Viral and developmental cell fusion mechanisms: conservation and divergence

Membrane fusion is a fundamental requirement in numerous developmental, physiological, and pathological processes in eukaryotes. So far, only a limited number of viral and cellular fusogens, proteins that fuse membranes, have been isolated and characterized. Despite the diversity in structures and functions of known fusogens, some common principles of action apply to all fusion reactions. These can serve as guidelines in the search for new fusogens, and may allow the formulation of a cross-species, unified theory to explain divergent and convergent evolutionary principles of membrane fusion.     

ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating

During yeast mating, two haploid nuclei fuse membranes to form a single diploid nucleus. However, the known proteins required for nuclear fusion are unlikely to function as direct fusogens (i.e., they are unlikely to directly catalyze lipid bilayer fusion) based on their predicted structure and localization. Therefore we screened known fusogens from vesicle trafficking (soluble N-ethylmaleimide–sensitive factor attachment protein receptors [SNAREs]) and homotypic endoplasmic reticulum (ER) fusion (Sey1p) for additional roles in nuclear fusion. Here we demonstrate that the ER-localized SNAREs Sec20p, Ufe1p, Use1p, and Bos1p are required for efficient nuclear fusion. In contrast, Sey1p is required indirectly for nuclear fusion; sey1Δ zygotes accumulate ER at the zone of cell fusion, causing a block in nuclear congression. However, double mutants of Sey1p and Sec20p, Ufe1p, or Use1p, but not Bos1p, display extreme ER morphology defects, worse than either single mutant, suggesting that retrograde SNAREs fuse ER in the absence of Sey1p. Together these data demonstrate that SNAREs mediate nuclear fusion, ER fusion after cell fusion is necessary to complete nuclear congression, and there exists a SNARE-mediated, Sey1p-independent ER fusion pathway.     

Lysophosphatidylcholine Reversibly Arrests Pore Expansion during Syncytium Formation Mediated by Diverse Viral Fusogens

Using lysophosphatidylcholine, a curvature-inducing lysolipid, we have isolated a reversible, “stalled pore” phenotype during syncytium formation induced by the p14 fusion-associated small transmembrane (FAST) protein and influenza virus hemagglutinin (HA) fusogens. This is the first evidence that lateral propagation of stable fusion pores leading to syncytiogenesis mediated by diverse viral fusogens is inhibited by promotion of positive membrane curvature in the outer leaflets of the lipid bilayer surrounding intercellular fusion pores.     

A virus-encoded cell-cell fusion machine dependent on surrogate adhesins

The reovirus fusion-associated small transmembrane (FAST) proteins function as virus-encoded cellular fusogens, mediating efficient cell-cell rather than virus-cell membrane fusion. With ectodomains of only approximately 20-40 residues, it is unclear how such diminutive viral fusion proteins mediate the initial stages (i.e. membrane contact and close membrane apposition) of the fusion reaction that precede actual membrane merger. We now show that the FAST proteins lack specific receptor-binding activity, and in their natural biological context of promoting cell-cell fusion, rely on cadherins to promote close membrane apposition. The FAST proteins, however, are not specifically reliant on cadherin engagement to mediate membrane apposition as indicated by their ability to efficiently utilize other adhesins in the fusion reaction. Results further indicate that surrogate adhesion proteins that bridge membranes as close as 13 nm apart enhance FAST protein-induced cell-cell fusion, but active actin remodelling is required for maximal fusion activity. The FAST proteins are the first example of membrane fusion proteins that have specifically evolved to function as opportunistic fusogens, designed to exploit and convert naturally occurring adhesion sites into fusion sites. The capacity of surrogate, non-cognate adhesins and active actin remodelling to enhance the cell-cell fusion activity of the FAST proteins are features perfectly suited to the structural and functional evolution of these fusogens as the minimal fusion component of a virus-encoded cellular fusion machine. These results also provide a basis for reconciling the rudimentary structure of the FAST proteins with their capacity to fuse cellular membranes.     

