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.     

Multifaceted Sequence-Dependent and -Independent Roles for Reovirus FAST Protein Cytoplasmic Tails in Fusion Pore Formation and Syncytiogenesis

Fusogenic reoviruses utilize the FAST proteins, a novel family of nonstructural viral membrane fusion proteins, to induce cell-cell fusion and syncytium formation. Unlike the paradigmatic enveloped virus fusion proteins, the FAST proteins position the majority of their mass within and internal to the membrane in which they reside, resulting in extended C-terminal cytoplasmic tails (CTs). Using tail truncations, we demonstrate that the last 8 residues of the 36-residue CT of the avian reovirus p10 FAST protein and the last 20 residues of the 68-residue CT of the reptilian reovirus p14 FAST protein enhance, but are not required for, pore expansion and syncytium formation. Further truncations indicate that the membrane-distal 12 residues of the p10 and 47 residues of the p14 CTs are essential for pore formation and that a residual tail of 21 to 24 residues that includes a conserved, membrane-proximal polybasic region present in all FAST proteins is insufficient to maintain FAST protein fusion activity. Unexpectedly, a reextension of the tail-truncated, nonfusogenic p10 and p14 constructs with scrambled versions of the deleted sequences restored pore formation and syncytiogenesis, while reextensions with heterologous sequences partially restored pore formation but failed to rescue syncytiogenesis. The membrane-distal regions of the FAST protein CTs therefore exert multiple effects on the membrane fusion reaction, serving in both sequence-dependent and sequence-independent manners as positive effectors of pore formation, pore expansion, and syncytiogenesis.The only examples of nonenveloped viruses that induce cell-cell fusion and syncytium formation occur within the family Orthoreoviridae, an extremely diverse group of viruses containing segmented double-stranded RNA genomes (9). In recent years, the viral proteins responsible for the syncytiogenic phenotype of the fusogenic orthoreoviruses and aquareoviruses have been identified and characterized (14, 18, 41, 46). These fusion-associated small transmembrane (FAST) proteins define a new family of viral fusogens with several unique biological and biophysical properties. Unlike the well-characterized enveloped virus fusion proteins, reovirus FAST proteins are nonstructural viral proteins and are therefore not involved in mediating virus-cell fusion and virus entry (18, 21, 46). The FAST proteins are instead dedicated to inducing cell-cell fusion and syncytium formation following their expression and trafficking to the plasma membrane of virus-infected or transfected cells (14, 17, 46). Data from previously reported studies also suggest that the FAST proteins serve as virulence factors for the fusogenic reoviruses, promoting virus dissemination and increased tissue destruction (6, 43). How this atypical family of viral fusogens functions to mediate cell-cell membrane fusion remains unclear.The unusual biological role of the FAST proteins as nonstructural, virus-encoded, “cellular” fusogens is embodied in structural features that clearly distinguish the FAST proteins from the membrane fusion proteins of enveloped viruses. There are currently four distinct members of the FAST protein family, named according to their molecular masses: the homologous p10 proteins of avian reovirus (ARV) and Nelson Bay reovirus and the unrelated p14, p15, and p22 proteins of reptilian reovirus (RRV), baboon reovirus, and Atlantic salmon aquareovirus, respectively (14, 18, 41, 46). These proteins are the smallest known fusogens, ranging from 95 to 198 amino acids in size, and assume an asymmetric topology in the plasma membrane, with a single transmembrane domain that separates small N-terminal ectodomains of ∼20 to 41 residues from equal-sized or considerably larger C-terminal endodomains of ∼36 to 141 residues (Fig. ​(Fig.1A).1A). A number of structural motifs in both the ecto- and endodomains of the FAST proteins have been identified, including sites of acylation, hydrophobic patches, a membrane-proximal polybasic region, and regions rich in proline, cysteine, or arginine, proline, and histidine. Each of the FAST proteins has its own signature repertoire and arrangement of these motifs. Determining how these various motifs contribute to the fusogenic activity of the FAST proteins remains an area of active investigation.Open in a separate windowFIG. 