JOURNAL OF VIROLOGY, Apr. 2009, p. 2941–2950 0022-538X/09/$08.00⫹0 doi:10.1128/JVI.01869-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 7
Reovirus FAST Protein Transmembrane Domains Function in a Modular, Primary Sequence-Independent Manner To Mediate Cell-Cell Membrane Fusion䌤 Eileen K. Clancy and Roy Duncan* Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5 Received 4 September 2008/Accepted 26 December 2008
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. Three distinct members of the FAST protein family have been identified, and they are named according to their predicted molecular masses: p14 of reptilian reovirus, p15 of baboon reovirus, and the p10 proteins of Nelson Bay reovirus and avian reovirus (ARV) (13, 18, 48). The FAST proteins share no significant amino acid identity, but they do share certain structural features. Each has a single transmembrane domain (TMD), which, in the natural absence of a cleavable N-terminal signal peptide in the FAST proteins, functions as a reverse signal-anchor sequence (24). The TMD/signal-anchor directs the cotranslational insertion of the FAST proteins into the membrane of the endoplasmic reticulum (ER) in a bitopic Nexoplasmic/Ccytoplasmic (Nexo/Ccyt) topology (Fig. 1). The spatial arrangement of the FAST protein TMDs results in ectodomains of just ⬃20 to 40 residues, with as much or more of the mass of the protein being comprised of the TMD and cytosolic endodomain. This unusual asymmetric membrane topology contrasts markedly with the topologies of the membrane fusion proteins encoded by most enveloped viruses, which generally position the majority of their mass external to the membrane (22). Dramatic structural remodeling of the complex ectodomains of the enveloped virus fusion proteins serves as a driving force for the membrane fusion reaction (8, 12). The small size of the FAST protein ectodomains, therefore, necessitates alternative models to explain how these diminutive viral fusion proteins mediate membrane merger. In addition to shared topologies, the FAST proteins all possess a repertoire of potential membrane interaction motifs that presumably function in concert to alter membrane structure and promote the merger of adjacent bilayers. Each FAST protein has its own signature arrangement of these motifs. In addition to their TMDs, all of the FAST proteins contain one
The fusion-associated small transmembrane (FAST) proteins are a unique family of membrane fusion proteins encoded by the fusogenic reoviruses (20). At 95 to 140 amino acids in size, the FAST proteins are the smallest known viral membrane fusion proteins. Rather than mediating virus-cell fusion, the FAST proteins are nonstructural viral proteins that are expressed on the surfaces of virus-infected or -transfected cells, where they induce cell-cell fusion and the formation of multinucleated syncytia. A purified FAST protein, when reconstituted into liposome membranes, induces liposome-cell and liposome-liposome fusion, indicating the FAST proteins are bona fide membrane fusion proteins (54). In their natural biological context as cell-cell fusogens, however, the FAST proteins exploit cellular adhesins and actin remodeling to maximize their cell-cell fusion potential (40). Studies further suggest that cell-cell fusion mediated by the FAST proteins may contribute to rapid localized dissemination of the infection, followed by apoptosis-induced disruption of the syncytia, resulting in a burst of infectious-progeny-virus release (19, 21, 41). This two-step process for virus dissemination mediated by the FAST proteins may contribute to the natural pathogenicity of the fusogenic reoviruses. How this remarkable family of virus-encoded fusogens induce membrane fusion and syncytium formation remains unclear, but several recent studies have defined specific subdomains and structural motifs likely to be involved in the fusion process.
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5. Phone: (902) 494-6770. Fax: (902) 494-5125. E-mail:
[email protected]. 䌤 Published ahead of print on 7 January 2009. 2941
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further suggest that a structural motif common to the TMDs of members of the FAST protein family may function in a modular, sequence-independent manner to drive cell-cell membrane fusion. MATERIALS AND METHODS
FIG. 1. Structural motifs of the FAST proteins. The linear arrangement of structural motifs present in the ARV p10, p15, and p14 FAST proteins is depicted. The N-terminal ectodomains and C-terminal endodomains are shown to the left and right, respectively, of the indicated TMDs. The numbers indicate amino acid residues. HP, hydrophobic patch; PB, polybasic region; myr, myristoylation; pal, palmitoylation; PP, polyproline; C, cysteine residue.
