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. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc

Reovirus FAST Protein Transmembrane Domains Function in a Modular, Primary Sequence-Independent Manner To Mediate Cell-Cell Membrane Fusion▿

The FAST proteins are a unique family of virus-encoded cell-cell membrane fusion proteins. In the absence of a cleavable N-terminal signal peptide, a single-pass transmembrane domain (TMD) functions as a reverse signal-anchor to direct the FAST proteins into the plasma membrane in an Nexo/Ccyt topology. There is little information available on the role of the FAST protein TMD in the cell-cell membrane fusion reaction. We show that in the absence of conservation in the length or primary amino acid sequence, the p14 TMD can be functionally exchanged with the TMDs of the p10 and p15 FAST proteins. This is not the case for chimeric p14 proteins containing the TMDs of two different enveloped viral fusion proteins or a cellular membrane protein; such chimeric proteins were defective for both pore formation and syncytiogenesis. TMD structural features that are conserved within members of the FAST protein family presumably play direct roles in the fusion reaction. Molecular modeling suggests that the funnel-shaped architecture of the FAST protein TMDs may represent such a conserved structural and functional motif. Interestingly, although heterologous TMDs exert diverse influences on the trafficking of the p14 FAST protein, these TMDs are capable of functioning as reverse signal-anchor sequences to direct p14 into lipid rafts in the correct membrane topology. The FAST protein TMDs are therefore not primary determinants of type III protein topology, but they do play a direct, sequence-independent role in the membrane fusion reaction

Unusual Topological Arrangement of Structural Motifs in the Baboon Reovirus Fusion-Associated Small Transmembrane Protein

Select members of the Reoviridae are the only nonenveloped viruses known to induce syncytium formation. The fusogenic orthoreoviruses accomplish cell-cell fusion through a distinct class of membrane fusion-inducing proteins referred to as the fusion-associated small transmembrane (FAST) proteins. The p15 membrane fusion protein of baboon reovirus is unique among the FAST proteins in that it contains two hydrophobic regions (H1 and H2) recognized as potential transmembrane (TM) domains, suggesting a polytopic topology. However, detailed topological analysis of p15 indicated only the H1 domain is membrane spanning. In the absence of an N-terminal signal peptide, the H1 TM domain serves as a reverse signal-anchor to direct p15 membrane insertion and a bitopic N(exoplasmic)/C(cytoplasmic) topology. This topology results in the translocation of the smallest ectodomain (∼20 residues) of any known viral fusion protein, with the majority of p15 positioned on the cytosolic side of the membrane. Mutagenic analysis indicated the unusual presence of an N-terminal myristic acid on the small p15 ectodomain is essential to the fusion process. Furthermore, the only other hydrophobic region (H2) present in p15, aside from the TM domain, is located within the endodomain. Consequently, the p15 ectodomain is devoid of a fusion peptide motif, a hallmark feature of membrane fusion proteins. The exceedingly small, myristoylated ectodomain and the unusual topological distribution of structural motifs in this nonenveloped virus membrane fusion protein necessitate alternate models of protein-mediated 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

ALPS motifs can functionally replace the p15 HP.

<p>(A) Helical wheel representations of the p15 HP and ALPS motifs from the indicated proteins. Numbers indicate the location of these motifs in their respective proteins. Color code: yellow, hydrophobic; purple, serine and threonine residues; grey, Gly and Ala residues; blue, basic residues; red, acidic residues; pink, asparagine; green, proline. Numbers inside the helical wheels indicate the mean hydrophobic moment calculated using HeliQuest (<a href="http://heliquest.ipmc.cnrs.fr/" target="_blank">http://heliquest.ipmc.cnrs.fr/</a>). (B) Giemsa-stained micrographs of QM5 cells expressing wt p15 protein or chimeric p15 containing the ArfGap1 ALPS replacing the p15 HP taken at 10 h post-transfection. (C) Relative fusogenicity of p15 proteins containing replacement of the p15 HP with the ALPS motifs indicated in panel A. Arf-HF and Kes-HF are ALPS motifs containing Ala substitutions of three residues in the hydrophibic face (L200, W211, and F221 in ArfGAP1; W10, F13, and F20 in Kes1p). Results from a representative experiment (n = 2) are presented as the levels of syncytium formation SD relative to wt p15 based on quantifying syncytial nuclei in triplicate samples. The inset shows a western blot of equivalent protein loads of lysates from cells expressing the indicated p15 constructs probed with anti-p15 or -actin.</p

ALPS motif can functionally replace an amphipathic helix in the p14 FAST protein.

