Fusogenic Activity Research Articles

Quantitative Analysis of Lipid Droplet Fusion: Inefficient Steady State Fusion but Rapid Stimulation by Chemical Fusogens

Lipid droplets (LDs) are dynamic cytoplasmic organelles containing neutral lipids and bounded by a phospholipid monolayer. Previous studies have suggested that LDs can undergo constitutive homotypic fusion, a process linked to the inhibitory effects of fatty acids on glucose transporter trafficking. Using strict quantitative criteria for LD fusion together with refined light microscopic methods and real-time analysis, we now show that LDs in diverse cell types show low constitutive fusogenic activity under normal growth conditions. To investigate the possible modulation of LD fusion, we screened for agents that can trigger fusion. A number of pharmacological agents caused homotypic fusion of lipid droplets in a variety of cell types. This provided a novel cell system to study rapid regulated fusion between homotypic phospholipid monolayers. LD fusion involved an initial step in which the two adjacent membranes became continuous (<10 s), followed by the slower merging (100 s) of the neutral lipid cores to produce a single spherical LD. These fusion events were accompanied by changes to the LD surface organization. Measurements of LDs undergoing homotypic fusion showed that fused LDs maintained their initial volume, with a corresponding decrease in surface area suggesting rapid removal of membrane from the fused LD. This study provides estimates for the level of constitutive LD fusion in cells and questions the role of LD fusion in vivo. In addition, it highlights the extent of LD restructuring which occurs when homotypic LD fusion is triggered in a variety of cell types.

A single amino acid substitution in the haemagglutinin-neuraminidase protein of Newcastle disease virus results in increased fusion promotion and decreased neuraminidase activities without changes in virus pathotype

Attachment of Newcastle disease virus (NDV) to the host cell is mediated by the haemagglutinin-neuraminidase (HN), a multifunctional protein that has receptor recognition, neuraminidase (NA) and fusion promotion activities. The process that connects receptor binding and fusion triggering is poorly understood and amino acid residues important for the functions of the protein remain to be fully determined. During the process of generating an infectious clone of the Anhinga strain of NDV, we were able to rescue a NDV with highly increased fusogenic activity in vitro and decreased haemagglutinating activity, as compared with the wild-type parental strain. Sequencing of this recombinant virus showed a single mutation at amino acid position 192 of the HN protein (Ile→Met). In the present study, we characterized that single amino acid substitution (I192M) in three strains of NDV by assessing the NA activity and fusogenic potential of the mutated versus wild-type proteins in cell cultures. The original recombinant NDV harbouring the mutation in the HN gene was also used to characterize the phenotype of the virus in cell cultures, embryonated chicken eggs and day-old chickens. Mutation I192M results in low NA activity and highly increased cell fusion in vitro, without changes in the viral pathotype of recombinant viruses harbouring the mutation in vivo. The results obtained suggest that multiple regions of the HN-protein globular head are important for fusion promotion, and that wild-type levels of NA activity are not absolutely required for viral infection.

All-or-None versus Graded: Single-Vesicle Analysis Reveals Lipid Composition Effects on Membrane Permeabilization

We report a single-vesicle approach to compare the all-or-none and graded mechanisms of lipid bilayer permeabilization by CpreTM and NpreTM, two peptides derived from the membrane-proximal external region of the HIV fusion glycoprotein gp41subunit. According to bulk requenching assays, these peptides permeabilize large unilamellar vesicles via all-or-none and graded mechanisms, respectively. Visualization of the process using giant unilamellar vesicles shows that the permeabilization of individual liposomes by these two peptides differs in kinetics, degree of dye filling, and stability of the permeabilized state. All-or-none permeabilization by CpreTM is characterized by fast and total filling of the individual vesicles. This process is usually accompanied by the formation of stably open pores, as judged from the capacity of the vesicles to incorporate a second dye added after several hours. In contrast, graded permeabilization by NpreTM is transient and exhibits slower kinetics, which leads to partial filling of the individual liposomes. Of importance, quantitative analysis of vesicle population distribution allowed the identification of mixed mechanisms of membrane permeabilization and the assessment of cholesterol effects. Specifically, the presence of this viral envelope lipid increased the stability of the permeating structures, which may have implications for the fusogenic activity of gp41.