1.ARV p10 and RRV p14 FAST protein topologies and tail truncations. (A) Diagrammatic representation of the p10 and p14 FAST proteins showing their topology in the plasma membrane. Both are single-pass transmembrane proteins with N-terminal ectodomains on the surface of cells and C-terminal endodomains in the cytoplasm. Structural motifs include hydrophobic patches (HP), polybasic motifs (PB), fatty acid modifications (indicated by squiggly lines) that are either the N-terminal myristoylation or palmitoylation of a dicysteine motif (CC), and a polyproline motif (PP). The total number of residues in each protein is indicated by the numbers. (B) The amino acid sequences of the p10 and p14 endodomains are shown, along with the motifs described above. Progressive truncations of the CTs were constructed (arrows), with the numbers indicating the last amino acid present in the full-length proteins or each truncation.Numerous studies of diverse fusion processes define five general steps of the pathway for membrane fusion and syncytium formation: membrane binding, close membrane apposition, hemifusion (i.e., the mixing of the outer leaflets of the two bilayers), stable pore formation, and pore expansion (12, 13, 44). The well-characterized enveloped virus fusion proteins utilize extensive structural rearrangement of their complex ectodomains to provide mechanical energy to draw membranes into close proximity and promote membrane merger (21, 53). The limited size of the FAST protein ectodomains precludes such a mechanical model for membrane fusion, necessitating the development of alternate models to explain how the diminutive FAST proteins breach the thermodynamic barriers that prevent the spontaneous merger of biological membranes. The FAST proteins are both necessary and sufficient to mediate membrane fusion (51). However, data from recent studies indicate that for maximal cell fusion activity, the FAST proteins rely on surrogate adhesins to mediate close membrane apposition (42). Data from recent studies also indicate that a small percentage of the p14 FAST protein expressed in virus-infected or transfected cells is proteolytically processed to generate a bioactive, soluble endodomain that recruits cellular pathways to drive the expansion of stable fusion pores into the extended fusion apertures needed for syncytium formation (50). The FAST proteins therefore utilize accessory proteins to mediate the prefusion (membrane binding and apposition) and postfusion (pore expansion) stages of syncytiogenesis, retaining within their rudimentary structures all that is required to mediate the actual process of membrane merger. This subdivision of the multistep process of syncytium formation is reflected in, and is perfectly suited to, the evolution of the FAST proteins as virus-encoded cellular fusogens.The small size of the FAST protein ectodomains and their donor membrane-focused topology contrast markedly with enveloped virus fusion proteins that position the majority of their mass external to the membrane. While the complex ectodomains of the enveloped virus fusion proteins clearly play an essential role in the fusion reaction, the involvement of their cytoplasmic tails (CTs) is far less certain, and no consistent picture of the role of these C-terminal tails has emerged. The CTs of many enveloped viral fusion proteins, including baculovirus (31), severe acute respiratory syndrome coronavirus (5), vesicular stomatitis virus (36), parainfluenza virus type 2 (56), and influenza A virus subtype H3 (10), play no role in the membrane fusion reaction. Of the fusion protein tails that do modulate the fusion reaction, the majority serve inhibitory roles, including the F proteins of measles virus and parainfluenza virus type 5 SER (7, 45, 52), glycoprotein B from several herpesviruses (22, 24, 28), and the fusion proteins of numerous retroviruses (1, 8, 30, 32, 34, 47, 48). These inhibitory cytoplasmic domains alter the conformation of the fusion protein ectodomains, thereby coupling virion maturation to fusion competence (1, 2, 35, 52, 54). In the few cases where extensive tail truncations adversely affect fusion, these truncations generally decrease but do not eliminate syncytiogenesis, and it is the membrane-proximal portion of the tail that promotes pore formation or pore expansion (20, 25, 26, 32).Since the FAST proteins are nonstructural viral proteins, their CTs (also referred to as endodomains) are not required to suppress fusion activity until after virus particle assembly. At the same time, the disproportionate size of their endodomains strongly suggests that these CTs play an important role in membrane fusion activity. Although one such role of the p14 CT is the generation of a soluble endodomain that recruits cellular factors involved in pore expansion, the majority of p14 is not proteolytically processed, suggesting that FAST protein CTs may serve additional roles as components of the intact protein (50). We now show that C-terminal truncations of the p10 and p14 FAST proteins reduced and eventually eliminated cell-cell fusion. Fluorescence-based pore formation assays coupled with tail reextension studies further revealed that FAST protein CTs drive fusion pore formation and expansion in both sequence-dependent and sequence-independent manners. The membrane-distal regions of FAST protein CTs therefore exert multiple effects on the mechanism of membrane fusion.     

