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• W2060818486 abstract "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. 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. The genus Orthoreovirus contains five recognized species, four of which induce cell-cell fusion and multinucleated syncytium formation (1Chappell J.D. Duncan R. Mertens P.P. Dermody T.S. Fauquet C. Mayo M.A. Maniloff J. Desselberger U. Ball L.A. Orthoreovirus, Reoviridae. Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses. Elsevier/Academic Press, London2005: 455-465Google Scholar). Additional fusogenic orthoreoviruses continue to be discovered (2Thalmann C.M. Cummins D.M. Yu M. Lunt R. Pritchard L.I. Hansson E. Crameri S. Hyatt A. Wang L.F. Broome virus, a new fusogenic Orthoreovirus species isolated from an Australian fruit bat.Virology. 2010; 402: 26-40Crossref PubMed Scopus (54) Google Scholar), and the related genus Aquareovirus also contains members that induce syncytium formation (3Attoui H. Fang Q. Mohd Jaafar F. Cantaloube J.F. Biagini P. de Micco P. de Lamballerie X. Common evolutionary origin of aquareoviruses and orthoreoviruses revealed by genome characterization of golden shiner reovirus, grass carp reovirus, striped bass reovirus, and golden ide reovirus (genus Aquareovirus, family Reoviridae).J. Gen. Virol. 2002; 83: 1941-1951Crossref PubMed Scopus (190) Google Scholar). Nonenveloped viruses lack a lipid membrane and virus entry into cells does not involve a membrane fusion event. Thus, nonenveloped viruses generally do not encode membrane fusion proteins and as a result do not induce syncytium formation. The fusogenic orthoreoviruses are a rare exception to this rule. Studies over the past few years have identified the orthoreovirus and aquareovirus proteins responsible for syncytiogenesis (2Thalmann C.M. Cummins D.M. Yu M. Lunt R. Pritchard L.I. Hansson E. Crameri S. Hyatt A. Wang L.F. Broome virus, a new fusogenic Orthoreovirus species isolated from an Australian fruit bat.Virology. 2010; 402: 26-40Crossref PubMed Scopus (54) Google Scholar, 4Corcoran J.A. Duncan R. Reptilian reovirus utilizes a small type III protein with an external myristylated amino terminus to mediate cell-cell fusion.J. Virol. 2004; 78: 4342-4351Crossref PubMed Scopus (71) Google Scholar, 5Dawe S. Duncan R. The S4 genome segment of baboon reovirus is bicistronic and encodes a novel fusion-associated small transmembrane protein.J. Virol. 2002; 76: 2131-2140Crossref PubMed Scopus (64) Google Scholar, 6Racine T. Hurst T. Barry C. Shou J. Kibenge F. Duncan R. Aquareovirus effects syncytiogenesis by using a novel member of the FAST protein family translated from a noncanonical translation start site.J. Virol. 2009; 83: 5951-5955Crossref PubMed Scopus (36) Google Scholar, 7Shmulevitz M. Duncan R. A new class of fusion-associated small transmembrane (FAST) proteins encoded by the non-enveloped fusogenic reoviruses.EMBO J. 2000; 19: 902-912Crossref PubMed Scopus (134) Google Scholar). These fusion-associated small transmembrane (FAST) 6The abbreviations used are: FASTfusion-associated small transmembranePRMproline-rich motifPPIIpolyproline type II helixFPfusion peptideHPhydrophobic patchDOPE1,2-dioleoyl-sn-glycero-3-phosphoethanolamineNBD-DOPE1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2–1,3-benzoxadiazol-4-yl)Rho-DOPE1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)DPCdodecyl phosphocholineDMSOdimethyl sulfoxideNOESYnuclear Overhauser effect spectroscopyTOCSYtotal correlation spectroscopyCDcircular dichroismTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. proteins define a new family of viral fusogens whose structural and functional features distinguish them from the well characterized fusion proteins of enveloped viruses, a paradigm for the general process of protein-mediated membrane fusion (8White J.M. Delos S.E. Brecher M. Schornberg K. Structures and mechanisms of viral membrane fusion proteins. Multiple variations on a common theme.Crit. Rev. Biochem. Mol. Biol. 2008; 43: 189-219Crossref PubMed Scopus (643) Google Scholar). The unique features of the FAST proteins indicate their mechanism of action is unlikely to adhere to the tenets of the enveloped virus membrane fusion model, suggesting there are alternate mechanisms of protein-mediated membrane fusion. fusion-associated small transmembrane proline-rich motif polyproline type II helix fusion peptide hydrophobic patch 