Modulation of entry of enveloped viruses by cholesterol and sphingolipids (Review)

Enveloped animal viruses infect host cells by fusion of viral and target membranes. This crucial fusion event occurs either with the plasma membrane of the host cells at the physiological pH or with the endosomal membranes at low pH and is triggered by specific glycoproteins in the virus envelope. Both lipids and proteins play critical and co-operative roles in the fusion process. Interactions of viral proteins with their receptors direct which membranes fuse and viral fusion proteins then drive the process. These fusion proteins operate on lipid assemblies, whose physical and mechanical properties are equally important to the proper functioning of the process. Lipids contribute to the viral fusion process by virtue of their distinct chemical structure, composition and/or their preferred partitioning into specific microdomains in the plasma membrane called 'rafts'. An involvement of lipid rafts in viral entry and membrane fusion has been examined recently. However, the mechanism(s) by which lipids as dynamic raft components control viral envelope-glycoprotein-triggered fusion is not clear. This paper will review literature findings on the contribution of the two raft-associated lipids, cholesterol and sphingolipids in viral entry.     

Modulation of entry of enveloped viruses by cholesterol and sphingolipids (Review)

Enveloped animal viruses infect host cells by fusion of viral and target membranes. This crucial fusion event occurs either with the plasma membrane of the host cells at the physiological pH or with the endosomal membranes at low pH and is triggered by specific glycoproteins in the virus envelope. Both lipids and proteins play critical and co-operative roles in the fusion process. Interactions of viral proteins with their receptors direct which membranes fuse and viral fusion proteins then drive the process. These fusion proteins operate on lipid assemblies, whose physical and mechanical properties are equally important to the proper functioning of the process. Lipids contribute to the viral fusion process by virtue of their distinct chemical structure, composition and/or their preferred partitioning into specific microdomains in the plasma membrane called 'rafts'. An involvement of lipid rafts in viral entry and membrane fusion has been examined recently. However, the mechanism(s) by which lipids as dynamic raft components control viral envelope-glycoprotein-triggered fusion is not clear. This paper will review literature findings on the contribution of the two raft-associated lipids, cholesterol and sphingolipids in viral entry.     

Membrane fusion in prokaryotes: bacteriophage phi 6 membrane fuses with the Pseudomonas syringae outer membrane.

Protein-triggered membrane fusion in the prokaryotic system is described using the lipid-containing enveloped bacterial virus phi 6 and its host, the Gram-negative bacterium Pseudomonas syringae. Bacteriophage particles can be fused to form multiple particles where two or more nucleocapsids are surrounded by a single membrane vesicle with a volume proportional to the number of fused particles. For fusion to occur, a fusogenic protein is required in the membrane of the participating phage particles. Upon infection of the host cell, fusion of the viral membrane with the bacterial membrane takes place without leakage of the periplasmic enzyme alkaline phosphatase to the extracellular supernatant. There is a time-dependent mixing of fluorescent phage phospholipids with the bacterial membrane lipids between 5 and 20 min post-infection. The phage membrane proteins and phospholipids co-purify with the bacterial outer membrane of infected cells. The fusion is independent of divalent cations and pH, resembling Sendai virus fusion with the plasma membrane. This is the first targeted, protein-dependent fusion event described in prokaryotes.     

Anionic lipids in Ca(2+)-triggered fusion

Anionic lipids are native membrane components that have a profound impact on many cellular processes, including regulated exocytosis. Nonetheless, the full nature of their contribution to the fast, Ca(2+)-triggered fusion pathway remains poorly defined. Here we utilize the tightly coupled quantitative molecular and functional analyses enabled by the cortical vesicle model system to elucidate the roles of specific anionic lipids in the docking, priming and fusion steps of regulated release. Studies with cholesterol sulfate established that effectively localized anionic lipids could contribute to Ca(2+)-sensing and even bind Ca(2+) directly as effectors of necessary membrane rearrangements. The data thus support a role for phosphatidylserine in Ca(2+) sensing. In contrast, phosphatidylinositol would appear to serve regulatory functions in the physiological fusion machine, contributing to priming and thus the modulation and tuning of the fusion process. We note the complexities associated with establishing the specific roles of (anionic) lipids in the native fusion mechanism, including their localization and interactions with other critical components that also remain to be more clearly and quantitatively defined.     