Protoplast fusion: a tool for intergeneric gene transfer in bacteria

Protoplasts can be isolated from bacterial cells by digestion of the cell wall with the help of lysozyme in presence of osmotic stabilizers. Fusion of protoplasts can be induced by chemical fusogens like polyethylene glycol. The electrofusion technique has been reported in bacteria in which the fusion frequency is much higher than that obtained by PEG induced protoplast fusion. This technology allows recombination to take place not only between related species but also between unrelated genera and is of great potential in the breeding and improvement of industrial strains. This review includes the information and developments on the protoplast fusion in bacteria with special reference to genetic recombination by protoplast fusion between phylogenetically unrelated bacteria.     

Biophysical and functional assays for viral membrane fusion peptides

Membrane fusion is a protein catalyzed biophysical reaction that involves the simultaneous intermixing of two phospholipid bilayers and of the aqueous compartments bound by their respective bilayers. In the case of enveloped virus fusogens, short hydrophobic or amphipathic fusion peptides that are components of the larger fusion complex are essential for the membrane merger event. The process of cell–cell membrane fusion and syncytium formation induced by the nonenveloped fusogenic orthoreoviruses is driven by the Fusion-Associated Small Transmembrane (FAST) proteins, which are similarly dependent on the action of fusion peptides. In this article, we describe some simple methods for the biophysical characterization of viral membrane fusion peptides. Liposomes serve as an ideal model system for characterizing peptide–membrane interactions because their size, shape and composition can be readily manipulated. We present details of fluorescence assays used to elucidate the kinetics of membrane fusion as well as complimentary assays used to characterize peptide-induced liposome binding and aggregation.     

Cell fusions in mammals

Cell fusions are important to fertilization, placentation, development of skeletal muscle and bone, calcium homeostasis and the immune defense system. Additionally, cell fusions participate in tissue repair and may be important to cancer development and progression. A large number of factors appear to regulate cell fusions, including receptors and ligands, membrane domain organizing proteins, proteases, signaling molecules and fusogenic proteins forming alpha-helical bundles that bring membranes close together. The syncytin family of proteins represent true fusogens and the founding member, syncytin-1, has been documented to be involved in fusions between placental trophoblasts, between cancer cells and between cancer cells and host cells. We review the literature with emphasis on the syncytin family and propose that syncytins may represent universal fusogens in primates and rodents, which work together with a number of other proteins to regulate the cell fusion machinery.     

YKT6 is a core constituent of membrane fusion machineries at the Arabidopsis trans-Golgi network

SNARE complex formation is essential for membrane fusion in exocytotic and vacuolar trafficking pathways. Vesicle-associated (v-) SNARE associates with a target membrane (t-) SNARE to form a SNARE complex bridging two membranes, which may facilitate membrane fusion. The Arabidopsis genome encodes a large number of predicted SNARE proteins that might function primarily as fusogens for vesicle transport in endomembrane systems. The SNAREs SYP41, SYP61 and VTI12 reside in the trans-Golgi network and have been proposed to function together in vesicle fusion with this organelle. Here, we use a liposome fusion assay to demonstrate that VTI12 and either SYP41 or SYP61, but not both, are required for membrane fusion. This indicates that SYP41 and SYP61 are likely to function in independent vesicle fusion reactions in Arabidopsis. In addition, we have identified two new functionally interchangeable components, YKT61 and YKT62, that show sequence similarity to the multifunctional yeast SNARE YKT6. Both YKT61 and YKT62 interact with SYP41 and are essential for membrane fusion mediated by either SYP41 or SYP61. These results therefore define the core constituents required for membrane fusion at the Arabidopsis trans-Golgi network.     

Calcium ions and cell fusion. Effects of chemical fusogens on the permeability of erythrocytes to calcium and other ions.