additional short stretch of moderately hydrophobic residues, termed the hydrophobic patch; this motif resides in the ectodomains of the p10 and p14 FAST proteins but in the endodomain of p15. Mutational and functional analyses of the p10 and p14 hydrophobic patches suggest this motif may function analogously to the fusion peptides or fusion loops of the enveloped virus fusion proteins (15, 17, 49). Each of the FAST proteins contains a membrane-proximal cluster of basic residues in its endodomain, which in the case of p10 has been shown to be essential for cell-cell fusion (50). The FAST proteins are also all modified by fatty acylation; essential N-terminal myristate moieties are present in p14 and p15, while p10 contains an essential palmitoylated dicysteine motif in its endodomain, immediately adjacent to the TMD (13, 18, 50). The TMDs of integral membrane proteins have been implicated in protein-protein and protein-lipid interactions that affect multimer formation, protein clustering in lipid microdomains, and topogenesis and membrane trafficking (3, 43, 51, 55, 57). Numerous studies also indicate a critical role for TMDs in membrane fusion (29, 44). While no clear consensus has emerged on the role of the TMDs of enveloped virus fusion proteins in membrane fusion, results suggest that the TMD exerts its effect on the pore formation stage of the fusion reaction, that the anchor sequence must be long enough to span the membrane and withstand the stresses of the hemifusion-to-fusion transition, and that undefined features of the TMD beyond its length contribute to the fusion reaction. In view of numerous studies implicating the TMDs of the enveloped virus fusion proteins in the membrane fusion reaction, the TMDs of the FAST proteins might also represent membrane-interactive motifs directly involved in the membrane fusion process. In contrast to the wealth of studies of the TMDs of the enveloped virus fusion proteins, only limited mutations of the ARV p10 TMD have been made, revealing a role for a triglycine motif in p10-mediated fusion (50), an observation consistent with the importance of glycine residues in the TMDs of certain enveloped virus fusion proteins (10, 33). To address this significant gap in our understanding of the FAST proteins, we examined the role of the p14 FAST protein TMD in protein topogenesis, protein trafficking, and membrane fusion. Our results indicate that, independent of its role as a reverse signal-anchor, the p14 FAST protein TMD is essential for pore formation and syncytiogenesis. The results
Cells and antibodies. QM5 and Vero cells were maintained at 37°C in a 5% CO2 atmosphere and grown in medium 199 with Earle’s salts and supplemented with 10% or 5% heat-inactivated fetal bovine serum, respectively. The rabbit polyclonal anti-p14 antiserum was previously described (13). Rabbit antiserum against the p14 ectodomain (residues 1 to 36) was prepared by New England Peptide (anti-p14ecto). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (KPL) and anti-green fluorescent protein (GFP) polyclonal antibody (Clonetech) were from the indicated commercial sources. Cloning and plasmids. The pcDNA3-p14 plasmid was described previously (13). This clone was used as a template for point substitutions in the p14 TMD, created using the Quick-Change (Stratagene) method according to the manufacturer’s specifications. The Excite (Stratagene) mutagenesis method was used, according to the manufacturer’s specifications, to create chimeric p14 constructs containing the TMD of either the p10 or p15 FAST protein (p14TM10 and p14TM15, respectively). The pcDNA3-p14 clone was used as a template for sequential PCRs using nested primers to generate p14 chimeras in which the TMD was replaced by that of influenza virus hemagglutinin (HA), vesicular stomatitis virus (VSV) G protein, or the human transferrin receptor (hTfR) (p14TMHA, p14TMVSV, and p14TMTfR, respectively). All mutants were subcloned into the pcDNA3 mammalian expression vector (Invitrogen) and confirmed by sequencing. Transfections, cell staining, and syncytial indexing. Cluster plates containing subconfluent monolayers of QM5 cells were