Mechanisms of membrane fusion: disparate players and common principles

Membrane fusion can occur between cells, between different intracellular compartments, between intracellular compartments and the plasma membrane and between lipid-bound structures such as viral particles and cellular membranes. In order for membranes to fuse they must first be brought together. The more highly curved a membrane is, the more fusogenic it becomes. We discuss how proteins, including SNAREs, synaptotagmins and viral fusion proteins, might mediate close membrane apposition and induction of membrane curvature to drive diverse fusion processes. We also highlight common principles that can be derived from the analysis of the role of these proteins.     

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.     

Ultrastructure of cell fusion and premature chromosome condensation (PCC) of thymocyte nuclei in metaphase II mouse oocytes

Following PEG (polyethylene glycol) treatment of ovulated metaphase II mouse oocytes aggregated with thymocytes, fusion of cell membranes occurs. Prerequisites for cell fusion are: close apposition of lectin-agglutinated (phytohemagglutinin-treated) membranes of both cells, formation of firm punctual adhesion sites, and expansion of adhesion sites over a certain area. Establishment of the firm cell-cell contact is associated with development of actin-like filaments along both of the adhering plasma membranes. Membrane fusion occurs at single or multiple sites, and is followed by internalization of thymocyte-oocyte membrane complexes decorated with actin filaments into the hybrid cell cytoplasm. A filamentous actin layer forms also along the inner surface of newly formed hybrid oocyte-thymocyte plasma membrane. Thymocyte nuclei incorporated into oocyte cytoplasm undergo nuclear envelope breakdown and premature chromosome condensation (PCC) leading, eventually, to formation of single chromatids complete with kinetochores. Concomitantly with chromatin condensation an extensive polymerization of microtubules starts in the center of the chromatin mass which leads to the formation of an apparently non-functional spindle-like structure.     

Morphological changes and spatial regulation of diacylglycerol kinase-zeta, syntrophins, and Rac1 during myoblast fusion

The fusion of mononuclear myoblasts into multinucleated myofibers is essential for the formation and growth of skeletal muscle. Myoblast fusion follows a well-defined sequence of cellular events, from initial recognition and adhesion, to alignment, and finally plasma membrane fusion. These processes depend upon coordinated remodeling of the actin cytoskeleton. Our recent studies suggest diacylglycerol kinase-zeta (DGK-zeta), an enzyme that metabolizes diacylglycerol to yield phosphatidic acid, plays an important role in actin reorganization. Here, we investigated whether DGK-zeta has a role in the fusion of cultured C2C12 myoblasts. We show that DGK-zeta and syntrophins, scaffold proteins of the dystrophin glycoprotein complex that bind directly to DGK-zeta, are spatially regulated during fusion. Both proteins accumulated with the GTPase Rac1 at sites where fine filopodia mediate the initial contact between myoblasts. In addition, DGK-zeta codistributed with the Ca(2+)-dependent cell adhesion molecule N-cadherin at nascent, but not previously established cell contacts. We provide evidence that C2 cells are pulled together at cell-cell junctions by N-cadherin-containing filopodia reminiscent of epithelial adhesion zippers, which guide the advance of lamellipodia from apposing cells. At later times, vesicles with properties of macropinosomes formed close to cell-cell junctions. Reconstruction of confocal optical sections showed these form dome-like protrusions from the dorsal surface of contacting cells. Collectively, these results suggest DGK-zeta and syntrophins play a role at multiple stages of the fusion process. Moreover, our findings provide a potential link between changes in the lipid content of the membrane bilayer and reorganization of the actin cytoskeleton during myoblast fusion.     

Extensive syncytium formation mediated by the reovirus FAST proteins triggers apoptosis-induced membrane instability