Cascade of Events Governing Cell-Cell Fusion Induced by Herpes Simplex Virus Glycoproteins gD, gH/gL, and gB

Herpesviruses minimally require the envelope proteins gB and gH/gL for virus entry and cell-cell fusion; herpes simplex virus (HSV) additionally requires the receptor-binding protein gD. Although gB is a class III fusion protein, gH/gL does not resemble any documented viral fusion protein at a structural level. Based on those data, we proposed that gH/gL does not function as a cofusogen with gB but instead regulates the fusogenic activity of gB. Here, we present data to support that hypothesis. First, receptor-positive B78H1-C10 cells expressing gH/gL fused with receptor-negative B78H1 cells expressing gB and gD (fusion in trans). Second, fusion occurred when gH/gL-expressing C10 cells preexposed to soluble gD were subsequently cocultured with gB-expressing B78 cells. In contrast, prior exposure of gB-expressing C10 cells to soluble gD did not promote subsequent fusion with gH/gL-expressing B78 cells. These data suggest that fusion involves activation of gH/gL by receptor-bound gD. Most importantly, soluble gH/gL triggered a low level of fusion of C10 cells expressing gD and gB; a much higher level was achieved when gB-expressing C10 cells were exposed to a combination of soluble gH/gL and gD. These data clearly show that gB acts as the HSV fusogen following activation by gD and gH/gL. We suggest the following steps leading to fusion: (i) conformational changes to gD upon receptor binding, (ii) alteration of gH/gL by receptor-activated gD, and (iii) upregulation of the fusogenic potential of gB following its interaction with activated gH/gL. The third step may be common to other herpesviruses.

Mitochondrial shape changes: orchestrating cell pathophysiology

Mitochondria are highly dynamic organelles, the location, size and distribution of which are controlled by a family of proteins that modulate mitochondrial fusion and fission. Recent evidence indicates that mitochondrial morphology is crucial for cell physiology, as changes in mitochondrial shape have been linked to neurodegeneration, calcium signalling, lifespan and cell death. Because immune cells contain few mitochondria, these organelles have been considered to have only a marginal role in this physiological context-which is conversely well characterized from the point of view of signalling. Nevertheless, accumulating evidence shows that mitochondrial dynamics have an impact on the migration and activation of immune cells and on the innate immune response. Here, we discuss the roles of mitochondrial dynamics in cell pathophysiology and consider how studying dynamics in the context of the immune system could increase our knowledge about the role of dynamics in key signalling cascades.

The ratio of unsaturated fatty acids in biosurfactants affects the efficiency of gene transfection

An unsaturated hydrocarbon chain in phospholipid was reported to affect a phase transition and a fusogenic activity after mixing membranes, and consequently to achieve a high DNA transfection efficiency. We previously showed that a biosurfactant mannosylerythritol lipid-A (MEL-A) enhances the gene transfection efficiency of cationic liposomes. Here, we have studied the effects of unsaturated fatty acid ratio of MEL-A on the physicochemical properties and gene delivery into cells of cationic liposomes using MEL-A with three different unsaturated fatty acid ratios (9.1%, 21.5%, and 46.3%). The gene transfer efficiency of cationic liposomes containing MEL-A (21.5%) was much higher than that of those containing MEL-A (9.1%) and MEL-A (46.3%). MEL-A (21.5%)-containing cationic liposomes induced highly efficient membrane fusion after addition of anionic liposomes and led to subsequent DNA release. Imaging analysis revealed that MEL-A (21.5%)-containing liposomes fused with the plasma membrane and delivered DNA into the nucleus of NIH-3T3 cells, MEL-A (46.3%)-containing liposomes fused with the plasma membrane did not deliver DNA into the nucleus, and MEL-A (9.1%)-containing liposomes neither fused with the plasma membrane nor delivered DNA into the nucleus. Thus, it is understandable that the unsaturated fatty acid ratio of MEL-A strongly influences the gene transfection efficiency of cationic liposomes.

Distinct mechanisms of lipid bilayer perturbation induced by peptides derived from the membrane-proximal external region of HIV-1 gp41

The conserved, membrane-proximal external region (MPER) of the human immunodeficiency virus type-1 envelope glycoprotein 41 subunit is required for fusogenic activity. It has been proposed that MPER functions by disrupting the virion membrane. Supporting its critical role in viral entry as a membrane-bound entity, MPER constitutes the target for broadly neutralizing antibodies that have evolved mechanisms to recognize membrane-inserted epitopes. We have analyzed here the molecular mechanisms of membrane permeabilization induced by N-preTM and PreTM-C, two peptides derived from MPER sequences showing a tendency to associate with the bilayer interface or to transfer into the hydrocarbon core, respectively. Both peptides contained the full epitope sequence recognized by the 4E10 monoclonal antibody (MAb4E10), which was subsequently used to probe peptide accessibility from the water phase. Capacities of N-preTM and PreTM-C for associating with vesicles and inducing their permeabilization were comparable. Howe...

