A Compact,Multifunctional Fusion Module Directs Cholesterol-Dependent Homomultimerization and Syncytiogenic Efficiency of Reovirus p10 FAST Proteins

The homologous p10 fusion-associated small transmembrane (FAST) proteins of the avian (ARV) and Nelson Bay (NBV) reoviruses are the smallest known viral membrane fusion proteins, and are virulence determinants of the fusogenic reoviruses. The small size of FAST proteins is incompatible with the paradigmatic membrane fusion pathway proposed for enveloped viral fusion proteins. Understanding how these diminutive viral fusogens mediate the complex process of membrane fusion is therefore of considerable interest, from both the pathogenesis and mechanism-of-action perspectives. Using chimeric ARV/NBV p10 constructs, the 36–40-residue ectodomain was identified as the major determinant of the differing fusion efficiencies of these homologous p10 proteins. Extensive mutagenic analysis determined the ectodomain comprises two distinct, essential functional motifs. Syncytiogenesis assays, thiol-specific surface biotinylation, and liposome lipid mixing assays identified an ∼25-residue, N-terminal motif that dictates formation of a cystine loop fusion peptide in both ARV and NBV p10. Surface immunofluorescence staining, FRET analysis and cholesterol depletion/repletion studies determined the cystine loop motif is connected through a two-residue linker to a 13-residue membrane-proximal ectodomain region (MPER). The MPER constitutes a second, independent motif governing reversible, cholesterol-dependent assembly of p10 multimers in the plasma membrane. Results further indicate that: (1) ARV and NBV homomultimers segregate to distinct, cholesterol-dependent microdomains in the plasma membrane; (2) p10 homomultimerization and cholesterol-dependent microdomain localization are co-dependent; and (3) the four juxtamembrane MPER residues present in the multimerization motif dictate species-specific microdomain association and homomultimerization. The p10 ectodomain therefore constitutes a remarkably compact, multifunctional fusion module that directs syncytiogenic efficiency and species-specific assembly of p10 homomultimers into cholesterol-dependent fusion platforms in the plasma membrane.     

Features of a Spatially Constrained Cystine Loop in the p10 FAST Protein Ectodomain Define a New Class of Viral Fusion Peptides

The reovirus fusion-associated small transmembrane (FAST) proteins are the smallest known viral membrane fusion proteins. With ectodomains of only ∼20–40 residues, it is unclear how such diminutive fusion proteins can mediate cell-cell fusion and syncytium formation. Contained within the 40-residue ectodomain of the p10 FAST protein resides an 11-residue sequence of moderately apolar residues, termed the hydrophobic patch (HP). Previous studies indicate the p10 HP shares operational features with the fusion peptide motifs found within the enveloped virus membrane fusion proteins. Using biotinylation assays, we now report that two highly conserved cysteine residues flanking the p10 HP form an essential intramolecular disulfide bond to create a cystine loop. Mutagenic analyses revealed that both formation of the cystine loop and p10 membrane fusion activity are highly sensitive to changes in the size and spatial arrangement of amino acids within the loop. The p10 cystine loop may therefore function as a cystine noose, where fusion peptide activity is dependent on structural constraints within the noose that force solvent exposure of key hydrophobic residues. Moreover, inhibitors of cell surface thioreductase activity indicate that disruption of the disulfide bridge is important for p10-mediated membrane fusion. This is the first example of a viral fusion peptide composed of a small, spatially constrained cystine loop whose function is dependent on altered loop formation, and it suggests the p10 cystine loop represents a new class of viral fusion peptides.     

Cell-cell membrane fusion induced by p15 fusion-associated small transmembrane (FAST) protein requires a novel fusion peptide motif containing a myristoylated polyproline type II helix

The p15 fusion-associated small transmembrane (FAST) protein is a nonstructural viral protein that induces cell-cell fusion and syncytium formation. The exceptionally small, myristoylated N-terminal ectodomain of p15 lacks any of the defining features of a typical viral fusion protein. NMR and CD spectroscopy indicate this small fusion module comprises a left-handed polyproline type II (PPII) helix flanked by small, unstructured N and C termini. Individual prolines in the 6-residue proline-rich motif are highly tolerant of alanine substitutions, but multiple substitutions that disrupt the PPII helix eliminate cell-cell fusion activity. A synthetic p15 ectodomain peptide induces lipid mixing between liposomes, but with unusual kinetics that involve a long lag phase before the onset of rapid lipid mixing, and the length of the lag phase correlates with the kinetics of peptide-induced liposome aggregation. Lipid mixing, liposome aggregation, and stable peptide-membrane interactions are all dependent on both the N-terminal myristate and the presence of the PPII helix. We present a model for the mechanism of action of this novel viral fusion peptide, whereby the N-terminal myristate mediates initial, reversible peptide-membrane binding that is stabilized by subsequent amino acid-membrane interactions. These interactions induce a biphasic membrane fusion reaction, with peptide-induced liposome aggregation representing a distinct, rate-limiting event that precedes membrane merger. Although the prolines in the proline-rich motif do not directly interact with membranes, the PPII helix may function to force solvent exposure of hydrophobic amino acid side chains in the regions flanking the helix to promote membrane binding, apposition, and fusion.     