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2–1,3-benzoxadiazol-4-yl) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) dodecyl phosphocholine dimethyl sulfoxide nuclear Overhauser effect spectroscopy total correlation spectroscopy circular dichroism N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. The three different classes of enveloped virus fusion proteins have dramatically different structures but remarkably conserved structural changes occur during the fusion reaction. These virus structural proteins contain large, multimeric ectodomains that undergo complex conformational changes to drive virus-cell membrane fusion (9Kielian M. Rey F.A. Virus membrane-fusion proteins. More than one way to make a hairpin.Nat. Rev. Microbiol. 2006; 4: 67-76Crossref PubMed Scopus (442) Google Scholar, 10Melikyan G.B. Common principles and intermediates of viral protein-mediated fusion. The HIV-1 paradigm.Retrovirology. 2008; 5: 111Crossref PubMed Scopus (144) Google Scholar, 11Roche S. Albertini A.A. Lepault J. Bressanelli S. Gaudin Y. Structures of vesicular stomatitis virus glycoprotein. Membrane fusion revisited.Cell. Mol. Life Sci. 2008; 65: 1716-1728Crossref PubMed Scopus (119) Google Scholar). An intermediate structure positions a fusion peptide (FP) at one end of the structure for partial insertion into the target membrane, whereas the opposite end is anchored in the viral membrane by the transmembrane domain. Collapse of the extended structure into a compact trimeric hairpin is presumed to pull the two membranes together and positions the fusion peptide and transmembrane domain at the same end of the structure, thereby driving membrane merger (8White J.M. Delos S.E. Brecher M. Schornberg K. Structures and mechanisms of viral membrane fusion proteins. Multiple variations on a common theme.Crit. Rev. Biochem. Mol. Biol. 2008; 43: 189-219Crossref PubMed Scopus (643) Google Scholar, 12Harrison S.C. Viral membrane fusion.Nat. Struct. Mol. Biol. 2008; 15: 690-698Crossref PubMed Scopus (933) Google Scholar). The helical N-terminal fusion peptides present in Class I viral fusion proteins, such as influenza hemagglutinin, form an amphipathic kinked helix or helical hairpin that exposes a hydrophobic face for membrane insertion and fusion (13Lai A.L. Park H. White J.M. Tamm L.K. Fusion peptide of influenza hemagglutinin requires a fixed angle boomerang structure for activity.J. Biol. Chem. 2006; 281: 5760-5770Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 14Li Y. Tamm L.K. Structure and plasticity of the human immunodeficiency virus gp41 fusion domain in lipid micelles and bilayers.Biophys. J. 2007; 93: 876-885Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 15Lorieau J.L. Louis J.M. Bax A. The complete influenza hemagglutinin fusion domain adopts a tight helical hairpin arrangement at the lipid:water interface.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 11341-11346Crossref PubMed Scopus (129) Google Scholar). Some of the Class I fusogens, and all of the Class II and III fusogens, have internal FPs at the tips of elongated β-strands. These fusion loops position hydrophobic and aromatic residues at the tips of the loops for shallow insertion into the outer leaflet of the target membrane (16Gibbons D.L. Vaney M.C. Roussel A. Vigouroux A. Reilly B. Lepault J. Kielian M. Rey F.A. Conformational change and protein-protein interactions of the fusion protein of Semliki Forest virus.Nature. 2004; 427: 320-325Crossref PubMed Scopus (298) Google Scholar, 17Gregory S.M. Harada E. Liang B. Delos S.E. White J.M. Tamm L.K. Structure and function of the complete internal fusion loop from Ebolavirus glycoprotein 2.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 11211-11216Crossref PubMed Scopus (90) Google Scholar, 18Kadlec J. Loureiro S. Abrescia N.G. Stuart D.I. Jones I.M. The postfusion structure of baculovirus gp64 supports a unified view of viral fusion machines.Nat. Struct. Mol. Biol. 2008; 15: 1024-1030Crossref PubMed Scopus (184) Google Scholar, 19Modis Y. Ogata S. Clements D. Harrison S.C. Structure of the dengue virus envelope protein after membrane fusion.Nature. 2004; 427: 313-319Crossref PubMed Scopus (900) Google Scholar, 20Rey F.A. Molecular gymnastics at the herpesvirus surface.EMBO Rep. 2006; 7: 1000-1005Crossref PubMed Scopus (52) Google Scholar, 21Roche S. Bressanelli S. Rey F.A. Gaudin Y. Crystal structure of the low pH form of the vesicular stomatitis virus glycoprotein G.Science. 