The Effect of Hydrophilic Ionic Liquids 1-Ethyl-3-Methylimidazolium Lactate and Choline Lactate on Lipid Vesicle Fusion

Ionic liquids (ILs) are room-temperature molten salts that have applications in both physical sciences and more recently in the purification of proteins and lipids, gene transfection and sample preparation for electron microscopy (EM) studies. Transfection of genes into cells requires membrane fusion between the cell membrane and the transfection reagent, thus, ILs may be induce a membrane fusion event. To clarify the behavior of ILs with cell membranes the effect of ILs on model membranes, i.e., liposomes, were investigated. We used two standard ILs, 1-ethyl-3-methylimidazolium lactate ([EMI][Lac]) and choline lactate ([Ch][Lac]), and focused on whether these ILs can induce lipid vesicle fusion. Fluorescence resonance energy transfer and dynamic light scattering were employed to determine whether the ILs induced vesicle fusion. Vesicle solutions at low IL concentrations showed negligible fusion when compared with the controls in the absence of ILs. At concentrations of 30% (v/v), both types of ILs induced vesicle fusion up to 1.3 and 1.6 times the fluorescence intensity of the control in the presence of [Ch][Lac] and [EMI][Lac], respectively. This is the first demonstration that [EMI][Lac] and [Ch][Lac] induce vesicle fusion at high IL concentrations and this observation should have a significant influence on basic biophysical studies. Conversely, the ability to avoid vesicle fusion at low IL concentrations is clearly advantageous for EM studies of lipid samples and cells. This new information describing IL-lipid membrane interactions should impact EM observations examining cell morphology.     

Lipid signaling on the mitochondrial surface

Regulated production and elimination of the signaling lipids phosphatidic acid (PA), diacylglycerol (DAG), and phosphatidylinositol 4,5-bisphosphate (PI4,5P₂) creates a complex and interconnected signaling network that modulates a wide variety of eukaryotic cell biological events. PA production at the plasma membrane and on trafficking membrane organelles by classical Phospholipase D (PLD) through the hydrolysis of phosphatidylcholine (PC) has been studied widely. In this chapter, we review a newly identified, non-canonical member of the PLD superfamily, MitoPLD, which localizes to the mitochondrial surface and plays a role in mitochondrial fusion via the hydrolysis of cardiolipin (CL) to generate PA. The role of PA in facilitating the mitochondrial fusion event carried out by proteins known as Mitofusins is intriguing in light of the role classic PLD-generated PA plays in facilitating SNARE-mediated fusion of secretory membrane vesicles into the plasma membrane. In addition, however, PA on the mitochondrial surface may also trigger a signaling cascade that elevates DAG, leading to downstream events that affect mitochondrial fission and energy production. PA production on the mitochondrial surface may also stimulate local production of PI4,5P₂ to facilitate mitochondrial fission and subcellular trafficking or facilitate Ca²⁺ influx.     

Alteration of lipid organization following fertilization of sea urchin eggs

The fluorescent probe merocyanine 540 was used to examine the organization of the lipids in the external leaflet of the plasma membrane after fertilization of sea urchin eggs. These lipids in unfertilized eggs are closely packed, as evidenced by their inability to bind the dye, whereas in fertilized eggs and cells of embryos up to at least the gastrula stage, the membrane becomes more loosely organized, and stains with bright ring fluorescence. Induction of late fertilization events with ammonia failed to induce this change in staining behavior. Sperm components are not required to induce this alteration since parthenogenetically activated eggs stained. However, treatment of eggs with procaine, which specifically inhibits the early event of cortical granule fusion, was effective in suppressing staining. These results indicate that cortical granule fusion after fertilization results in a change in the organization of the lipids of the plasma membrane of sea urchin eggs.     