Computational insight in the role of fusogenic lipopeptides at the onset of liposome fusion

We performed an extensive computational study to obtain insight in the molecular mechanisms that take place prior to membrane fusion. We focused on membrane-anchored hybrid macromolecules (lipid–polymer–oligopeptide) that mimic biological SNARE proteins in terms of liposome fusion characteristics [H. Robson Marsden et al., 2009]; efficient micro-second simulation was enabled by combining validated MARTINI force fields for the molecular building blocks in coarse-grained molecular dynamics (CGMD). We find that individual peptide domains in the hybrid macromolecules bind and partially integrate parallel to the membrane surface, in agreement with experimental findings. By varying several experimental design parameters, we observe that peptide domains remain in the solvent phase only in two cases: (1) for solitary lipopeptides (low concentration), below a threshold area per lipid in the membrane, and (2) when the lipopeptide concentration is high enough for the peptide domains to self-assemble into tetrameric homo-complexes. The peptide–membrane binding is not affected by solvent-induced peptide unfolding, which we mimicked by relaxing the usual MARTINI helix constraints. Remarkably, in this case, a reverse transition to a helical secondary structure is observed after binding, highlighting the role of the membrane as a template (partitioning–folding coupling). Our findings undermine the current view of the initial stages towards fusion, in which membranes are thought to be kept in close apposition via dimerization of individual complementary peptides in the solvent phase. Although we did not study actual fusion, our simulations show that the formation of homomers, which is suppressed in experimental peptide-pair design and therefore believed to be insignificant for fusion, by peptides anchored to the same membrane does play a key role in this locking mechanism and potentially also in membrane destabilization that precede fusion.     

Myristoylation, a protruding loop, and structural plasticity are essential features of a nonenveloped virus fusion peptide motif

Members of the fusion-associated small transmembrane (FAST) protein family are a distinct class of membrane fusion proteins encoded by nonenveloped fusogenic reoviruses. The 125-residue p14 FAST protein of reptilian reovirus has an approximately 38-residue myristoylated N-terminal ectodomain containing a moderately apolar N-proximal region, termed the hydrophobic patch. Mutagenic analysis indicated sequence-specific elements in the N-proximal portion of the p14 hydrophobic patch affected cell-cell fusion activity, independent of overall effects on the relative hydrophobicity of the motif. Circular dichroism (CD) of a myristoylated peptide representing the majority of the p14 ectodomain suggested this region is mostly disordered in solution but assumes increased structure in an apolar environment. From NMR spectroscopic data and simulated annealing, the soluble nonmyristoylated p14 ectodomain peptide consists of an N-proximal extended loop flanked by two proline hinges. The remaining two-thirds of the ectodomain peptide structure is disordered, consistent with predictions based on CD spectra of the myristoylated peptide. The myristoylated p14 ectodomain peptide, but not a nonmyristoylated version of the same peptide nor a myristoylated scrambled peptide, mediated extensive lipid mixing in a liposome fusion assay. Based on the lipid mixing activity, structural plasticity, environmentally induced conformational changes, and kinked structures predicted for the p14 ectodomain and hydrophobic patch (all features associated with fusion peptides), we propose that the majority of the p14 ectodomain is composed of a fusion peptide motif, the first such motif dependent on myristoylation for membrane fusion activity.     