1. Fusogenic and non-fusogenic chemicals were tesetd for their ability to allow 45Ca2+ and 3H2O to enter hen and human erythrocytes. 2. The ratio of 45Ca2+/3H2O in treated cells to that in untreated cells is referred to as the entry ratio. 3. Within 1 min at 37 degrees C both water-soluble and lipid-soluble fusogens increased the value of the entry ratio, which reached maximum values in 5--10 min. 4. Values of the entry ratio in the range of 4--12 were found under conditions that led to cell fusion. 5. Closely related but non-fusogenic chemicals did not significantly alter the entry ratio. 6. The entry ratios for 86Rb+, 22Na+ and 35SO42- were also significantly increased by both lipid-soluble and water-soluble fusogens, though the increases were not as large as those for 45Ca2+. 7. It is suggested that fusogenic compounds increase the permeability of biological membranes to ions, and that an increase in the concentration of intracellular Ca2+ initiates or facilitates events that lead to the chemically induced fusion of erythrocytes.     

Determinants of membrane activity from mutational analysis of the HIV fusion peptide

We have synthesized a small library of 38 variants of the 23-residue fusion peptide domain found at the N-terminus of gp41 glycoprotein of HIV. This hydrophobic, glycine-rich sequence is critical for viral infectivity and is thought to be central in the membrane fusion of viral envelope with the host membrane. There has been extensive discussion regarding the origin of fusogenicity in this viral fusion sequence. Our library of fusion peptide variants was designed to address the biophysical importance of secondary structure, peptide flexibility, glycine content, and placement. We assayed each peptide for its ability to induce lipid mixing and membrane permeablization in synthetic vesicles. We find that the viral fusion peptide may be greatly simplified while retaining fusogenic function and minimizing membrane-permeablizing function; to the best of our knowledge, this is the first attempt to optimize fusogenic function of the HIV fusion peptide through sequence variation. Our data show that many flexible, linear, minimally hydrophobic peptides may achieve the biophysical function of fusion; glycine does not appear to be essential. These findings will be useful in the design of synthetic fusogens for cellular delivery.     

The type I membrane protein EFF-1 is essential for developmental cell fusion

Multinucleate cells are widespread in nature, yet the mechanism by which cells fuse their plasma membranes is poorly understood. To identify animal fusogens, we performed new screens for mutations that abolish cell fusion within tissues of C. elegans throughout development. We identified the gene eff-1, which is expressed as cells acquire fusion competence and encodes a novel integral membrane protein. EFF-1 sequence motifs suggest physicochemical actions that could cause adjacent bilayers to fuse. Mutations in the extracellular domain of EFF-1 completely block epithelial cell membrane fusion without affecting other perfusion events such as cell generation, patterning, differentiation, and adhesion. Thus, EFF-1 is a key component in the mechanism of cell fusion, a process essential to normal animal development.     

Use of a fluorescence assay to monitor the kinetics of fusion between erythrocyte ghosts,as induced by sendai virus

The kinetics of the fusion process between erythrocyte ghosts, as induced by Sendal virus, were readily revealed by a simple fluorescence procedure previously employed to characterize the fusion of viruses with biological membranes. The method relies on the relief of fluorescence selfquenching of the membrane-inserted probe octadecyl Rhodamine B chloride (R₁₈) as occurs when labeled membranes fuse with unlabeled counterparts. The kinetics of R₁₈ insertion into ghost membranes, the non-exchangeable properties of the fluorophore and the kinetics, and some characteristics of Sendai virus-induced fusion of ghosts, are described. We propose that the experimental approach may be particularly advantageous to obtain insight into the efficiency and mechanism of a wide range of fusogens, capable of inducing fusion of erythrocyte membranes.     