transfected with expression plasmids using Lipofectamine (Invitrogen) according to the manufacturer’s instructions. Cotransfections with GFP were carried out at a 1:1 ratio. At various times posttransfection, the monolayers were fixed with methanol and stained using Wright-Giemsa stain, and a syncytial index was determined by counting the syncytial nuclei in random microscopic fields, as previously described (13). Alternatively, cells were methanol fixed and immunostained using 1:1,000 anti-p14 antibody, as described previously (13). Membrane fractionation, detergent-resistant membrane analysis, and Western blotting. The membrane and soluble fractions from transfected QM5 cells were obtained by vesiculating cells through a 29-gauge syringe, followed by ultracentrifugation at 100,000 ⫻ g for 45 min, as described previously (48). The membrane and soluble fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 15% acrylamide gels. Detergent-resistant membranes (DRMs) were analyzed as previously described (14). Briefly, p14-, p14TMTfR-, placental alkaline phosphatase (PLAP)-, or hTfRtransfected QM5 cells were lysed with 0.5% (vol/vol) Triton X-100 at 4°C; the cell lysates were mixed with 2.4 M sucrose and overlaid with 0.9, 0.8, 0.7, and 0.1 M sucrose solutions; and the sucrose density gradients were subjected to high ultracentrifugation and fractionated into 0.5-ml aliquots. Proteins were visualized using 1:20,000 anti-p14 or 1:10,000 anti-GFP polyclonal or 1:2,000 anti-TfR monoclonal antibody with 1:10,000 goat anti-rabbit immunoglobulin G (H⫹L) peroxidase-labeled antibody. Western blots were imaged on a Typhoon 9410 Variable Mode Imager (Amersham Biosciences). Fluorescent cell staining. QM5 cells were transfected in culture plates containing coverslips, as described above. At 6 or 24 h posttransfection, the cells were washed twice with Hanks balanced salt solution (HBSS), fixed for 20 min in 3.7% formaldehyde, and permeabilized in 0.1% Triton X-100 in phosphatebuffered saline (PBS) for staining of intracellular p14. The cells were stained with 1:1,000 anti-p14 primary antibody and 1:200 Alexa-Fluor 488 secondary antibody, as described previously (13). For surface staining, cells were transfected for 6 or 24 h and then stained with 1:200 anti-p14ecto and 1:200 Alexa-Fluor 488 secondary antibody and fixed with 3.7% formaldehyde, as described previously (13). Cells were mounted on glass slides using fluorescence mounting medium (Dako) and then visualized and photographed using a Zeiss LSM510 scanning argon laser confocal microscope and the ⫻100 objective. Fluorescence-activated cell sorter surface expression. QM5 cells were transfected in culture plates at ⬃70% confluence, as described above. At 24 h posttransfection, the cells were washed three times in cold HBSS, blocked for 30 min (1% bovine serum albumin, 0.02% NaN3, 5% normal goat serum), and then incubated with primary antibody (1:200 anti-p14ecto in HBSS, 1% bovine serum albumin, 0.02% NaN3, 5% normal goat serum) for 1 h at 4°C. The cells were washed six times with cold HBSS and then secondary antibody (1:2,000 Alexa 647
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in HBSS, 1% bovine serum albumin, 0.02% NaN3, 5% normal goat serum) for 1 h at 4°C and washed again as described above. The cells were lifted using 50 mM EDTA in PBS, resuspended in PBS, and then fixed in 3.7% formaldehyde. Pore formation assay. QM5 cells at low cell densities were cotransfected with pEGFP and pcDNA3, p14TMHA, p14TMTfR, or authentic p14 for 6 (p14) or 24 h. An excess of Vero cells were stained with 20 g/ml of calcein red for 30 min at 37°C, suspended with 0.1% trypsin, and then seeded onto the transfected QM5 cells 3 h posttransfection. The cells were cocultured at 37°C for ⬃3 h until small syncytia were visible in wells transfected with wild-type p14. Cells were harvested with 0.1% trypsin and fixed in 3.7% paraformaldehyde, and cofluorescent cells were analyzed by flow cytometry (FACSCalibur [Becton Dickinson]) using appropriate filter sets. The cells were gated for GFP expression, and 10,000 GFPpositive cells were counted and analyzed using Cell Quest software.