Differential Association of Syntrophin Pairs with the Dystrophin Complex

While the specificity and timing of membrane fusion in diverse physiological reactions, including virus–cell fusion, is determined by proteins, fusion always involves the merger of membrane lipid bilayers. We have isolated a lipid-dependent stage of cell–cell fusion mediated by influenza hemagglutinin and triggered by cell exposure to mildly acidic pH. This stage preceded actual membrane merger and fusion pore formation but was subsequent to a low pH–induced change in hemagglutinin conformation that is required for fusion. A low pH conformation of hemagglutinin was required to achieve this lipid-dependent stage and also, downstream of it, to drive fusion to completion. The lower the pH of the medium applied to trigger fusion and, thus, the more hemagglutinin molecules activated, the less profound was the dependence of fusion on lipids. Membrane-incorporated lipids affected fusion in a manner that correlated with their dynamic molecular shape, a characteristic that determines a lipid monolayer's propensity to bend in different directions. The lipid sensitivity of this stage, i.e., inhibition of fusion by inverted cone–shaped lysophosphatidylcholine and promotion by cone-shaped oleic acid, was consistent with the stalk hypothesis of fusion, suggesting that fusion proteins begin membrane merger by promoting the formation of a bent, lipid-involving, stalk intermediate.     

Phospholipase D in calcium-regulated exocytosis: Lessons from chromaffin cells

Membrane fusion remains one of the less well-understood processes in cell biology. A variety of mechanisms have been proposed to explain how the generation of fusogenic lipids at sites of exocytosis facilitates secretion in mammalian cells. Over the last decade, chromaffin cells have served as an important cellular model to demonstrate a key role for phospholipase D1 (PLD1) generated phosphatidic acid in regulated exocytosis. The current model proposes that phosphatidic acid plays a biophysical role, generating a negative curvature and thus promoting fusion of secretory vesicles with the plasma membrane. Moreover, multiple signaling pathways converging on PLD1 regulation have been unraveled in chromaffin cells, suggesting a complex level of regulation dependant on the physiological context.     

Mechanical coupling via the membrane fusion SNARE protein syntaxin 1A: a molecular dynamics study

SNARE trans complexes between membranes likely promote membrane fusion. For the t-SNARE syntaxin 1A involved in synaptic transmission, the secondary structure and bending stiffness of the five-residue juxtamembrane linker is assumed to determine the required mechanical energy transfer from the cytosolic core complex to the membrane. These properties have here been studied by molecular dynamics and annealing simulations for the wild-type and a C-terminal-prolongated mutant within a neutral and an acidic bilayer, suggesting linker stiffnesses above 1.7 but below 50 x 10(-3) kcal mol(-1) deg(-2). The transmembrane helix was found to be tilted by 15 degrees and tightly anchored within the membrane with a stiffness of 4-5 kcal mol(-1) A(-2). The linker turned out to be marginally helical and strongly influenced by its lipid environment. Charged lipids increased the helicity and H3 helix tilt stiffness. For the wild type, the linker was seen embedded deeply within the polar region of the bilayer, whereas the prolongation shifted the linker outward. This reduced its helicity and increased its average tilt, thereby presumably reducing fusion efficiency. Our results suggest that partially unstructured linkers provide considerable mechanical coupling; the energy transduced cooperatively by the linkers in a native fusion event is thus estimated to be 3-8 kcal/mol, implying a two-to-five orders of magnitude fusion rate increase.     

A cell free system reveals that capacitation is a prerequisite for membrane fusion during the acrosome reaction.

Plasma and outer acrosomal membranes were extracted from bovine spermatozoa and used in an in vitro fusion assay. Fusion was revealed by monitoring the merging of lipids using the chlorophyll a-N,N'-dioctadecyloxacarbocyanine-p-toluene sulfonate (DCY) method [(1984) Biochim. Biophys. Acta 769, 531-542]. The requirement for capacitation, as well as the effects of pH, calcium and spermine, on membrane fusion in our cell-free system were similar to those observed in vivo on the acrosomal reaction. This demonstrates for the first time that capacitation and alterations in intracellular pH and calcium concentration, which must precede the acrosomal reaction, are required for the membrane fusion event.     