2-Methacryloyloxyethyl phosphorylcholine polymer (MPC)-coating improves the transfection activity of GALA-modified lipid nanoparticles by assisting the cellular uptake and intracellular dissociation of plasmid DNA in primary hepatocytes

We previously reported that modification of GALA peptide on the surface of liposomes enhanced fusion with endosomal membrane, and cytoplasmic release of encapsulated macromolecules. We report herein that an additional coating of GALA-modified liposomes with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer resulted in a two order of magnitude enhancement in the transfection activity of encapsulating plasmid DNA (pDNA). Quantification of the delivered gene copies in whole cells and isolated nuclei revealed that the increase of transfection activity can be attributed to improved efficiencies in cellular uptake and post-nuclear delivery processes. Imaging studies revealed that the intracellular dissociation of pDNA from the lipid envelope is enhanced by GALA modification and further coating with MPC polymer in a stepwise manner. The MPC polymer-coating decreased the ζ-potential of GALA-modified liposomes, suggesting that it assisted in the functional display of negatively charged GALA on the cationic liposomes by providing shielding from mutual electrostatic interactions. Collectively, these data indicate that MPC polymer-coating induced the fusogenic activity of the GALA-modified envelope with endosomes, leading to a more effective cytoplasmic release pDNA. The extensive fusion of the lipid envelope may also reduce electrostatic interactions between mRNA and cationic lipid components, thereby resulting in an enhancement in the translation process.

GCM1 Regulation of the Expression of Syncytin 2 and Its Cognate Receptor MFSD2A in Human Placenta1

Syncytin 2 is a newly identified placental membrane protein with fusogenic and immunosuppressive activities. Major facilitator superfamily domain containing 2A (MFSD2A) is the cognate receptor for syncytin 2-mediated cell-cell fusion. Both syncytin 2 and MFSD2A are highly expressed in placenta. In this study to understand the regulation of syncytin 2 and MFSD2A expression in placenta, we found that syncytin 2 gene is epigenetically silenced in nonplacental cells by cytosine-phosphate-guanine (CpG) dinucleotide methylation and that expression of syncytin 2 and MFSD2A genes are regulated by the placental transcription factor GCM1 in placental cells. Functional GCM1-binding sites were identified in syncytin 2 and MFSD2A promoters based on electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Because GCM1 activity is decreased in hypoxic placental cells, we further confirmed that expression of MFSD2A is downregulated in hypoxic BeWo choriocarcinoma cells. Interestingly, ectopic expression of GCM1 activated syncytin 2 and MFSD2A expression in MCF-7 breast cancer cells and facilitated MCF-7 cell fusion. The expression of syncytin 2 in MCF-7 cells was partly attributed to CpG demethylation in the syncytin 2 promoter in the presence of GCM1. Our results suggest that GCM1 is a critical factor in controlling placental cell fusion through transcriptional regulation of syncytin 2 and MFSD2A gene expression in placenta. In addition, GCM1 may also play an important role in the epigenetic regulation of syncytin 2 gene expression.

The SM protein Vps33 and the t-SNARE Habc domain promote fusion pore opening

Intracellular membrane fusion proceeds via distinct stages of membrane docking, hemifusion and fusion pore opening and depends on interacting families of Rab, SNARE and SM proteins. Trans-SNARE complexes dock the membranes in close apposition. Efficient fusion requires further SNARE-associated proteins. They might increase the number of trans-SNARE complexes or the fusogenic potential of a single SNARE complex. We investigated the contributions of the SM protein Vps33 to hemifusion and pore opening between yeast vacuoles. Mutations in Vps33 that weaken its interactions with the SNARE complex allowed normal trans-SNARE pairing and lipid mixing but retarded content mixing. Deleting the H(abc) domain of the vacuolar t-SNARE Vam3, which interacts with Vps33, had the same effect. This suggests that SM proteins promote fusion pore opening by enhancing the fusogenic activity of a SNARE complex. They should thus be considered integral parts of the fusion machinery.

Structure of the Newcastle disease virus F protein in the post-fusion conformation.