2006; 313: 187-191Crossref PubMed Scopus (353) Google Scholar). Whether FPs serve merely as membrane anchors or function to destabilize the lamellar structure of the outer leaflet of the bilayer to induce the first stage of membrane fusion is still unclear (22Epand R.M. Fusion peptides and the mechanism of viral fusion.Biochim. Biophys. Acta. 2003; 1614: 116-121Crossref PubMed Scopus (224) Google Scholar, 23Chernomordik L.V. Kozlov M.M. Mechanics of membrane fusion.Nat. Struct. Mol. Biol. 2008; 15: 675-683Crossref PubMed Scopus (729) Google Scholar). In contrast to the enveloped virus fusion proteins, the FAST proteins are all small (95–198 amino acids), single pass membrane proteins with very small N-terminal ecto- and C-terminal endodomains that function from both sides of the membrane to drive the fusion process (24Boutilier J. Duncan R. Chernomordik L.V. Koslov M.M. Membrane Fusion. Vol. 68. Elsevier, San Diego, CA2011: 107-140Google Scholar). Unlike the enveloped virus fusogens, the FAST proteins are nonstructural viral proteins that are expressed inside virus-infected cells where they traffic to the plasma membrane to induce fusion of virus-infected cells with neighboring uninfected cells (5Dawe S. Duncan R. The S4 genome segment of baboon reovirus is bicistronic and encodes a novel fusion-associated small transmembrane protein.J. Virol. 2002; 76: 2131-2140Crossref PubMed Scopus (64) Google Scholar, 7Shmulevitz M. Duncan R. A new class of fusion-associated small transmembrane (FAST) proteins encoded by the non-enveloped fusogenic reoviruses.EMBO J. 2000; 19: 902-912Crossref PubMed Scopus (134) Google Scholar). The FAST proteins have therefore specifically evolved to induce cell-cell, rather than virus-cell, membrane fusion. The structural limitations of the FAST protein ectodomains (only 19–43 residues) means they cannot induce membrane merger using complex structural rearrangements to pull membranes into close proximity. Instead, mutational studies indicate the FAST protein ectodomains, endodomains, and transmembrane domains are all directly involved in the fusion process (25Barry C. Duncan R. Multifaceted sequence-dependent and -independent roles for reovirus FAST protein cytoplasmic tails in fusion pore formation and syncytiogenesis.J. Virol. 2009; 83: 12185-12195Crossref PubMed Scopus (25) Google Scholar, 26Clancy E.K. Duncan R. Reovirus FAST protein transmembrane domains function in a modular, primary sequence-independent manner to mediate cell-cell membrane fusion.J. Virol. 2009; 83: 2941-2950Crossref PubMed Scopus (33) Google Scholar, 27Shmulevitz M. Salsman J. Duncan R. Palmitoylation, membrane-proximal basic residues, and transmembrane glycine residues in the reovirus p10 protein are essential for syncytium formation.J. Virol. 2003; 77: 9769-9779Crossref PubMed Scopus (40) Google Scholar). Specific functional motifs have been identified in each of these fusion modules, although each FAST protein fusion module has its own unique repertoire of these motifs. Chimeric studies demonstrate that various combinations of the fusion modules from the different FAST proteins can be assembled to generate functional chimeras, but not all combinations are tolerated (24Boutilier J. Duncan R. Chernomordik L.V. Koslov M.M. Membrane Fusion. Vol. 68. Elsevier, San Diego, CA2011: 107-140Google Scholar, 26Clancy E.K. Duncan R. Reovirus FAST protein transmembrane domains function in a modular, primary sequence-independent manner to mediate cell-cell membrane fusion.J. Virol. 2009; 83: 2941-2950Crossref PubMed Scopus (33) Google Scholar, 28Clancy E.K. Duncan R. Helix-destabilizing, β-branched, and polar residues in the baboon reovirus p15 transmembrane domain influence the modularity of FAST proteins.J. Virol. 2011; 85: 4707-4719Crossref PubMed Scopus (14) Google Scholar). The FAST proteins are therefore modular fusogens, but specific combinations of functional motifs must be present in the different fusion modules on either side of the membrane to generate an active cell-cell fusogen. Defining the roles of these functional motifs is the focus of ongoing research efforts. One such functional motif is the hydrophobic patches (HPs) present in each of the FAST proteins. These motifs are the only regions of the FAST proteins that possess an overall hydrophobic character (aside from the transmembrane domain), although they are actually more amphiphilic than hydrophobic (2Thalmann C.M. Cummins D.M. Yu M. Lunt R. Pritchard L.I. Hansson E. Crameri S. Hyatt A. Wang L.F. Broome virus, a new fusogenic Orthoreovirus species isolated from an Australian fruit bat.Virology. 