Membrane fusion correlates with surface charge in exocytic vesicles

Stimulation of gastric parietal cells results in exocytic recruitment of the proton pump (H(+),K(+)-ATPase) from a pool of intracellular membranes (tubulovesicles) to the apical plasma membrane. We have previously reconstituted a step in this process, the homotypic fusion of tubulovesicles, and shown that they also fuse with liposomes in a protein-dependent manner [Duman, J. G., Singh, G., Lee, G. Y., Machen, T. E., and Forte, J. G. (2002) Traffic 3, 203-17]. Further, the lipid composition of the liposomes affects their ability to undergo fusion with tubulovesicles. In the present study, we investigated the lipid requirements for tubulovesicular membrane fusion using a fluorescent probe relaxation assay as well as transfer of protein between tubulovesicles and liposomes of defined composition. Initially, we tested the ability of tubulovesicles to undergo fusion with a panel of synthetic phosphatidylcholine-based liposomes containing a variety of common membrane lipids of various shapes and charges. We found that anionic lipids such as phosphatidylserine, phosphatidic acid, and phosphoinositides were best able to enhance tubulovesicle-liposome fusion and that they did it in a dose-dependent, apparently saturable manner. Next, we altered the lipid compositions of actual tubulovesicles and observed that addition of anionic lipids was able to enhance tubulovesicle-tubulovesicle fusion in vitro; thus, we hypothesized that the charge imparted by the lipids, per se, was responsible for the enhancement of membrane fusion. Accordingly, addition of negative charges to one of two pools of tubulovesicles in a fusion assay using anionic detergents increased membrane fusion; whereas, addition of positively charged cationic detergent decreased membrane fusion and could be used to back-titrate the anionic effects. Surprisingly, when both pools of fusing membranes were loaded with anionic detergents, fusion was markedly increased. The ability of anionic charges to enhance fusion was diminished as the ionic strength of the fusion medium was increased, suggesting that the mechanism of fusion enhancement depends on the surface charge of the membranes. Finally, the fusion reaction was highly dependent on temperature, and anionic charge appears to lower the activation energy of the fusion reaction. Taken together, these data suggest that (1) tubulovesicular fusion is enhanced by an increase in membrane surface negative charge associated with a lower activation energy and (2) neutralization or reversal of the surface charge prevents tubulovesicular fusion.     

Molecular and mesoscopic geometries in autophagosome generation. A review

Autophagy is an essential process in cell self-repair and survival. The centre of the autophagic event is the generation of the so-called autophagosome (AP), a vesicle surrounded by a double membrane (two bilayers). The AP delivers its cargo to a lysosome, for degradation and re-use of the hydrolysis products as new building blocks. AP formation is a very complex event, requiring dozens of specific proteins, and involving numerous instances of membrane biogenesis and architecture, including membrane fusion and fission. Many stages of AP generation can be rationalised in terms of curvature, both the molecular geometry of lipids interpreted in terms of ‘intrinsic curvature’, and the overall mesoscopic curvature of the whole membrane, as observed with microscopy techniques. The present contribution intends to bring together the worlds of biophysics and cell biology of autophagy, in the hope that the resulting cross-pollination will generate abundant fruit.     

Dibucaine-induced synchronous mucocyst secretion in Tetrahymena

Synchronous secretion of all available mature mucocysts was induced in late log phase cultures of Tetrahymena thermophilia (B III) by the local anaesthetic dibucaine. No assembled fusion rosettes were seen within the plasma membrane after release until 2-3 hrs of regrowth, thus proving that the rosettes are not permanent sites within the plasma membrane but have to be reassembled each time for a new fusion event to occur. Concomitant with the reappearance of assembled fusion rosettes, the cell cytoplasm fills up with precursors of new mucocysts thus linking the two events together.     