RESULTS Transmembrane glycine residues are not important for p14mediated fusion. Previous studies indicated that glycine residues in the TMD are important in the fusion activities of various enveloped virus fusion proteins and the p10 FAST protein of ARV (10, 33, 36, 50). While no specific role for glycine residues has been defined, their potential involvement in promoting either TMD-TMD interactions or destabilization of the TMD helix has been posited (29, 44). To determine if glycine residues were similarly important to the p14 fusion process, alanines were substituted for either or both of the p14 transmembrane glycine residues to create the p14G42A, p14G53A, and p14G42/53A constructs (Fig. 2A). These constructs were transfected into QM5 cells, and their cell-cell fusion activities were assessed by microscopic examination of cell monolayers for the presence of multinucleated syncytia. As shown qualitatively by light microscopy of Giemsa-stained monolayers (Fig. 2B) and quantitatively using a syncytial-indexing assay (Fig. 2C), all three p14 mutants containing glycine-alanine substitutions retained the full fusion capacity of the wild-type protein. Therefore, unlike the p10 FAST protein and several enveloped virus fusion proteins, the mechanism of membrane fusion mediated by the p14 FAST protein is not dependent on the presence of glycine residues in the TMD. The FAST protein TMDs are interchangeable. The discordance between the effects of glycine substitutions on the fusion activities of the p14 FAST protein (Fig. 2) and the p10 FAST protein (50) raised the possibility that the TMDs of the various FAST proteins might have different roles during the fusion reaction. If so, then one might predict that the different FAST protein TMDs would not be interchangeable. To test this hypothesis, we created chimeric p14 proteins in which the native TMD was replaced with that of the p15 or p10 FAST protein. The N termini of the FAST protein TMDs were set as the first residue following a charged glutamic acid (p14 and p15) or, lacking a charged residue, a tyrosine adjacent to an upstream proline (p10). The C termini of the p14 and p15 TMDs were set at the last residue preceding the first downstream positively charged residue (Fig. 3A). For p10, the C-terminal boundary was set immediately upstream of two palmitoylated cysteine residues (which are absent from p14 and p15) that occur adjacent to the first positively charged residue (50). All of these boundaries were identified by one or more of the algorithms used to predict the locations of TMDs. Using these margins, the 19-residue p14 TMD was replaced with either the 19-residue TMD of p10 (p14TM10) or the 23-residue p15 TMD (p14TM15). These chimeric p14 constructs were as-
FIG. 2. Transmembrane glycine residues are not required for p14mediated fusion. (A) Linear representation of the p14 TMD indicating alanine substitutions for glycine residues. (B) QM5 cells were transfected with wild-type p14 (a) or a TMD mutant with a glycine substitution, p14G42A (b), p14G53A (c), or p14G42/53A (d), and then Giemsa stained to detect multinucleated-syncytium formation. Scale bars ⫽ 100 m. (C) QM5 cells were transfected with p14, p14G42A, p14G53A, or p14G42/53A, and the average number of syncytial nuclei per field was determined from Giemsa-stained monolayers at 6 h posttransfection. The results are expressed as the means plus standard deviations of a representative experiment in triplicate.
sessed for their cell-cell fusion activities in transfected QM5 cells. A time course analysis of syncytiogenesis revealed that the p14 constructs containing the heterologous TMDs of the other FAST proteins retained cell-cell fusion capacity; both constructs fused monolayers to completion (i.e., all cells were incorporated into syncytia), although the kinetics of syncytiogenesis was reduced relative to that of the authentic p14 protein (Fig. 3B). The p14TM10 construct, which exchanged TMDs of equal lengths, displayed a more pronounced reduction in the rate of cell-cell fusion compared to the p14TM15
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FIG. 3. The p14 TMD has no specific primary sequence requirements. (A) Linear representation of the p14 TMD and substituted TMDs of p10 and p15. The boldface characters indicate the TMDs, and the flanking sequences are indicated in lightface. (B) QM5 cells were transfected with wild-type p14 or with chimeric p14TM10 or p14TM15, and the average numbers of syncytial nuclei per field were determined from Giemsa-stained monolayers at the indicated times posttransfection. Cells transfected with p14 or p14TM15 could not be quantified at 12 h posttransfection due to syncytium-induced destruction of the monolayer. The results are expressed as the means ⫾ standard deviations of a representative experiment in triplicate.