Structural basis of viral invasion: lessons from paramyxovirus F

The structures of glycoproteins that mediate enveloped virus entry into cells have revealed dramatic structural changes that accompany membrane fusion and provided mechanistic insights into this process. The group of class I viral fusion proteins includes the influenza hemagglutinin, paramyxovirus F, HIV env, and other mechanistically related fusogens, but these proteins are unrelated in sequence and exhibit clearly distinct structural features. Recently determined crystal structures of the paramyxovirus F protein in two conformations, representing pre-fusion and post-fusion states, reveal a novel protein architecture that undergoes large-scale, irreversible refolding during membrane fusion, extending our understanding of this diverse group of membrane fusion machines.     

Cytoskeleton reorganization in influenza hemagglutinin-initiated syncytium formation

Little is known about the mechanisms of cell-cell fusion in development and diseases and, especially, about fusion stages downstream of an opening of nascent fusion pore(s). Earlier works on different cell-cell fusion reactions have indicated that cytoskeleton plays important role in syncytium formation. However, due to complexity of these reactions and multifaceted contributions of cytoskeleton in cell physiology, it has remained unclear whether cytoskeleton directly drives fusion pore expansion or affects preceding fusion stages. Here we explore cellular reorganization associated with fusion pore expansion in syncytium formation using relatively simple experimental system. Fusion between murine embryonic fibroblasts NIH3T3-based cells is initiated on demand by well-characterized fusogen influenza virus hemagglutinin. We uncouple early fusion stages dependent on protein fusogens from subsequent fusion pore expansion stage and establish that the transition from local fusion to syncytium requires metabolic activity of living cells. Effective syncytium formation for cells with disorganized actin and microtubule cytoskeleton argues against hypothesis that cytoskeleton drives fusion expansion.     

A novel function for the sperm adhesion protein IZUMO1 in cell–cell fusion

Mammalian sperm–egg adhesion depends on the trans-interaction between the sperm-specific type I glycoprotein IZUMO1 and its oocyte-specific GPI-anchored receptor JUNO. However, the mechanisms and proteins (fusogens) that mediate the following step of gamete fusion remain unknown. Using live imaging and content mixing assays in a heterologous system and structure-guided mutagenesis, we unveil an unexpected function for IZUMO1 in cell-to-cell fusion. We show that IZUMO1 alone is sufficient to induce fusion, and that this ability is retained in a mutant unable to bind JUNO. On the other hand, a triple mutation in exposed aromatic residues prevents this fusogenic activity without impairing JUNO interaction. Our findings suggest a second function for IZUMO1 as a unilateral mouse gamete fusogen.     

Exocytosis reconstituted from the sea urchin egg is unaffected by calcium pretreatment of granules and plasma membrane

Micromolar calcium ion concentrations stimulate exocytosis in a reconstituted system made by recombining in the plasma membrane and cortical secretory granules of the sea urchin egg. The isolated cortical granules are unaffected by calcium concentrations up to 1 mM, nor do granule aggregates undergo any mutual fusion at this concentration. Both isolated plasma membrane and cortical granules can be pretreated with 1 mM Ca before reconstitution without affecting the subsequent exocytosis of the reconstituted system in response to micromolar calcium concentrations. On reconstitution, aggregated cortical granules will fuse with one another in response to micromolar calcium provided that one of their number is in contact with the plasma membrane. If exocytosis involves the generation of lipid fusogens, then these results suggest that the calcium-stimulated production of a fusogen can occur only when contiguity exists between cortical granules and plasma membrane. They also suggest that a substance involved in exocytosis can diffuse and cause piggy-back fusion of secretory granules that are in contact with the plasma membrane. Our results are also consistent with a scheme in which calcium ions cause a reversible, allosteric activation of an exocytotic protein.     