Interplay between lipids and the proteinaceous membrane fusion machinery

For membrane fusion to occur, opposed lipid bilayers initially establish a fusion pore, often followed by complete mixing of the fusing membranes. Contemporary views suggest that during fusion lipid bilayers are continuous passive platforms that are disrupted and remodeled by catalytic proteins. Some models propose that even the architecture and composition of the fusion pore might be dominated by proteins rather than lipids. Hence, lipids have no regulatory contribution to this process; they simply adapt their shape passively for filling space between otherwise autonomous protein machineries.However, an increasing number of experimental findings indicate that membrane fusion critically depends on a variety of lipids and lipid derivatives. Therefore, a purely proteocentric view describes fusion mechanisms insufficiently. Instead, lipids have functions probably at different levels, as (i) a general influence on the propensity of lipid bilayers to fuse, (ii) a role in recruiting exocytotic proteins to the plasma membrane, (iii) a role in organizing membrane domains for fusion and (iv) direct regulatory effects on fusion protein complexes. In this review we have made an attempt to bring together the large body of evidence supporting a major role for lipids in membrane fusion either directly or indirectly.     

The Lipid Composition and Physical Properties of the Yeast Vacuole Affect the Hemifusion–Fusion Transition

Yeast vacuole fusion requires the formation of SNARE bundles between membranes. Although the function of vacuolar SNAREs is controlled in part by regulatory lipids, the exact role of the membrane in regulating fusion remains unclear. Because SNAREs are membrane‐anchored and transmit the force required for fusion to the bilayer, we hypothesized that the lipid composition and curvature of the membrane aid in controlling fusion. Here, we examined the effect of altering membrane fluidity and curvature on the functionality of fusion‐incompetent SNARE mutants that are thought to generate insufficient force to trigger the hemifusion–fusion transition. The hemifusion–fusion transition was inhibited by disrupting the 3Q:1R stoichiometry of SNARE bundles with the mutant SNARE Vam7p^Q283R. Similarly, replacing the transmembrane domain of the syntaxin homolog Vam3p with a lipid anchor allowed hemifusion, but not content mixing. Hemifusion‐stalled reactions containing either of the SNARE mutants were stimulated to fuse with chlorpromazine, an amphipathic molecule that alters membrane fluidity and curvature. The activity of mutant SNAREs was also rescued by the overexpression of SNAREs, thus multiplying the force transferred to the membrane. Thus, we conclude that either increasing membrane fluidity, or multiplying SNARE‐generated energy restored the fusogenicity of mutant SNAREs that are stalled at hemifusion. We also found that regulatory lipids differentially modulated the complex formation of wild‐type SNAREs. Together, these data indicate that the physical properties and the lipid composition of the membrane affect the function of SNAREs in promoting the hemifusion–fusion transition.     

The Fusion of Membranes and Vesicles: Pathway and Energy Barriers from Dissipative Particle Dynamics

The fusion of lipid bilayers is studied with dissipative particle dynamics simulations. First, to achieve control over membrane properties, the effects of individual simulation parameters are studied and optimized. Then, a large number of fusion events for a vesicle and a planar bilayer are simulated using the optimized parameter set. In the observed fusion pathway, configurations of individual lipids play an important role. Fusion starts with individual lipids assuming a splayed tail configuration with one tail inserted in each membrane. To determine the corresponding energy barrier, we measure the average work for interbilayer flips of a lipid tail, i.e., the average work to displace one lipid tail from one bilayer to the other. This energy barrier is found to depend strongly on a certain dissipative particle dynamics parameter, and, thus, can be adjusted in the simulations. Overall, three subprocesses have been identified in the fusion pathway. Their energy barriers are estimated to lie in the range 8-15 k_BT. The fusion probability is found to possess a maximum at intermediate tension values. As one decreases the tension, the fusion probability seems to vanish before the tensionless membrane state is attained. This would imply that the tension has to exceed a certain threshold value to induce fusion.     