construct, which contained the longer TMD of the p15 FAST protein yet fused monolayers with kinetics very similar to those of authentic p14 (Fig. 3B). The FAST protein TMDs are therefore interchangeable and capable of functioning in a modular fashion with ecto- and endodomains that differ extensively in their sequences, repertoires, and arrangements of structural motifs (Fig. 1). The different lengths and lack of sequence identity in the FAST protein TMDs implied the absence of a strict sequence- or length-dependent role of the TMD in p14mediated membrane fusion. The p14 TMD is required for pore formation and cell-cell fusion. While lacking any obvious sequence similarity, it was possible that the FAST protein TMDs all shared some undefined structural feature(s) required for membrane fusion. To test this hypothesis, chimeric p14 proteins were constructed in which the native p14 TMD was replaced with those of other viral fusion proteins, either the 27-residue TMD of influenza virus HA (p14TMHA) or the 20-residue TMD of VSV G protein (p14TMVSV) (Fig. 4A). A third construct was produced in which the p14 TMD was replaced with the 26-residue TMD of a nonfusogenic membrane protein, hTfR (p14TMTfR). All of these chimeric p14 constructs were expressed in transfected cells, as determined by immunostaining using polyclonal antisera that recognized the p14 ecto- and endodomains present in the chimeric proteins (Fig. 4B). However, when assessed for their cell-cell fusion activities, all of these chimeric p14 constructs failed to induce syncytium formation, as evidenced by the presence of single antigen-positive cells (Fig. 4B). Giemsa staining of monolayers to clearly reveal cell nuclei
FIG. 4. The p14 TMD cannot be functionally replaced by heterologous TMDs. (A) Linear representation of the p14 TMD and substituted TMDs of hTfR, the VSV G protein, and influenza virus HA. (B) QM5 cells were transfected with wild-type p14 (a), p14TMTfR (b), p14TMVSV (c), or p14TMHA (d) and immunostained using polyclonal anti-p14. The arrows in image a indicate antigen-positive syncytia. Scale bars ⫽ 100 m. (C) Dual-color pore formation assay. QM5 cells were cotransfected with GFP and pcDNA3 vector, p14, p14TMHA, or p14TMTfR and overseeded with calcein red-labeled Vero cells 3 h posttransfection. The cells were resuspended and fixed 9 h posttransfection, and GFP-positive cells were gated. The percentages of GFP-positive cells that acquired the aqueous calcein red fluor due to pore formation (indicated above the horizontal gating line) were quantified and are shown relative to the forward scatter (FSC-H). au, arbitrary units.
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also failed to detect any evidence of cell-cell fusion, even after prolonged incubation of the transfected cells (data not shown). The p14 TMD can therefore be functionally replaced with the TMDs of other FAST proteins, but not with the TMDs from heterologous proteins. Numerous studies implicate the TMDs of enveloped virus fusion proteins in the transition from hemifusion to stable pore formation (29, 30, 35, 44). Repeated attempts to identify hemifusion mutants of the FAST proteins, using the diversity of approaches applied to analyze hemifusion mutants of the enveloped virus fusion proteins, have so far been unsuccessful (unpublished data). Instead, a quantitative pore formation assay recently developed for the FAST proteins (40) was used to determine whether the loss of syncytiogenic activity of the chimeric p14 constructs was due to a deficiency in stable pore formation and/or the subsequent expansion of fusion pores into syncytia. Subconfluent monolayers of QM5 cells were cotransfected with plasmids expressing enhanced GFP and authentic p14 or the chimeric p14TMHA or p14TMTfR protein. Vero cells were labeled with the small aqueous fluor calcein red-orange and then trypsinized and seeded onto the transfected QM5 cells. The two cell populations were cocultured for 3 h to allow cell-cell fusion to progress, and resuspended cells were analyzed by flow cytometry to determine the percentage of GFP-expressing cells that acquired calcein red (Fig. 4C). Pore formation induced by authentic p14 was clearly evident from the fourfold increase in the number of cofluorescent cells relative to the background level observed in vector-transfected cells. In contrast, in three separate experiments conducted in duplicate, cells expressing the p14TMHA or p14TMTfR protein yielded no increase in the number of cofluorescent cells relative to vector-transfected cells (Fig. 4C). The TMDs from