2010; 402: 26-40Crossref PubMed Scopus (54) Google Scholar, 4Corcoran J.A. Duncan R. Reptilian reovirus utilizes a small type III protein with an external myristylated amino terminus to mediate cell-cell fusion.J. Virol. 2004; 78: 4342-4351Crossref PubMed Scopus (71) Google Scholar, 5Dawe S. Duncan R. The S4 genome segment of baboon reovirus is bicistronic and encodes a novel fusion-associated small transmembrane protein.J. Virol. 2002; 76: 2131-2140Crossref PubMed Scopus (64) Google Scholar, 6Racine T. Hurst T. Barry C. Shou J. Kibenge F. Duncan R. Aquareovirus effects syncytiogenesis by using a novel member of the FAST protein family translated from a noncanonical translation start site.J. Virol. 2009; 83: 5951-5955Crossref PubMed Scopus (36) Google Scholar, 7Shmulevitz M. Duncan R. A new class of fusion-associated small transmembrane (FAST) proteins encoded by the non-enveloped fusogenic reoviruses.EMBO J. 2000; 19: 902-912Crossref PubMed Scopus (134) Google Scholar). The HP motifs are located in the ectodomains of the p10 and p14 FAST proteins, but in the cytosolic endodomains of the p13, p15 and p22 FAST proteins. In the case of p10 and p14, the ectodomain HPs possess functional features of FPs. Synthetic peptides of both motifs induce liposome-liposome lipid mixing indicating they have membrane destabilizing properties, and amino acid substitutions in these motifs have adverse effects on cell-cell fusion activity of the full-length FAST protein (29Barry C. Key T. Haddad R. Duncan R. Features of a spatially constrained cystine loop in the p10 FAST protein ectodomain define a new class of viral fusion peptides.J. Biol. Chem. 2010; 285: 16424-16433Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 30Corcoran J.A. Syvitski R. Top D. Epand R.M. Epand R.F. Jakeman D. Duncan R. Myristoylation, a protruding loop, and structural plasticity are essential features of a non-enveloped virus fusion peptide motif.J. Biol. Chem. 2004; 279: 51386-51394Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 31Shmulevitz M. Epand R.F. Epand R.M. Duncan R. Structural and functional properties of an unusual internal fusion peptide in a non-enveloped virus membrane fusion protein.J. Virol. 2004; 78: 2808-2818Crossref PubMed Scopus (38) Google Scholar). The p14 HP contains a myristoylated, proline-hinged loop, whereas the p10 HP contains a cystine loop, and the apices of both of these loops contain a Phe-Val dipeptide, similar to the residues located at the tips of the enveloped virus fusion loops. The p10 and p14 FPs are not sequestered within a complex protein tertiary structure, nor are they likely to serve as membrane anchors because the FAST proteins cannot use mechanical energy provided by complex ectodomain structural rearrangements to promote close membrane apposition. Instead, the Phe and Val residues in the p10 and p14 FPs may be positioned to directly interact with a closely apposed target membrane, thereby inducing curvature changes or other alterations to the interfacial region of the membrane required for merger of the outer leaflets. The p15 FAST protein of baboon reovirus contains the hallmark features of a FAST protein, including a single transmembrane domain that functions as a reverse signal anchor to direct an Nexternal/Cinternal membrane topology, an essential fatty acid modification (i.e. N-terminal myristoylation), a membrane-proximal polybasic motif of no known function, and a HP (Fig. 1A). The p15 FAST protein has the smallest ectodomain (19 residues), and this fusion module has a proline-rich motif (PRM) and an overall hydrophilic nature with no apparent FP motif (5Dawe S. Duncan R. The S4 genome segment of baboon reovirus is bicistronic and encodes a novel fusion-associated small transmembrane protein.J. Virol. 2002; 76: 2131-2140Crossref PubMed Scopus (64) Google Scholar, 32Dawe S. Corcoran J.A. Clancy E.K. Salsman J. Duncan R. Unusual topological arrangement of structural motifs in the baboon reovirus fusion-associated small transmembrane protein.J. Virol. 