Structural and functional properties of an unusual internal fusion peptide in a nonenveloped virus membrane fusion protein

The avian and Nelson Bay reoviruses are two of only a limited number of nonenveloped viruses capable of inducing cell-cell membrane fusion. These viruses encode the smallest known membrane fusion proteins (p10). We now show that a region of moderate hydrophobicity we call the hydrophobic patch (HP), present in the small N-terminal ectodomain of p10, shares the following characteristics with the fusion peptides of enveloped virus fusion proteins: (i) an abundance of glycine and alanine residues, (ii) a potential amphipathic secondary structure, (iii) membrane-seeking characteristics that correspond to the degree of hydrophobicity, and (iv) the ability to induce lipid mixing in a liposome fusion assay. The p10 HP is therefore predicted to provide a function in the mechanism of membrane fusion similar to those of the fusion peptides of enveloped virus fusion peptides, namely, association with and destabilization of opposing lipid bilayers. Mutational and biophysical analysis suggested that the internal fusion peptide of p10 lacks alpha-helical content and exists as a disulfide-stabilized loop structure. Similar kinked structures have been reported in the fusion peptides of several enveloped virus fusion proteins. The preservation of a predicted loop structure in the fusion peptide of this unusual nonenveloped virus membrane fusion protein supports an imperative role for a kinked fusion peptide motif in biological membrane fusion.     

Computational insight in the role of fusogenic lipopeptides at the onset of liposome fusion

We performed an extensive computational study to obtain insight in the molecular mechanisms that take place prior to membrane fusion. We focused on membrane-anchored hybrid macromolecules (lipid–polymer–oligopeptide) that mimic biological SNARE proteins in terms of liposome fusion characteristics [H. Robson Marsden et al., 2009]; efficient micro-second simulation was enabled by combining validated MARTINI force fields for the molecular building blocks in coarse-grained molecular dynamics (CGMD). We find that individual peptide domains in the hybrid macromolecules bind and partially integrate parallel to the membrane surface, in agreement with experimental findings. By varying several experimental design parameters, we observe that peptide domains remain in the solvent phase only in two cases: (1) for solitary lipopeptides (low concentration), below a threshold area per lipid in the membrane, and (2) when the lipopeptide concentration is high enough for the peptide domains to self-assemble into tetrameric homo-complexes. The peptide-membrane binding is not affected by solvent-induced peptide unfolding, which we mimicked by relaxing the usual MARTINI helix constraints. Remarkably, in this case, a reverse transition to a helical secondary structure is observed after binding, highlighting the role of the membrane as a template (partitioning-folding coupling). Our findings undermine the current view of the initial stages towards fusion, in which membranes are thought to be kept in close apposition via dimerization of individual complementary peptides in the solvent phase. Although we did not study actual fusion, our simulations show that the formation of homomers, which is suppressed in experimental peptide pair design and therefore believed to be insignificant for fusion, by peptides anchored to the same membrane does play a key role in this locking mechanism and potentially also in membrane destabilization that precedes fusion.     

Architecture of a nascent viral fusion pore

Enveloped viruses use specialized protein machinery to fuse the viral membrane with that of the host cell during cell invasion. In influenza virus, hundreds of copies of the haemagglutinin (HA) fusion glycoprotein project from the virus surface. Despite intensive study of HA and its fusion activity, the protein's modus operandi in manipulating viral and target membranes to catalyse their fusion is poorly understood. Here, the three‐dimensional architecture of influenza virus–liposome complexes at pH 5.5 was investigated by electron cryo‐tomography. Tomographic reconstructions show that early stages of membrane remodeling take place in a target membrane‐centric manner, progressing from punctate dimples, to the formation of a pinched liposomal funnel that may impinge on the apparently unperturbed viral envelope. The results suggest that the M1 matrix layer serves as an endoskeleton for the virus and a foundation for HA during membrane fusion. Fluorescence spectroscopy monitoring fusion between liposomes and virions shows that leakage of liposome contents takes place more rapidly than lipid mixing at pH 5.5. The relation of ‘leaky’ fusion to the observed prefusion structures is discussed.     

Sequential Partially Overlapping Gene Arrangement in the Tricistronic S1 Genome Segments of Avian Reovirus and Nelson Bay Reovirus: Implications for Translation Initiation