The paramyxovirus F protein is a class I viral membrane fusion protein which undergoes a significant refolding transition during virus entry. Previous studies of the Newcastle disease virus, human parainfluenza virus 3 and parainfluenza virus 5 F proteins revealed differences in the pre- and post-fusion structures. The NDV Queensland (Q) F structure lacked structural elements observed in the other two structures, which are key to the refolding and fusogenic activity of F. Here we present the NDV Australia-Victoria (AV) F protein post-fusion structure and provide EM evidence for its folding to a pre-fusion form. The NDV AV F structure contains heptad repeat elements missing in the previous NDV Q F structure, forming a post-fusion six-helix bundle (6HB) similar to the post-fusion hPIV3 F structure. Electrostatic and temperature factor analysis of the F structures points to regions of these proteins that may be functionally important in their membrane fusion activity.

The Presence of a Single N-terminal Histidine Residue Enhances the Fusogenic Properties of a Membranotropic Peptide Derived from Herpes Simplex Virus Type 1 Glycoprotein H

Herpes simplex virus type 1 (HSV-1)-induced membrane fusion remains one of the most elusive mechanisms to be deciphered in viral entry. The structure resolution of glycoprotein gB has revealed the presence of fusogenic domains in this protein and pointed out the key role of gB in the entry mechanism of HSV-1. A second putative fusogenic glycoprotein is represented by the heterodimer comprising the membrane-anchored glycoprotein H (gH) and the small secreted glycoprotein L, which remains on the viral envelope in virtue of its non-covalent interaction with gH. Different domains scattered on the ectodomain of HSV-1 gH have been demonstrated to display membranotropic characteristics. The segment from amino acid 626 to 644 represents the most fusogenic region identified by studies with synthetic peptides and model membranes. Herein we have identified the minimal fusogenic sequence present on gH. An enlongation at the N terminus of a single histidine (His) has proved to profoundly increase the fusogenic activity of the original sequence. Nuclear magnetic resonance (NMR) studies have shown that the addition of the N-terminal His contributes to the formation and stabilization of an alpha-helical domain with high fusion propensity.

Modification of the pH‐dependent function of the HMPV F protein

Paramyxoviruses are a diverse class of negative‐strand RNA viruses that contain numerous clinically relevant human pathogens, including human metapneumovirus (HMPV), a recently identified respiratory pathogen for which there is currently no treatment. Amidst the open reading frames encoded within the HMPV genome is the fusion (F) protein, an integral membrane protein responsible for fusion of the viral and host membranes, resulting in delivery of a viral particle into the host cell and subsequent infection. In this study, we sought to further examine the novel pH‐dependent mechanism of the HMPV F protein recently described in our lab. Prior studies of other paramyxoviral fusion proteins have identified moieties in the HRA and F2 regions of the protein that interact in the pre‐fusion state to form a putative trigger for membrane fusion. Site‐directed mutagenesis of key residues in these regions of HMPV F resulted in dramatic effects fusogenic activity in vitro, including effects on the dependency of low pH. We hypothesize that these mutations may have effects on F pre‐fusion stability and conformational change to the post‐fusion state. The data suggest that alanine mutations of some of these residues may lower stability of the pre‐fusion state, leading to increased triggering, and in some cases, may be sufficient to overcome the requirement for low pH for transition to the post‐fusion conformation. Research supported by NIH R01 AI051517.

Carboxylated hyperbranched poly(glycidol)s for preparation of pH-sensitive liposomes

Previous reports by the authors described intracellular delivery using liposomes modified with various carboxylated poly(glycidol) derivatives. These linear polymer-modified liposomes exhibited a pH-dependent membrane fusion behavior in cellular acidic compartments. However, the effect of the backbone structure on membrane fusion activity remains unknown. Therefore, this study specifically investigated the backbone structure to obtain pH-sensitive polymers with much higher fusogenic activity and to reveal the effect of the polymer backbone structure on the interaction with the membrane. Hyperbranched poly(glycidol) (HPG) derivatives were prepared as a new type of pH-sensitive polymer and used for the modification of liposomes. The resultant HPG derivatives exhibited high hydrophobicity and intensive interaction with the membrane concomitantly with the increasing degree of polymerization (DP). Furthermore, HPG derivatives showed a stronger interaction with the membrane than the linear polymers show. Liposomes modified with HPG derivatives of high DP delivered contents into the cytosol of DC2.4 cells, a dendritic cell line, more effectively than the linear polymer-modified liposomes do. Results show that the backbone structure of pH-sensitive polymers affected their pH-sensitivity and interaction with liposomal and cellular membranes.