2005; 79: 6216-6226Crossref PubMed Scopus (34) Google Scholar). It is not at all apparent how this fusion module could function to promote cell-cell membrane fusion. To examine this issue, we used a combination of NMR and CD spectroscopy, syncytium formation, peptide-induced lipid mixing assays, and peptide-liposome binding studies. Results indicate the p15 ectodomain functions as a novel FP motif to induce lipid mixing, dependent on both the N-terminal myristate and the presence of a polyproline type II (PPII) helix. Although residues in the PPII helix do not directly associate with membranes, this structure appears to be required to force exposure of flanking hydrophobic residues that do interact with membranes, stabilizing the weak myristate-membrane interactions and driving the fusion reaction. QM5 and Vero cells were maintained as previously described (4Corcoran J.A. Duncan R. Reptilian reovirus utilizes a small type III protein with an external myristylated amino terminus to mediate cell-cell fusion.J. Virol. 2004; 78: 4342-4351Crossref PubMed Scopus (71) Google Scholar). Cells growing in 12-well plates were transfected with Lipofectamine using the manufacturer's protocol (Invitrogen), and supplemented with serum-containing medium 5 h post-transfection and incubated for the indicated periods of time. The generation and specificity of the rabbit polyclonal p15-specific antisera has been previously described (32Dawe S. Corcoran J.A. Clancy E.K. Salsman J. Duncan R. Unusual topological arrangement of structural motifs in the baboon reovirus fusion-associated small transmembrane protein.J. Virol. 2005; 79: 6216-6226Crossref PubMed Scopus (34) Google Scholar). The Stratagene QuikChange mutagenesis method was used to replace individual residues within the proline-rich motif (PPAPPP; residues 10–15). Authentic p15 was used as a template to make the following substitutions: Q9A, A12P, P13A, P15A, P11A/A12P, P10A/P14A, p15pro1 (AAAAAA), and p15pro2 (PAAAPA). The sequence of all constructs was confirmed. The p15 ectodomain proline-rich motif (VQPPAPPPNA) and a mutant version of this peptide (VQPAAAPANA) were obtained from Dalton Chemical Laboratories, Inc. at 95% purity. A poly-l-proline peptide was purchased from Sigma. All CD spectra were acquired using a Jasco J-810 spectropolarimeter with a 0.1-cm optical path. The p15 peptides were dissolved in phosphate buffer (10 mm, pH 7.0) to a final concentration of 0.2 mg/ml, unless otherwise noted. Spectra were obtained between 250 and 200 nm, and 10 scans were acquired and summed for each sample and the resulting spectrum was smoothed to remove residual noise. Measurements requiring different buffers were modified to include either 3 m urea or 4 m NaCl. Because of the absorbance and light scattering of the buffer components in these solutions at short wavelengths, measurements were taken to 210 nm. Transfected monolayers were fixed and stained with Wright-Giemsa stain (Diff-Quik), and the syncytiogenic activity of various p15 mutants was quantified using a syncytial index assay as previously described (4Corcoran J.A. Duncan R. Reptilian reovirus utilizes a small type III protein with an external myristylated amino terminus to mediate cell-cell fusion.J. Virol. 2004; 78: 4342-4351Crossref PubMed Scopus (71) Google Scholar), based on microscopic quantification of the numbers of syncytial nuclei present in five random fields of view. Transfected monolayers were methanol-fixed and immunostained using primary rabbit polyclonal anti-p15 antiserum as previously described (32Dawe S. Corcoran J.A. Clancy E.K. Salsman J. Duncan R. Unusual topological arrangement of structural motifs in the baboon reovirus fusion-associated small transmembrane protein.J. Virol. 2005; 79: 6216-6226Crossref PubMed Scopus (34) Google Scholar). For immunoblot analysis, cells were grown in 10-cm dishes and lysed in RIPA buffer 8 h post-transfection. Equivalent protein loads of sample (as determined by a Lowry assay) were separated by SDS-PAGE and p15 was detected by immunoblotting as described previously (5Dawe S. Duncan R. The S4 genome segment of baboon reovirus is bicistronic and encodes a novel fusion-associated small transmembrane protein.J. Virol. 2002; 76: 2131-2140Crossref PubMed Scopus (64) Google Scholar). Synthetic p15 peptides were dissolved in buffered 95:5 or 0:100% H2O/D2O with and without 300 mm perdeuterated dodecylphosphocholine (DPC-d38; 98 atom %D). The aqueous buffer used for sample preparation contained 50 mm K2HPO4/KH2PO4 at pH 6.0 or 8.0. For all samples, the concentration of peptide was 5 mm. One- and two-dimensional 1H NMR data sets were collected on a Bruker AVANCE 500 spectrometer and processed as previously described (33Syvitski R.T. Burton I. Mattatall N.R. Douglas S.E. Jakeman D.L. Structural characterization of the antimicrobial peptide pleurocidin from winter flounder.Biochemistry. 