Previous studies of the avian reovirus strain S1133 (ARV-S1133) S1 genome segment revealed that the open reading frame (ORF) encoding the final sigmaC viral cell attachment protein initiates over 600 nucleotides distal from the 5' end of the S1 mRNA and is preceded by two predicted small nonoverlapping ORFs. To more clearly define the translational properties of this unusual polycistronic RNA, we pursued a comparative analysis of the S1 genome segment of the related Nelson Bay reovirus (NBV). Sequence analysis indicated that the 3'-proximal ORF present on the NBV S1 genome segment also encodes a final sigmaC homolog, as evidenced by the presence of an extended N-terminal heptad repeat characteristic of the coiled-coil region common to the cell attachment proteins of reoviruses. Most importantly, the NBV S1 genome segment contains two conserved ORFs upstream of the final sigmaC coding region that are extended relative to the predicted ORFs of ARV-S1133 and are arranged in a sequential, partially overlapping fashion. Sequence analysis of the S1 genome segments of two additional strains of ARV indicated a similar overlapping tricistronic gene arrangement as predicted for the NBV S1 genome segment. Expression analysis of the ARV S1 genome segment indicated that all three ORFs are functional in vitro and in virus-infected cells. In addition to the previously described p10 and final sigmaC gene products, the S1 genome segment encodes from the central ORF a 17-kDa basic protein (p17) of no known function. Optimizing the translation start site of the ARV p10 ORF lead to an approximately 15-fold increase in p10 expression with little or no effect on translation of the downstream final sigmaC ORF. These results suggest that translation initiation complexes can bypass over 600 nucleotides and two functional overlapping upstream ORFs in order to access the distal final sigmaC start site.     

A role of complexin-lipid interactions in membrane fusion

Complexins (Cpxs) and synaptotagmins regulate calcium-dependent exocytosis. A central helix in Cpx confers specific binding to the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) fusion machinery. An accessory helix in the amino-terminal region inhibits membrane fusion by blocking SNAREpin zippering. We now show that an amphipathic helix in the carboxy-terminal region of CpxI binds lipid bilayers and affects SNARE-mediated lipid mixing in a liposome fusion assay. The substitution of a hydrophobic amino acid within the helix by a charged residue abolishes the lipid interaction and the stimulatory effect of CpxI in liposome fusion. In contrast, the introduction of the bulky hydrophobic amino acid tryptophan stimulates lipid binding and liposome fusion. This data shows that local Cpx-lipid interactions can play a role in membrane fusion.     

Membrane fusion by proline-rich Rz1 lipoprotein, the bacteriophage lambda Rz1 gene product.

The fusogenic properties of Rz1, the proline-rich lipoprotein that is the bacteriophage lambda Rz1 gene product, were studied. Light scattering was used to monitor Rz1-induced aggregation of artificial neutral (dipalmitoylphosphatidylcholine/cholesterol) and negatively charged (dipalmitoylphosphatidylcholine/cholesterol/dioleoylphosphatidylserin e) liposomes. Fluorescence assays [the resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)dihexadecanol-sn-glycero-3-phosphoethanolamine lipid fluorescent probes, as well as fluorescent complex formation between terbium ions and dipicolinic acid encapsulated in two liposome populations and calcein fluorescence] were used to monitor Rz1-induced lipid mixing, contents mixing and leakage of neutral and negatively charged liposomes. The results demonstrated that Rz1 caused adhesion of neutral and negatively charged liposomes with concomitant lipid mixing; membrane distortion, leading to the fusion of liposomes and hence their internal content mixing; and local destruction of the membrane accompanied by leakage of the liposome contents. The use of artificial membranes showed that Rz1 induced the fusion of membranes devoid of any proteins. This might mean that the proline stretch of Rz1 allowed interaction with membrane lipids. It is suggested that Rz1-induced liposome fusion was mediated primarily by the generation of local perturbation in the bilayer lipid membrane and to a lesser extent by electrostatic forces.     

Liposome reconstitution of a minimal protein-mediated membrane fusion machine

Biological membrane fusion is dependent on protein catalysts to mediate localized restructuring of lipid bilayers. A central theme in current models of protein-mediated membrane fusion involves the sequential refolding of complex homomeric or heteromeric protein fusion machines. The structural features of a new family of fusion-associated small transmembrane (FAST) proteins appear incompatible with existing models of membrane fusion protein function. While the FAST proteins function to induce efficient cell-cell fusion when expressed in transfected cells, it was unclear whether they function on their own to mediate membrane fusion or are dependent on cellular protein cofactors. Using proteoliposomes containing the purified p14 FAST protein of reptilian reovirus, we now show via liposome-cell and liposome-liposome fusion assays that p14 is both necessary and sufficient for membrane fusion. Stoichiometric and kinetic analyses suggest that the relative efficiency of p14-mediated membrane fusion rivals that of the more complex cellular and viral fusion proteins, making the FAST proteins the simplest known membrane fusion machines.     