Fusogenic Activity of Cationic Lipids Correlates with Lipid Shape Distribution

Cationic lipids are efficient and popular agents to transport anionic molecules (DNA, RNA, anionic proteins) into cells. Addition of the co-lipid DOPE into cationic lipid formulations is considered as promoting cell delivery of DNA by enhancing fusion processes with cell membranes. Here we demonstrate, by combining FRET and confocal microscopy, that some cationic lipids do not require a co-lipid to fuse efficiently with cells. These cationic lipids are able to self-organize into bilayers that are stable enough to form liposomes, while presenting some destabilizing properties reminiscent of the conically-shaped fusogenic co-lipid, DOPE.

Fusogenic Activity of PLA2-IIA and SMase in PEG-Mediated Membrane Fusion

Phospholipase A2-IIA (PLA2-IIA) and sphingomyelinase (SMase) have been shown to induce fusion of membranes both in vivo and in vitro. The fusogenic potential of these two enzymes has been observed, for example, during neurotransmitter release. It is also believed that the products of hydrolysis of these two enzymes induce a reduction in the free energy cost of the fusion event. Assessment of the biophysical mechanisms by which these enzymes propel membrane fusion may provide relevant information for a variety of biological scenarios including neurotransmitter release. Poly(ethylene glycol)-(PEG-) mediated fusion of small unilamellar vesicles (SUVs) was studied in the presence of PLA2-IIA and SMase at varying temperatures between 17C and 40C. Lipid composition was dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), porcine brain sphingomyelin (SM), and cholesterol (CH) in a ratio of 35:30:15:20. Content mixing (CM), lipid mixing (LM) and leakage (L) time courses were obtained and globally fitted to a 3 or 4-state sequential vesicle fusion model introduced by Weinberg and Lentz (Weinberg and Lentz, Biophys. J., 2007, 92; 4012). Based on the global fits, rate constants for the transitions between states, and LM, CM and L occurrence probabilities are obtained. We observe that both LM and CM are enhanced by enzyme activity in SUVs. In addition, the exogenous addition of hydrolysis products also promotes LM and CM in the absence of the enzymes. A careful study of the temperature dependent plots is used to explore alterations in the energy barriers between intermediate states. These findings may shed light into the biological mechanisms wherein specific vesicle lipid structure and enzyme catalysis become interrelated to induce membrane fusion.

Cholesterol Effect on The Lipid Bilayer Perturbation Induced by Peptides Derived from the Membrane-Proximal External Region of HIV-1 gp41

The conserved, membrane-proximal external region (MPER) of the human immunodeficiency virus type-1 envelope glycoprotein 41 subunit is required for fusogenic activity. It has been proposed that MPER functions by disrupting the cholesterol-enriched virion membrane. We have compared the effects of cholesterol on the membrane perturbations induced by N-preTM and PreTM-C, two peptides derived from MPER sequences showing tendency to associate with the bilayer interface or to transfer into the hydrocarbon-core, respectively. Capacities of N-preTM and PreTM-C for associating with lipid vesicles were comparable. However, supporting the existence of different membrane-bound structures, N-preTM established unstable pores that induced permeabilization following a graded mechanism, whereas PreTM-C pores were stable and permeabilized LUVs and GUVs following an all-or-none mechanism. Cholesterol did not alter these permeabilization mechanisms, but affected differently the lytic capacities of the peptides. N-preTM partitioning and induced leakage decreased as the bilayer area compressibility modulus (KA) increased. In contrast, cholesterol highly stimulated PreTM-C-induced leakage under conditions that did not affect partitioning. Finally, fluid phase co-existence stimulated leakage induced by both peptides, which were confined within liquid disordered domains. These results support specific roles for cholesterol in modulating MPER membrane-disrupting effects that are not dependent on raft formation.