<p>(A) Schematic of the p14 FAST protein indicating the location of the ectodomain hydrophobic patch (HP), transmembrane domain (TM), polybasic cluster (PB), and amphipathic helix (AH). Numbers indicate residue position in p14, and numbers inside the helical wheels indicate the mean hydrophobic moment calculated using HeliQuest (<a href="http://heliquest.ipmc.cnrs.fr/" target="_blank">http://heliquest.ipmc.cnrs.fr/</a>). Sequence of the AH is depicted below. (B) Helical wheel representation of the p14 AH, color coded as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004962#ppat.1004962.g007" target="_blank">Fig 7</a>. (C, D) Fusogenicity of wt p14 and p14 containing the ArfGAP1 ALPS motif in place of the p14 AH (C), and wt p15 and p15 containing the p14 AH in place of the p15 HP (D). Results are mean number of syncytial nuclei per microscopic field SD for triplicate samples from a representative experiment (n = 2).</p

NMR structural determination of a helix-loop-helix conformation in p15HPpep.

<p>(A) Ensemble of the 50 lowest energy structures (out of 100 calculated) of wt p15HPpep with the C-terminal P81-I86 helix superposed. (B) Ensemble of the 50 lowest energy structures with the N-terminal L70-G74 helix superposed. (C) Backbone superposition of the two terminal p15HPpep helices (L70-G74 and P81-I86) of all 50 ensemble members onto the lowest-energy conformer. (D) Lowest energy conformer of the wt p15HPpep. In this conformer, the α-helical segments span residues L71-G74 and P81-I86 at the termini, connected by an unstructured turn/loop. Residues in panels C and D are color-coded as indicated.</p

Model of the role of the FLiPS motif in membrane fusion.

<p>Depicted is a FAST protein structure based on composite NMR structures of p14 ectodomain and transmembrane domain conformers, and a p15 endodomain FLiPS conformer. Linker regions and the extended C-terminal tail (which is intrinsically disordered) are modelled as unstructured loops. Residues are colour-coded: green, hydrophobic; blue, polar/charged; orange, neutral. Fusion peptide motifs in FAST protein ectodomains are predicted to combine with essential residues in the external interfacial region of the transmembrane domain [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004962#ppat.1004962.ref034" target="_blank">34</a>] to alter membrane curvature leading to dimple formation and merger of outer bilayer leaflets creating a presumed hemifusion intermediate (left). Negative curvature stresses in the outer monolayer create corresponding positive curvature stresses in the inner monolayer, rendering this transient intermediate energetically unfavorable and likely to revert to two planar bilayers. However, FLiPS partitioning into regions of increasing positive curvature, such as those in the inner monolayer at the base of the stalk-like hemifusion intermediate, would stabilize curvature stresses and promote further positive curvature making the forward reaction to pore formation (right) a more energetically favorable means to resolve the unstable hemifusion intermediate curvature. POPC molecules show insertion of the FLiPS into a hydrophobic defect generated by curvature stresses forcing apart lipid headgroups.</p

The p15HPpep peptide preferentially partitions into highly curved membranes.

<p>(A) Purified wt p15HPpep dissolved in DMSO was resolved by reverse-phase HPLC using a water:acetonitrile gradient and eluted with the indicated retention time. The chromatogram presents the area under the peaks in arbitrary units of absorbance at 215 nm. (B) The wt p15HPpep was mixed with liposomes (1:1:1 DOPC:DOPE:cholesterol) of the indicated diameters at a fixed peptide:lipid molar ratio (1:500), and liposomes were separated from free peptide by flotation on sucrose gradients. The peptides present in the top liposome and bottom free peptide fractions were detected by HPLC. Chromatograms from a representative experiment are shown in arbitrary fluorescence units at the same scale for the retention time corresponding to the p15HPpep. The No Liposome chromatograms are for p15HPpep treated exactly as above but incubated in the absence of liposomes before sucrose gradient fractionation; the chromatogram indicates the low level of free peptide contaminating the top sucrose fraction. (C) The liposome (top) and free peptide (bottom) sucrose fractions were analyzed by HPLC as in panel B, and the relative peptide concentration in each fraction quantified by integrating the area under the peak in the HPLC chromatograms. Percent peptide = Area_Top / Total Area_Top+Bottom. Bars represent the mean SEM of three experiments.</p