Phosphatidylinositol and phosphatidylinositol-3-phosphate activate HOPS to catalyze SNARE assembly,allowing small headgroup lipids to support the terminal steps of membrane fusion

Intracellular membrane fusion requires Rab GTPases, tethers, SNAREs of the R, Qa, Qb, and Qc families, and SNARE chaperones of the Sec17 (SNAP), Sec18 (NSF), and SM (Sec1/Munc18) families. The vacuolar HOPS complex combines the functions of membrane tethering and SM catalysis of SNARE assembly. HOPS is activated for this catalysis by binding to the vacuolar lipids and Rab. Of the eight major vacuolar lipids, we now report that phosphatidylinositol and phosphatidylinositol-3-phosphate are required to activate HOPS for SNARE complex assembly. These lipids plus ergosterol also allow full trans-SNARE complex assembly, yet do not support fusion, which is reliant on either phosphatidylethanolamine (PE) or on phosphatidic acid (PA), phosphatidylserine (PS), and diacylglycerol (DAG). Fusion with a synthetic tether and without HOPS, or even without SNAREs, still relies on either PE or on PS, PA, and DAG. These lipids are thus required for the terminal bilayer rearrangement step of fusion, distinct from the lipid requirements for the earlier step of activating HOPS for trans-SNARE assembly.     

Protein-induced fusion can be modulated by target membrane lipids through a structural switch at the level of the fusion peptide

Regulatory features of protein-induced membrane fusion are largely unclear, particularly at the level of the fusion peptide. Fusion peptides being part of larger protein complexes, such investigations are met with technical limitations. Here, we show that the fusion activity of influenza virus or Golgi membranes is strongly inhibited by minor amounts of (lyso)lipids when present in the target membrane but not when inserted into the viral or Golgi membrane itself. To investigate the underlying mechanism, we employ a membrane-anchored peptide system and show that fusion is similarly regulated by these lipids when inserted into the target but not when present in the peptide-containing membrane. Peptide-induced fusion is regulated by a reversible switch of secondary structure from a fusion-permissive alpha-helix to a nonfusogenic beta-sheet. The "on/off" activation of this switch is governed by minor amounts of (lyso)-phospholipids in targets, causing a drop in alpha-helix and a dramatic increase in beta-sheet contents. Concomitantly, fusion is inhibited, due to impaired peptide insertion into the target membrane. Our observations in biological fusion systems together with the model studies suggest that distinct lipids in target membranes provide a means for regulating membrane fusion by causing a reversible secondary structure switch of the fusion peptides.     

Fusion of liposomes with the plasma membrane of epithelial cells: fate of incorporated lipids as followed by freeze fracture and autoradiography of plastic sections

The fusion of liposomes with the plasma membrane of influenza virus- infected monolayers of an epithelial cell line, Madin-Darby canine kidney cells (van Meer et al., 1985. Biochemistry. 24:3593-3602), has been analyzed by morphological techniques. The distribution of liposomal lipids over the apical and basolateral plasma membrane domains after fusion was assessed by autoradiography of liposomal [3H]dipalmitoylphosphatidylcholine after rapid freezing or chemical fixation and further processing by freeze substitution and low temperature embedding. Before fusion, radioactivity was solely detected on the apical cell surface, indicating the absence of redistribution artifacts and demonstrating the reliability of lipid autoradiography on both a light and electron microscopical level. After induction of fusion by a low pH treatment, the basolateral plasma membrane domain became progressively labeled, indicative of rapid lateral diffusion of [3H]dipalmitoylphosphatidylcholine in the plasma membrane. Analysis of individual fusion events by freeze fracture after rapid freezing confirmed the rapid diffusion of the liposomal lipids into the plasma membrane, as intramembrane particle-free lipid patches were never observed. After the induction of liposome-cell fusion, well-defined intramembrane particles were present on the otherwise smooth liposomal fracture faces and on the fracture faces of the plasma membrane. Morphological evidence thus was obtained in favor of a local point fusion mechanism with an intramembrane particle as a specific structural fusion intermediate.