Synthetic Fusion Peptides of Tick-Borne Encephalitis Virus as Models for Membrane Fusion

The fusion peptide of TBEV is a short segment of the envelope protein that mediates viral and host cell membrane fusion at acidic pH. Previous studies on the E protein have shown that mutations at L107 have an effect on fusogenic activity. Structural studies have also suggested that during the fusion process the E protein rearranges to form a trimer. In the present study, a number of short peptides were synthesized, and their structure/activity was examined: (1) monomers consisting of residues 93-113 of the wild-type E protein with Leu at position 107 (WT) and two mutants, namely, L107F and L107T; (2) a monomer consisting of residues 93-113 of the E protein with a C105A mutation (TFPmn); (3) a trimer consisting of three monomers described in (2), linked at the C-terminus via 1 Lys (TFPtr); (4) a monomer consisting of residues 93-113 of the E protein plus six additional Lys at the C-terminus; and (5) a trimer consisting of three monomers described in (3), linked via the side chain of the sixth lysine. The secondary structure content of all peptides was investigated using circular dichroism (CD). Approximately seven of the residues were in beta-strand conformation, in the presence of POPC/POPE/cholesterol. The structures did not depend on pH significantly. The fusogenicity of the peptides was measured by FRET and photon correlation spectroscopy. The data suggest that TFPtr is the most fusogenic at acidic pH and that the mutation from L107 to T reduces activity. Molecular dynamics simulations of WT, L107T, and L107F suggest that this reduction in activity may be related to the fact that the mutations disrupt trimer stability. Finally, tryptophan fluorescence experiments were used to localize the peptides in the membrane. It was found that WT, L107F, TFPmn, and TFPtr could penetrate better into the acyl chain region of the lipids than the other peptides tested. The implications of these results on the fusion mechanism of TBEV E protein will be presented.

Identification of an endogenous retroviral envelope gene with fusogenic activity and placenta-specific expression in the rabbit: a new "syncytin" in a third order of mammals

BackgroundSyncytins are envelope genes of retroviral origin that have been co-opted by the host to mediate a specialized function in placentation. Two of these genes have already been identified in primates, as well as two distinct, non orthologous genes in rodents.ResultsHere we identified within the rabbit Oryctolagus cuniculus-which belongs to the lagomorpha order- an envelope (env) gene of retroviral origin with the characteristic features of a bona fide syncytin, that we named syncytin-Ory1. An in silico search for full-length env genes with an uninterrupted open reading frame within the rabbit genome first identified two candidate genes that were tested for their specific expression in the placenta by quantitative RT-PCR of RNA isolated from a large set of tissues. This resulted in the identification of an env gene with placenta-specific expression and belonging to a family of endogenous retroelements present at a limited copy number in the rabbit genome. Functional characterization of the identified placenta-expressed env gene after cloning in a CMV-driven expression vector and transient transfection experiments, demonstrated both fusogenic activity in an ex vivo cell-cell fusion assay and infectivity of pseudotypes. The receptor for the rabbit syncytin-Ory1 was found to be the same as that for human syncytin-1, i.e. the previously identified ASCT2 transporter. This was demonstrated by a co-culture fusion assay between hamster A23 cells transduced with an expression vector for ASCT2 and A23 cells transduced with syncytin-Ory1. Finally, in situ hybridization of rabbit placenta sections with a syncytin-Ory1 probe revealed specific expression at the level of the junctional zone between the placental lobe and the maternal decidua, where the invading syncytial fetal tissue contacts the maternal decidua to form the labyrinth, consistent with a role in the formation of the syncytiotrophoblast. The syncytin-Ory1 gene is found in Leporidae but not in Ochotonidae, and should therefore have entered the lagomorpha order 12-30 million years ago.ConclusionThe identification of a novel syncytin gene within a third order of mammals displaying syncytiotrophoblast formation during placentation strongly supports the notion that on several occasions retroviral infections have resulted in the independent capture of genes that have been positively selected for a convergent physiological role.

Structural features of fusogenic model transmembrane domains that differentially regulate inner and outer leaflet mixing in membrane fusion

The transmembrane domains of fusion proteins are known to be important for their fusogenic activity. In an effort to systematically investigate the structure/function relationships of transmembrane domains we had previously designed LV-peptides that mimic natural fusion protein TMDs in their ability to drive fusion after incorporation into liposomal membranes. Here, we investigate the impact of different structural features of LV-peptide TMDs on inner and outer leaflet mixing. We find that fusion driven by the helical peptides involves a hemifusion intermediate as previously seen for natural fusion proteins. Helix backbone dynamics enhances fusion by selectively promoting outer leaflet mixing. Furthermore, the hydrophobic length of the peptides as well as covalent attachment of long acyl chains affects outer and inner leaflet mixing to different extents. Different structural features of transmembrane domains thus appear to differentially influence the rearrangements of lipids in fusion initiation and the hemifusion-to-fusion transition. The relevance of these findings in respect to the function of natural fusion proteins is discussed.