heterologous proteins were therefore incapable of supporting not only p14-induced syncytium formation, but also the formation of small stable pores. Heterologous TMDs can function as signal-anchors to direct p14 membrane insertion. The FAST proteins are representatives of a minor subset of membrane proteins, the type III class of integral membrane proteins, which lack cleavable Nterminal signal peptides (24). Instead, type III proteins use their TMDs as reverse signal-anchors, which serve as both a membrane anchor and a membrane insertion signal to direct an Nexo/Ccyt membrane topology. In contrast, the TfR TMD functions as a typical signal-anchor sequence to direct native TfR into an opposite Cexo/Ncyt membrane topology (59), while the TMDs of the influenza virus HA and VSV G proteins serve as stop-transfer anchor sequences after an N-cleavable signal peptide directs membrane insertion. In view of the distinct roles of these TMDs in membrane insertion and topogenesis, the loss of fusion activity of chimeric p14 proteins containing these heterologous TMDs might reflect the inability of the TMDs to function as signal-anchors. To address whether heterologous TMDs can direct p14 into the membrane, the subcellular localization of the chimeric p14 proteins was assessed by fluorescence microscopy and subcellular fractionation. Permeabilized cells expressing authentic p14 or the chimeric p14 proteins were immunostained using a polyclonal antiserum that recognizes both the ecto- and endodomains of p14. Immunofluorescence microscopy revealed that the chimeric proteins displayed a punctate intra-
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FIG. 5. Heterologous TMDs function as signal-anchors to direct p14 membrane insertion. (A) QM5 cells were transfected with p14 (a), p14TMTfR (b), p14TMVSV (c), or p14TMHA (d); fixed 6 (p14) or 24 h posttransfection; and immunostained with polyclonal anti-p14 and Alexa 488-conjugated secondary antibody. Scale bars ⫽ 10 m. (B) Transfected QM5 cell extracts were fractionated into soluble (S) and membrane (M) fractions. The presence of p14, p14TMTfR (TfR), p14TMVSV (VSV), or p14TMHA (HA) in each fraction was detected by SDS-PAGE and immunoblotting with polyclonal anti-p14 antibody.
cellular staining pattern throughout the cytosol (Fig. 5A), consistent with a membrane protein trafficking through the ERGolgi apparatus pathway. Subcellular fractionation and Western blot analysis of the soluble and membrane fractions from transfected cells indicated that the chimeric proteins were expressed at levels equal to or greater than that of authentic p14 and that all of the chimeric p14 proteins exist as integral membrane proteins (Fig. 5B). The heterologous TMDs were therefore capable of functioning as signal-anchors to direct p14 membrane insertion. The HA and TfR TMDs function as reverse signal-anchors to direct p14 topogenesis. To confirm that heterologous TMDs can serve as reverse signal-anchors, directing the chimeric proteins to the plasma membrane in the correct NexoCcyt topology, nonpermeabilized, transfected QM5 cells expressing p14 or the various chimeric p14 proteins were immunostained using a polyclonal antiserum raised against a synthetic peptide representing the N-terminal p14 ectodomain. Confocal microscopy clearly revealed detection of p14, p14TMHA, and p14TMTfR on the cell surface (Fig. 6A), indicating that both the HA and TfR TMDs serve as reverse signal-anchors to traffic p14 in the correct membrane topology to the cell surface. The qualitative cell surface microscopy results were quantitatively confirmed by flow cytometry, using nonpermeabilized cells probed with a p14 ectodomain-specific antiserum (Fig. 6B). The rapid induction of p14-induced syncytium formation within 4 to 5 h posttransfection complicates flow cytometry due to the size of the syncytia. At these early time points, p14 steady-state protein levels on the cell surface (i.e., surface
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FIG. 7. p14TMTfR associates with DRMs. Lysates of QM5 cells transfected with wild-type p14, p14TMTfR, hTfR, or PLAP were subjected to sucrose density gradient ultracentrifugation. Sucrose fractions were analyzed for the presence of proteins by SDS-PAGE and Western blotting with polyclonal anti-p14, anti-TfR, or anti-PLAP antibody. The percent sucrose in each fraction is indicated below each row, and the locations of the detergent-resistant lipid rafts, intermediate-density fractions (inter), and high-density fractions containing solubilized proteins (sol) are indicated above each row.