Engineering cell surfaces via liposome fusion

In this study, we have rewired cell surfaces with ketone and oxyamine molecules based on liposome fusion for applications in cell-surface engineering. Lipid vesicles, functionalized with ketone and oxyamine molecules, display complementary chemistry and undergo recognition, docking, and subsequent fusion upon covalent oxime bond formation. Liposome fusion was characterized by several techniques including matrix-assisted laser-desorption/ionization mass spectrometry (MALDI-MS), light scattering, fluorescence resonance energy transfer (FRET), and transmission electron microscopy (TEM). When cultured with cells, ketone- and oxyamine-containing liposomes undergo spontaneous membrane fusion to present the respective molecules from cell surfaces. Ketone-functionalized cell surfaces serve as sites for chemoselective ligation with oxyamine-conjugated molecules. We tailored and fluorescently labeled cell surfaces with an oxyamine-conjugated rhodamine dye. As an application of this cell-surface engineering strategy, ketone- and oxyamine-functionalized cells were patterned on oxyamine- and ketone-presenting surfaces, respectively. Cells adhered, spread, and proliferated in the patterned regions via interfacial oxime linkage. The number of ketone molecules on the cell surface was also quantified by flow cytometry.     

The p14 fusion-associated small transmembrane (FAST) protein effects membrane fusion from a subset of membrane microdomains

The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX- and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipid-anchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.     

Atypical fusion peptide of Nelson Bay virus fusion-associated small transmembrane protein

A 10-kDa nonstructural transmembrane protein (p10) encoded by a reovirus, Nelson Bay virus, has been shown to induce syncytium formation (34). Sequence analysis and structural studies identified p10 as a type I membrane protein with a central transmembrane domain, a cytoplasmic basic region, and an N-terminal hydrophobic domain (HD) that was hypothesized to function as a fusion peptide. We performed mutational analysis on this slightly hydrophobic motif to identify possible structural requirements for fusion activity. Bulky aliphatic residues were found to be essential for optimal fusion, and an aromatic or highly hydrophobic side chain was found to be required at position 12. The requirement for hydrophilic residues within the HD was also examined: substitution of 10-Ser or 14-Ser with hydrophobic residues was found to reduce cell surface expression of p10 and delayed the onset of syncytium formation. Nonconservative substitutions of charged residues in the HD did not have an effect on fusion activity. Taken together, our results suggest that the HD is involved in both syncytium formation and in determining p10 transport and surface expression.     

Overexpression of thiol/disulfide isomerases enhances membrane fusion directed by the Newcastle disease virus fusion protein

Newcastle disease virus (NDV) fusion (F) protein directs membrane fusion, which is required for virus entry and cell-cell fusion. We have previously shown that free thiols are present in cell surface-expressed NDV F protein and that blocking the production of free thiols by thiol-disulfide exchange inhibitors inhibited the membrane fusion mediated by F protein (J Virol. 81:2328-2339, 2007). Extending these observations, we evaluated the role of the overexpression of two disulfide bond isomerases, protein disulfide isomerase (PDI) and ERdj5, in cell-cell fusion mediated by NDV glycoproteins. The overexpression of these isomerases resulted in significantly increased membrane fusion, as measured by syncytium formation and content mixing. The overexpression of these isomerases enhanced the production of free thiols in F protein when expressed without hemagglutination-neuraminidase (HN) protein but decreased free thiols in F protein expressed with HN protein. By evaluating the binding of conformation-sensitive antibodies, we found that the overexpression of these isomerases favored a postfusion conformation of surface-expressed F protein in the presence of HN protein. These results suggest that isomerases belonging to the PDI family catalyze the production of free thiols in F protein, and free thiols in F protein facilitate membrane fusion mediated by F protein.     

Membrane perturbation and fusion pore formation in influenza hemagglutinin-mediated membrane fusion. A new model for fusion

Low pH-induced fusion mediated by the hemagglutinin (HA) of influenza virus involves conformational changes in the protein that lead to the insertion of a "fusion peptide" domain of this protein into the target membrane and is thought to perturb the membrane, triggering fusion. By using whole virus, purified HA, or HA ectodomains, we found that shortly after insertion, pores of less than 26 A in diameter were formed in liposomal membranes. As measured by a novel assay, these pores stay open, or continue to close and open, for minutes to hours and persist after pH neutralization. With virus and purified HA, larger pores, allowing the leakage of dextrans, were seen at times well after insertion. For virus, dextran leakage was simultaneous with lipid mixing and the formation of "fusion pores," allowing the transfer of dextrans from the liposomal to the viral interior or vice versa. Pores did not form in the viral membrane in the absence of a target membrane. Based on these data, we propose a new model for fusion, in which HA initially forms a proteinaceous pore in the target, but not in the viral membrane, before a lipidic hemifusion intermediate is formed.     

Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor

Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation.     

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.     

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.