2005; 44: 7282-7293Crossref PubMed Scopus (59) Google Scholar). For structure calculations, distance restraints determined from the integration of NOESY cross-peaks (80 ms for myristoylated p15 peptide and 250 ms for non-myristoylated p15 peptide) were classified into four groups: strong, medium, weak, and very weak, corresponding to inter-proton distance ranges of <2.3, 2.0–3.5, 3.3–5.0, and 4.8–6.0 Å, respectively. All structural calculations were based on previous studies (34Nilges M. Kuszewski J. Brunger A.T. Computational Aspects of the Study of Biological Macromolecules by NMR. Plenum Press, New York1991Google Scholar) and were performed using the XPLOR 3.1 software package. The overall quality of these refined structures was examined with the program PROCHECK. Except for random-coil sections, all backbone dihedral angles resided in the well defined, acceptable regions of the Ramachandran plot. For structural determination, spin systems were identified through chemical shifts and characteristic TOCSY cross-peak patterns (60 ms for myristoylated p15 and 120 ms for non-myristoylated p15). Sequence-specific assignments were determined as described previously (30Corcoran J.A. Syvitski R. Top D. Epand R.M. Epand R.F. Jakeman D. Duncan R. Myristoylation, a protruding loop, and structural plasticity are essential features of a non-enveloped virus fusion peptide motif.J. Biol. Chem. 2004; 279: 51386-51394Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 34Nilges M. Kuszewski J. Brunger A.T. Computational Aspects of the Study of Biological Macromolecules by NMR. Plenum Press, New York1991Google Scholar). The structure of the non-myristoylated p15 ectodomain at pH 6.0 was deposited with the Protein Data base (PDB code 2LKW). Liposomes were prepared as previously described (30Corcoran J.A. Syvitski R. Top D. Epand R.M. Epand R.F. Jakeman D. Duncan R. Myristoylation, a protruding loop, and structural plasticity are essential features of a non-enveloped virus fusion peptide motif.J. Biol. Chem. 2004; 279: 51386-51394Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) using a 1:1:1 molar ratio of 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol (Avanti Polar Lipids) and extruded through a polycarbonate filter in a hand-held extruder (Avestin, Ottawa) to generate 100 nm liposomes. Fluorescent liposomes were similarly generated, except 4 mol % of DOPE in each liposome was replaced with 2 mol % each of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2–1,3-benzoxadiazol-4-yl) (NBD-DOPE) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rho-DOPE). The lipid concentration of each preparation was confirmed using a lipid-phosphorus assay (35Stewart J.C. Colorimetric determination of phospholipids with ammonium ferrothiocyanate.Anal. Biochem. 1980; 104: 10-14Crossref PubMed Scopus (1536) Google Scholar). Non-fluorescent and fluorescent liposomes were mixed in a 9:1 ratio in HEPES-buffered saline (HBS: 150 mm NaCl, 10 mm HEPES, pH 7.4) to obtain a final concentration of 100 μm lipid. The peptides were dissolved in DMSO and assayed for lipid mixing in the indicated final concentrations, using the same volume of DMSO as a solvent control. For temperature shift experiments, peptide and liposomes were mixed together and incubated at 20 °C for 0 or 10 min, then rapidly heated to 37 °C by addition of an equal volume of buffer preheated to 56 °C. Measurements were made at 37 °C using an excitation wavelength of 460 nm and an emission wavelength of 535 nm. Maximum lipid mixing was determined as previously described (36Pereira F.B. Goñi F.M. Muga A. Nieva J.L. Permeabilization and fusion of uncharged lipid vesicles induced by the HIV-1 fusion peptide adopting an extended conformation. Dose and sequ" @default.
• W2060818486 created "2016-06-24" @default.
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• W2060818486 creator A5015018538 @default.
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• W2060818486 date "2012-01-01" @default.
• W2060818486 modified "2023-09-26" @default.
• W2060818486 title "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" @default.
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