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.

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.

Piscine reovirus encodes a cytotoxic, non-fusogenic, integral membrane protein and previously unrecognized virion outer-capsid proteins.

Piscine reovirus (PRV) is a tentative new member of the family Reoviridae and has been linked to heart and skeletal muscle inflammation in farmed Atlantic salmon (Salmo salar L.). Recent sequence-based evidence suggests that PRV is about equally related to members of the genera Orthoreovirus and Aquareovirus. Sequence similarities have also suggested that PRV might encode a fusion-associated small transmembrane (FAST) protein, which in turn suggests that PRV might be the prototype of a new genus with syncytium-inducing potential. In previous support of this designation has been the absence of identifiable PRV-encoded homologues of either the virion outer-clamp protein of ortho- and aquareoviruses or the virion outer-fibre protein of most orthoreoviruses. In the current report, we have provided experimental evidence that the putative p13 FAST protein of PRV lacks the defining feature of the FAST protein family - the ability to induce syncytium formation. Instead, p13 is the first example of a cytosolic, integral membrane protein encoded by ortho- or aquareoviruses, and induces cytotoxicity in the absence of cell-cell fusion. Sequence analysis also identified signature motifs of the outer-clamp and outer-fibre proteins of other reoviruses in two of the predicted PRV gene products. Based on these findings, we conclude that PRV does not encode a FAST protein and is therefore unlikely to be a new fusogenic reovirus. The presence of a novel integral membrane protein and two previously unrecognized, essential outer-capsid proteins has important implications for the biology, evolution and taxonomic classification of this virus.

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.

Sterol Regulatory Element Binding Protein (SREBP)-1 is a novel regulator of the Transforming Growth Factor (TGF)-ß receptor I (TßRI) through exosomal secretion.

Accumulation of matrix in the glomerulus is a classic hallmark of diabetic nephropathy. The profibrotic cytokine transforming growth factor beta 1 (TGF-ß1) plays a central role in the development of glomerular sclerosis. Recent studies have demonstrated that the transcription factor sterol regulatory element binding protein (SREBP)-1 is an important regulator of glomerular sclerosis through both induction of TGF-ß1 as well as facilitation of its signaling. Here we have identified that SREBP-1 is also a novel regulator of TGF-ß receptor I (TßRI) expression in kidney mesangial cells. Inhibition of SREBP activation with fatostatin or downregulation of SREBP-1 using siRNA inhibited the expression of the receptor. SREBP-1 did not regulate TßRI transcription, nor did it induce its proteasomal or lysosomal degradation or proteolytic cleavage. Disruption of lipid rafts with cyclodextrin, however, prevented TßRI downregulation. This was not dependent on caveolae since SREBP-1 inhibition could induce TßRI downregulation in caveolin-1 knockout mesangial cells. SREBP-1 associated with TßRI, and SREBP-1 inhibition led to the secretion of TßRI in exosomes. Thus, we have identified a novel role for SREBP-1 as a cell surface retention factor for TßRI in mesangial cells, preventing its secretion in exosomes. Inhibition of SREBP-1 in vivo may thus provide a novel therapeutic strategy for diabetic nephropathy which targets multiple aspects of TGFß signaling and matrix upregulation.

Nuclear transportation of exogenous epidermal growth factor receptor and androgen receptor via extracellular vesicles.

Epidermal growth factor receptor (EGFR) plays a central role in the progression of several human malignancies. Although EGFR is a membrane receptor, it undergoes nuclear translocation, where it has a distinct signalling pathway. Herein, we report a novel mechanism by which cancer cells can directly transport EGFR to the nucleus of other cells via extracellular vesicles (EVs). The transported receptor is active and stimulates the nuclear EGFR pathways. Interestingly, the translocation of EGFR via EVs occurs independently of the nuclear localisation sequence that is required for nuclear translocation of endogenous EGFR. Also, we found that the mutant receptor EGFRvIII could be transported to the nucleus of other cells via EVs. To assess the role of EVs in the regulation of an actual nuclear receptor, we studied the regulation of androgen receptor (AR). We found that full-length AR and mutant variant ARv7 are secreted in EVs derived from prostate cancer cell lines and could be transported to the nucleus of AR-null cells. The EV-derived AR was able to bind the androgen-responsive promoter region of prostate specific antigen, and recruit RNA Pol II, an indication of active transcription. The nuclear-translocated AR via EVs enhanced the proliferation of acceptor cells in the absence of androgen. Finally, we provide evidence that nuclear localisation of AR could occur in vivo via orthotopically-injected EVs in male SCID mice prostate glands. To our knowledge, this is the first study showing the nuclear translocation of nuclear receptors via EVs, which significantly extends the role of EVs as paracrine transcriptional regulators.

Analysis of Extracellular Vesicles in the Tumor Microenvironment.

Extracellular vesicles (ECV) are membrane compartments shed from all types of cells in various physiological and pathological states. In recent years, ECV have gained an increasing interest from the scientific community for their role as an intercellular communicator that plays important roles in modifying the tumor microenvironment. Multiple techniques have been established to collect ECV from conditioned media of cell culture or physiological fluids. The gold standard methodology is differential centrifugation. Although alternative techniques exist to collect ECV, these techniques have not proven suitable as a substitution for the ultracentrifugation procedure.