The fusion-associated small transmembrane (FAST) proteins of the fusogenic reoviruses are the only known examples of membrane fusion proteins encoded by non-enveloped viruses. While the involvement of the FAST proteins in mediating extensive syncytium formation in virus-infected and -transfected cells is well established, the nature of the fusion reaction and the role of cell-cell fusion in the virus replication cycle remain unclear. To address these issues, we analyzed the syncytial phenotype induced by four different FAST proteins: the avian and Nelson Bay reovirus p10, reptilian reovirus p14, and baboon reovirus p15 FAST proteins. Results indicate that FAST protein-mediated cell-cell fusion is a relatively non-leaky process, as demonstrated by the absence of significant [3H]uridine release from cells undergoing fusion and by the resistance of these cells to treatment with hygromycin B, a membrane-impermeable translation inhibitor. However, diminished membrane integrity occurred subsequent to extensive syncytium formation and was associated with DNA fragmentation and chromatin condensation, indicating that extensive cell-cell fusion activates apoptotic signaling cascades. Inhibiting effector caspase activation or ablating the extent of syncytium formation, either by partial deletion of the avian reovirus p10 ecto-domain or by antibody inhibition of p14-mediated cell-cell fusion, all resulted in reduced membrane permeability changes. These observations suggest that the FAST proteins do not possess intrinsic membrane-lytic activity. Rather, extensive FAST protein-induced syncytium formation triggers an apoptotic response that contributes to altered membrane integrity. We propose that the FAST proteins have evolved to serve a dual role in the replication cycle of these fusogenic non-enveloped viruses, with non-leaky cell-cell fusion initially promoting localized cell-cell transmission of the infection followed by enhanced progeny virus release from apoptotic syncytia and systemic dissemination of the infection.     

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.     

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.     

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.     

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.     

Changes in membrane lipid composition cause alterations in epithelial cell-cell adhesion structures in renal papillary collecting duct cells

In epithelial tissues, adherens junctions (AJ) mediate cell-cell adhesion by using proteins called E-cadherins, which span the plasma membrane, contact E-cadherin on other cells and connect with the actin cytoskeleton inside the cell. Although AJ protein complexes are inserted in detergent-resistant membrane microdomains, the influence of membrane lipid composition in the preservation of AJ structures has not been extensively addressed. In the present work, we studied the contribution of membrane lipids to the preservation of renal epithelial cell-cell adhesion structures. We biochemically characterized the lipid composition of membranes containing AJ complexes. By using lipid membrane-affecting agents, we found that such agents induced the formation of new AJ protein-containing domains of different lipid composition. By using both biochemical approaches and fluorescence microscopy we demonstrated that the membrane phospholipid composition plays an essential role in the in vivo maintenance of AJ structures involved in cell-cell adhesion structures in renal papillary collecting duct cells.     

Structures and Mechanisms of Viral Membrane Fusion Proteins: Multiple Variations on a Common Theme

Recent work has identified three distinct classes of viral membrane fusion proteins based on structural criteria. In addition, there are at least four distinct mechanisms by which viral fusion proteins can be triggered to undergo fusion-inducing conformational changes. Viral fusion proteins also contain different types of fusion peptides and vary in their reliance on accessory proteins. These differing features combine to yield a rich diversity of fusion proteins. Yet despite this staggering diversity, all characterized viral fusion proteins convert from a fusion-competent state (dimers or trimers, depending on the class) to a membrane-embedded homotrimeric prehairpin, and then to a trimer-of-hairpins that brings the fusion peptide, attached to the target membrane, and the transmembrane domain, attached to the viral membrane, into close proximity thereby facilitating the union of viral and target membranes. During these conformational conversions, the fusion proteins induce membranes to progress through stages of close apposition, hemifusion, and then the formation of small, and finally large, fusion pores. Clearly, highly divergent proteins have converged on the same overall strategy to mediate fusion, an essential step in the life cycle of every enveloped virus.     

Structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme

Recent work has identified three distinct classes of viral membrane fusion proteins based on structural criteria. In addition, there are at least four distinct mechanisms by which viral fusion proteins can be triggered to undergo fusion-inducing conformational changes. Viral fusion proteins also contain different types of fusion peptides and vary in their reliance on accessory proteins. These differing features combine to yield a rich diversity of fusion proteins. Yet despite this staggering diversity, all characterized viral fusion proteins convert from a fusion-competent state (dimers or trimers, depending on the class) to a membrane-embedded homotrimeric prehairpin, and then to a trimer-of-hairpins that brings the fusion peptide, attached to the target membrane, and the transmembrane domain, attached to the viral membrane, into close proximity thereby facilitating the union of viral and target membranes. During these conformational conversions, the fusion proteins induce membranes to progress through stages of close apposition, hemifusion, and then the formation of small, and finally large, fusion pores. Clearly, highly divergent proteins have converged on the same overall strategy to mediate fusion, an essential step in the life cycle of every enveloped virus.     

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.     

Arf GAPs as regulators of the actin cytoskeleton.

The Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are a family of proteins with a common catalytic domain that induces hydrolysis of GTP bound to Arf GTP-binding proteins. At least three groups of multidomain Arf GAPs affect the actin cytoskeleton and cellular activities, such as migration and movement, that depend on the cytoskeleton. One role of the Arf GAPs is to regulate membrane remodelling that accompanies actin polymerization. Regulation of membrane remodelling is mediated in part by the regulation of Arf proteins. However, Arf GAPs also regulate actin independently of effects on membranes or Arf. These functions include acting as upstream regulators of Rho family proteins and providing a scaffold for Rho effectors and exchange factors. With multiple functional elements, the Arf GAPs could integrate signals and biochemical activities that result in co-ordinated changes in actin and membranes necessary for a wide range of cellular functions.     

Arf GAPs as regulators of the actin cytoskeleton

The Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are a family of proteins with a common catalytic domain that induces hydrolysis of GTP bound to Arf GTP-binding proteins. At least three groups of multidomain Arf GAPs affect the actin cytoskeleton and cellular activities, such as migration and movement, that depend on the cytoskeleton. One role of the Arf GAPs is to regulate membrane remodelling that accompanies actin polymerization. Regulation of membrane remodelling is mediated in part by the regulation of Arf proteins. However, Arf GAPs also regulate actin independently of effects on membranes or Arf. These functions include acting as upstream regulators of Rho family proteins and providing a scaffold for Rho effectors and exchange factors. With multiple functional elements, the Arf GAPs could integrate signals and biochemical activities that result in co-ordinated changes in actin and membranes necessary for a wide range of cellular functions.     

Actin cytoskeletal reorganizations and coreceptor-mediated activation of rac during human immunodeficiency virus-induced cell fusion

The membrane fusion events which initiate human immunodeficiency virus type 1 (HIV-1) infection and promote cytopathic syncytium formation in infected cells commence with the binding of the HIV envelope glycoprotein (Env) to CD4 and an appropriate coreceptor. Here, we show that HIV Env-coreceptor interactions activate Rac-1 GTPase and stimulate the actin filament network reorganizations that are requisite components of the cell fusion process. Disrupting actin filament dynamics with jasplakinolide or latrunculin A arrested fusion at a late step in the formation of Env-CD4-coreceptor complexes. Time-lapse confocal microscopy of living cells revealed vigorous activity of actin-based, target cell membrane extensions at the target cell-Env-expressing cell interface. The expression of dominant-negative forms of actin-regulating Rho-family GTPases established that HIV Env-mediated syncytium formation relies on Rac-1 but not on Cdc42 or Rho activation in target cells. Similar dependencies were found when cell fusion was induced by Env expressed on viral or cellular membranes. Additionally, Rac activity was specifically upregulated in a coreceptor-dependent manner in fusion reaction cell lysates. These results define a role for HIV Env-coreceptor interactions in activating the cellular factors essential for virus-cell and cell-cell fusion and provide evidence for the participation of pertussis toxin-insensitive signaling pathways in HIV-induced membrane fusion.     

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.     

The p10 FAST protein fusion peptide functions as a cystine noose to induce cholesterol-dependent liposome fusion without liposome tubulation

The reovirus p10 fusion-associated small transmembrane (FAST) proteins are the smallest known membrane fusion proteins, and evolved specifically to mediate cell–cell, rather than virus–cell, membrane fusion. The 36–40-residue ectodomains of avian reovirus (ARV) and Nelson Bay reovirus (NBV) p10 contain an essential intramolecular disulfide bond required for both cell–cell fusion and lipid mixing between liposomes. To more clearly define the functional, biochemical and biophysical features of this novel fusion peptide, synthetic peptides representing the p10 ectodomains of ARV and NBV were analyzed by solution-state NMR spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy-based hydrophobicity analysis, and liposome binding and fusion assays. Results indicate that disulfide bond formation promotes exposure of hydrophobic residues, as indicated by bis-ANS binding and time-dependent peptide aggregation under aqueous conditions, implying the disulfide bond creates a small, geometrically constrained, cystine noose. Noose formation is required for peptide partitioning into liposome membranes and liposome lipid mixing, and electron microscopy revealed that liposome–liposome fusion occurs in the absence of liposome tubulation. In addition, p10 fusion peptide activity, but not membrane partitioning, is dependent on membrane cholesterol.