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

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

Helical properties and hydrophobicity influence the function of the p15 HP.

<p>(A) Secondary structure predictions (left panel) of the wt p15 HP sequence and the indicated p15 HP Ala substitutions (GAG contains a double substitution of G74/76A) as predicted using Phyre² analysis (<a href="http://www.sbg.bio.ic.ac.uk/phyre2" target="_blank">http://www.sbg.bio.ic.ac.uk/phyre2</a>) of the complete p15 endodomain. Residues predicted to be -helical are bolded and underscored with a cylinder. Far-ultraviolet CD spectra (right panel) of wt p15HPpep or the Ala-substituted GAG or G74A p15HPpep peptides depicted on the left were obtained at 37°C in the presence of LPPG micelles. Measurements were collected from three runs in each of two independent experiments and converted to mean residue ellipticity (MRE). (B) QM5 cells expressing wt p15 protein or mutant p15 proteins (HP1-5) containing asparagine substitutions of HP residues indicated on the right were fixed at 10 h post transfection, Giemsa-stained, and quantified for the average number of syncytial nuclei. Results are mean SEM of triplicate samples from n = 3 independent experiments.</p

Centrally-located glycine and proline residues in the p15 HP are essential for cell-cell fusion.

<p>(A) Schematic of the p15 protein indicating locations of the transmembrane domain (TM), polybasic cluster (PB) and hydrophobic patch (HP). Numbers indicate residue positions in p15. Sequence of the HP is depicted below. (B) Transfected QM5 cells expressing wt p15 or p15 mutant proteins containing triple Ala substitutions of the indicated three consecutive HP residues were Giemsa-stained at 9 h post-transfection and the mean ± SEM of syncytial nuclei per microscopic field were quantified by bright field microscopy. (C) QM5 cells co-transfected with EGFP and wt p15 or the indicated p15 HP mutant proteins as described in panel B were co-cultured with non-transfected cells labeled with calcein red, then sorted by flow cytometry to quantify the percentage of dual fluorescent cells indicative of pore formation. Results are the means ± SEM of Overton subtractions using duplicate samples from n = 2 independent experiments. (D) QM5 cells expressing wt p15 (p15) or p15 mutant proteins containing Ala substitutions of the indicated HP residues constructs were processed as in panel B to quantify syncytium formation. (E) Western blot of QM5 cell lysates expressing wt p15 or p15 mutant proteins containing triple Ala substitutions of the indicated HP residues HP were probed with anti-p15 antiserum and anti-actin. (F) Cell surface fluorescence of QM5 cells expressing wt p15 (p15) or p15 mutant proteins containing Ala substitutions of the indicated HP residues was quantified by flow cytometry using anti-p15 antiserum and Alexa Fluor 647-conjugated secondary antibody. Results are percent surface fluorescence relative to wt p15. Bar graphs in panels B, D and E are the mean ± SEM for triplicate samples from n = 3 experiments.</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

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

The p15 HP cannot be replaced with heterologous membrane-interactive amphipathic helices.

<p>(A) Helical wheel projections of the indicated amphipathic helices, 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>. Numbers indicate the location of these motifs in their respective proteins, 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>). (B) Syncytium formation in Giemsa-stained, transfected QM5 cell monolayers expressing wt p15 or chimeric p15 constructs where the p15 HP was replaced by the indicated amphipathic helices. Images were acquired at 10 h post-transfection. (C) Western blot of equivalent protein loads of lysates from cells expressing the indicated p15 constructs probed with anti-p15 or anti-actin.</p

Mutant p15HPpep containing substitutions that disrupt the helix-loop-helix conformation partition into liposome membranes indiscriminate of membrane curvature.

<p>(A) Purified mutant p15HPpep peptides (G74A and G74/76A [GAG]) dissolved in DMSO were resolved by reverse-phase HPLC using a water:acetonitrile gradient and eluted with the indicated retention times (left panel). Peptides were 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. Peptides present in the top liposome and bottom free peptide fractions were detected by HPLC (right panel). Chromatograms from a representative experiment are shown in arbitrary fluorescence units at the same scale. (B) The liposome (top) and free peptide (bottom) sucrose fractions were analyzed by HPLC as in panel A, 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

Partitioning of p15HPpep into liposomes is based on curvature-dependent hydrophobic defects.

<p>(A) Partitioning curves of bis-ANS in 100 mM suspensions of 50 nm, 100 nm, and 400 nm liposomes. bis-ANS fluorescence was fit to one-site binding hyperbola nonlinear regression using GraphPad Prism. Data points represent the mean of three experiments, with error bars contained within the symbols on the graph. (B) HPLC chromatograms in arbitrary units of absorbance at 215 nm of the top (liposomes) and bottom (free peptide) fractions from sucrose gradients following partitioning of wt p15HPpep into liposomes of the indicated diameter as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004962#ppat.1004962.g004" target="_blank">Fig 4A</a>, except liposomes were pre-treated with 10 mM bis-ANS before p15HPpep addition. Note that retention times of the wt p15HPpep differ from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004962#ppat.1004962.g004" target="_blank">Fig 4</a> due to use of a different HPLC column. (C) As in panel B, quantifying percent p15HPpep associated with top (black bars) and bottom (white bars) sucrose fractions based on integrating the area under the chromatogram peaks. Results are mean SEM from three independent experiments.</p