SNARE regulators: matchmakers and matchbreakers

SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) are membrane-associated proteins that participate in the fusion of internal membranes in eukaryotic cells. SNAREs comprise three distinct and well-conserved families of molecules that act directly as membrane fusogens or, at the least, as elements that bring membranes into close apposition and allow for subsequent fusion events to occur. While the molecular events leading to fusion are still under debate, it is clear that a number of additional factors are required to bring about SNARE-mediated membrane fusion in vivo. Many of these factors, which collectively can be called SNARE regulators (e.g. Sec1/Munc18, synaptotagmin, GATE-16, LMA1, Munc13/UNC-13, synaptophysin, tomosyn, Vsm1, etc.), bind directly to SNAREs and are involved in the regulation of SNARE assembly as well as the ability of SNAREs to participate in trafficking events. In addition, recent studies have suggested a role for posttranslational modification (e.g., phosphorylation) in the regulation of SNARE functions. In this review the possible role of SNARE regulators in SNARE assembly and the involvement of SNARE phosphorylation in the regulation of intracellular membrane trafficking will be discussed.     

Residues within the C-terminal arm of the herpes simplex virus 1 glycoprotein B ectodomain contribute to its refolding during the fusion step of virus entry

Herpesvirus entry into cells requires coordinated interactions among several viral glycoproteins. The final membrane fusion step of entry is executed by glycoprotein B (gB), a class III viral fusion protein that is conserved across all herpesviruses. Fusion proteins are metastable proteins that mediate fusion by inserting into a target membrane and refolding from a prefusion to postfusion conformation to bring the viral and cell membranes together. Although the structure of gB has been solved in a conformation that likely represents its postfusion form, its prefusion structure and the details of how it refolds to execute fusion are unknown. The postfusion gB structure contains a trimeric coiled-coil at its core and a long C-terminal arm within the ectodomain packs against this coil in an antiparallel manner. This coil-arm complex is reminiscent of the six-helix bundle that provides the energy for fusion in class I fusogens. To determine the role of the coil-arm complex, we individually mutated residues in the herpes simplex virus 1 gB coil-arm complex to alanine and assessed the contribution of each residue to cell-cell and virus-cell fusion. Several coil mutations resulted in a loss of cell surface expression, indicating that the coil residues are important for proper processing of gB. Three mutations in the arm region (I671A, H681A, and F683A) reduced fusion without affecting expression. Combining these three arm mutations drastically reduced the ability of gB to execute fusion; however, fusion function could be restored by adding known hyperfusogenic mutations to the arm mutant. We propose that the formation of the coil-arm complex drives the gB transition to a postfusion conformation and the coil-arm complex performs a function similar to that of the six-helix bundle in class I fusion. Furthermore, we suggest that these specific mutations in the arm may energetically favor the prefusion state of gB.     

Enhanced Fusion Pore Expansion Mediated by the Trans-Acting Endodomain of the Reovirus FAST Proteins

The reovirus fusion-associated small transmembrane (FAST) proteins are virus-encoded membrane fusion proteins that function as dedicated cell–cell fusogens. The topology of these small, single-pass membrane proteins orients the majority of the protein on the distal side of the membrane (i.e., inside the cell). We now show that ectopic expression of the endodomains of the p10, p14, and p15 FAST proteins enhances syncytiogenesis induced by the full-length FAST proteins, both homotypically and heterotypically. Results further indicate that the 68-residue cytoplasmic endodomain of the p14 FAST protein (1) is endogenously generated from full-length p14 protein expressed in virus-infected or transfected cells; (2) enhances syncytiogenesis subsequent to stable pore formation; (3) increases the syncytiogenic activity of heterologous fusion proteins, including the differentiation-dependent fusion of murine myoblasts; (4) exerts its enhancing activity from the cytosol, independent of direct interactions with either the fusogen or the membranes being fused; and (5) contains several regions with protein–protein interaction motifs that influence enhancing activity. We propose that the unique evolution of the FAST proteins as virus-encoded cellular fusogens has allowed them to generate a trans-acting, soluble endodomain peptide to harness a cellular pathway or process involved in the poorly understood process that facilitates the transition from microfusion pores to macrofusion and syncytiogenesis.