), a selective carrier for Li⁺ among alkali cations, was found to be capable of mediating the transport of NH₄⁺ and of biogenic amines (catechols and indoles) across lipid bilayer membranes also. Ionophore-mediated electrical properties of planar lipid bilayers were studied under experimental conditions where the positively-charged amine species was dominant. The ionophore was found to act as a selective carrier of the biogenic amines, mediating their electrogenic transport across the membrane, forming 2:1 carrier-amine permeant complexes, carrying a net-charge of +1. Selectively among the amines corresponding to the following sequence: tryptamine (35) > Li⁺ (1) > serotonin (0.60) > dopamine (0.19) > norepinephrine (0.13) > epinephrine (0.05) > NH₄⁺ (0.05). The molecular factors involved in determining these selectivities are assessed.     

Soft perforation of planar bilayer lipid membranes of dipalmitoylphosphatidylcholine at the temperature of the phase transition from the liquid crystalline to the gel state

In contrast to the widely used method of electroporation, the method of soft perforation of lipid bilayers is proposed. It is based on the structural rearrangement of the lipid bilayer formed from disaturated phospholipids at the temperature of the phase transition from the liquid crystalline state to the gel state. This allows us to obtain a lipid pore population without the use of a strong electric field. It is shown that the planar lipid bilayer membrane (pBLM) formed from dipalmitoylphosphatidylcholine in 1 M LiCl aqueous solution exhibits the appearance of up to 50 lipid pores per 1 mm² of membrane surface, with an average single pore conductivity of 31±13 nS. The estimation of a single pore radius carried out with water-soluble poly(ethylene glycol)s (PEGs) showed that the average pore radius ranged between 1.0–1.7 nm. It was found experimentally that PEG-1450, PEG-2000, and PEG-3350 should be in a position to block the single pore conductivity completely, while PEG-6000 fully restored the ionic conductivity. The similarity of these PEG effects to ionic conductivity in protein pores makes it possible to suggest that the partition of the PEG molecules between the pore and the bulk solution does not depend on the nature of the chemical groups located in the pore wall.     

Histamine,theophylline and tryptamine transport through lipid bilayer membranes

Diffusion of histamine, theophylline and tryptamine through planar lipid bilayer membranes was studied as a function of pH. Membranes were made of egg phosphatidylcholine plus cholesterol (1 : 1 mol ratio) in tetradecane. Tracer fluxes and electrical conductances were used to estimate the permeabilities to nonionic and ionic species. Only the nonionic forms crossed the membrane at a significant rate. The membrane permeabilities to the nonionic species were: histamine, $\text{3.5 · 10}{}^{\mathrm{?5}}\text{cm}\text{· s}{}^{\mathrm{?1}}$; theophylline, $\text{2.9 · 10}{}^{\mathrm{?4}}\text{cm}\text{· s}{}^{\mathrm{?1}}$; and tryptamine, $\text{1.8 · 10}{}^{\mathrm{?1}}\text{cm}\text{· s}{}^{\mathrm{?1}}$. Chemical reactions in the unstirred layers are important in the transport of tryptamine and theophylline, but not histamine. For example, as pH decreased from 10.0 to 7.5 the ratio of nonionic (B) to ionic (BH⁺) tryptamine decreased by 300-fold, but the total tryptamine permeability decreased only 3-fold. The relative insensitivity of the total tryptamine permeability to the ratio, [B]/[BH⁺], is due to the rapid interconversion of B and BH⁺ in the instirred layers. Our model describing diffusion and reaction in the unstirred layers can explain some ‘anomalous’ relationships between pH and weak acid/base transport through lipid bilayer and biological membranes.     

An X-ray study of the cytoplasmic membranes of two Gram-positive bacteria

X-ray diffraction diagrams of partially disordered one-dimensional lattices of isolated bacterial cytoplasmic membranes are described and they provide a basis for suggesting possible molecular structures of bacterial membranes.Biochemical and electron microscope evidence points towards a lipid bilayer with a high degree of fluidity. The protein molecules are in a disordered configuration in the membrane.     

The concanavalin a receptor from human erythrocytes in lipid bilayer membranes. Interaction with concanavalin A and succinyl-concanavalin A

The concanavalin A receptor from human erythrocyte membranes has been isolated by affinity chromatography using the mild, readily-dialyzable detergent dodecyltrimethylammonium bromide. The purified protein has been reincorporated into large unilamellar phospholipid vesicles using a detergent dialysis technique. The mean diameter of these vesicles increases as the lipid: protein ratio decreases. Binding of succinyl-concanavalin A to these vesicles was quantitated using ¹²⁵I-labelled lectin in a filtration assay. The concanavalin A receptor in lipid bilayer vesicles provides specific high affinity binding sites for succinyl-concanavalin A with an association constant of 2.13·10⁶ M^?1. Scatchard plots indicate positive cooperativity of binding at very low lectin concentrations, a characteristic also seen in concanavalin A binding to intact human erythrocytes. The presence of bovine serum albumin has little effect on lectin binding and is not required for expression of cooperativity. Concanavalin A effectively competes with succinyl-concanavalin A for binding to the vesicles with an association constant of 4.83·10⁶ M^?1. Receptor-bearing vesicles are readily agglutinated by concanavalin A but not by its succinylated derivative. The kinetics of vesicle agglutination are biphasic, with an initial rapid phase followed by a pseudo-first order process. We suggest that studies on reassembled receptor proteins in lipid bilayers can provide valuable insight into receptor involvement in transmembrane signalling events and the factors involved in cell membrane behaviour and cell agglutination.     

Discovery and synthesis of novel benzofurazan derivatives as inhibitors of influenza A virus

The identification of a novel hit compound inhibitor of the protein–protein interaction between the influenza RNA-polymerase PA and PB1 subunits has been accomplished by means of high-throughput screening. A small family of structurally related molecules has been synthesized and biologically evaluated with most of the compounds showing micromolar potency of inhibition against viral replication.     

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.     

Hydrophobic segment of dengue virus C protein. Interaction with model membranes

Abstract

Dengue virus (DENV) C protein is essential for viral assembly. DENV C protein associates with intracellular membranes through a conserved hydrophobic domain and accumulates around endoplasmic reticulum-derived lipid droplets which could provide a platform for capsid formation during assembly. In a previous work we described a region in DENV C protein which induced a nearly complete membrane rupture of several membrane model systems, which was coincident with the theoretically predicted highly hydrophobic region of the protein. In this work we have carried out a study of the binding to and interaction with model biomembranes of a peptide corresponding to this DENV C region, DENV2_C6. We show that DENV2_C6 partitions into phospholipid membranes, is capable of rupturing membranes even at very low peptide-to-lipid ratios and its membrane-activity is modulated by lipid composition. These results identify an important region in the DENV C protein which might be directly implicated in the DENV life cycle through the modulation of membrane structure.