FIG. 6. The HA and TfR TMDs function as reverse signal-anchors to direct p14 topogenesis. (A) The presence of p14 (a), p14TMTfR (b), p14TMVSV (c), or p14TMHA (d) on the surfaces of transfected QM5 cells was detected 6 (p14) or 24 h posttransfection by immunostaining unfixed cells using anti-p14ecto antibody and Alexa 488-conjugated secondary antibody. Scale bars ⫽ 10 m. (B) Surface expression was determined by staining QM5 cells transfected with 0.5 g of p14G2A (black), p14TMTfR (red), p14TMVSV (blue), or p14TMHA (purple) (a) or 0.5 g of p14TMTfR and 0.01 g of p14G2A (b) with antip14ecto antibody and Alexa 647-conjugated secondary antibody, followed by fluorescence-activated cell sorter analysis. The gray histograms are mock-transfected cells similarly stained. (C) Giemsa stains of QM5 cells transfected with 0.5 g of p14TMTfR (a), 0.01 g of p14 (b), or 0.005 g of p14 (c). Scale bars ⫽ 100 m.
expression) are difficult to quantify. A previously characterized nonmyristoylated and nonfusogenic mutant of p14 that correctly traffics to the cell surface, referred to as p14G2A (13), was therefore used as a positive control, and cells transfected with the p14G2A, p14TMHA, and p14TMTfR constructs were analyzed for cell surface expression by flow cytometry at 24 h posttransfection. As shown in Fig. 6B, the HA TMD directed p14 to the plasma membrane in the correct topology as efficiently as the p14 TMD. Similar analysis revealed that cell surface levels of p14TMTfR in the correct topology were markedly reduced relative to those of authentic p14. Since
decreased cell surface levels of enveloped virus fusion proteins result in a loss of cell-cell fusion activity (16), the fusiondefective phenotype of p14TMTfR might reflect decreased cell surface expression rather than the inability of the heterologous TMD to function in the membrane fusion reaction. Flow cytometry was used to determine the plasmid dose that decreased p14G2A cell surface expression to levels equivalent to those observed in p14TMTfR-transfected cells (Fig. 6B, histogram b), and the same doses of plasmid expressing authentic p14 were used to transfect cells. Even at these reduced levels of surface expression, p14 induced extensive syncytium formation (Fig. 6C), although with delayed kinetics. At the same time points, p14TMTfR was still incapable of inducing cell-cell fusion. The fusion defect of the p14TMTfR construct therefore reflects the inability of this TMD to support the membrane fusion activity of p14. The p14 TMD is not required for localization to DRMs. It was recently reported that p14 localizes to DRMs, a property that correlates with p14-induced cell-cell fusion (14). For some proteins, the TMD is essential for DRM localization (2, 11, 23, 28, 43). Since TfR does not associate with DRMs, the decreased surface expression and/or the loss of fusion activity of the p14TMTfR protein might reflect a loss of chimeric p14 localization to DRMs. To examine this possibility, transfected QM5 cells were lysed at 4°C using 0.05% TX-100, and the DRM fraction was isolated by sucrose density gradient centrifugation. As previously reported (14), p14 was broadly distributed through the various sucrose density fractions, with a subpopulation isolated in the low-density fractions that correspond to DRMs (Fig. 7). PLAP, a prototypical DRM-associated protein, served as a positive control for DRM isolation and was detected in the same low-density sucrose fractions. Authentic TfR, which does not associate with DRMs, was found exclusively in high-density fractions that correspond to solubilized protein. Interestingly, the p14TMTfR protein displayed a broad sucrose density distribution similar to that of
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FIG. 8. p14TMVSV is not trafficked to the plasma membrane in the correct or reverse topology. Surface immunofluorescence of nonpermeabilized (a, b, and c) and intracellular fluorescence of permeabilized (d, e, and f) QM5 cells transfected with p14G2A, p14TMVSV, or 15end14, using anti-p14 antibody and Alexa 488-conjugated secondary antibody. Scale bar ⫽ 10 m.
authentic p14 (Fig. 7), indicating that the p14 TMD is not required for association with DRMs and that the TfR TMD does not target proteins to the soluble membrane fraction. The decreased surface expression and loss of fusion activity displayed by the p14TMTfR protein are therefore not due to an inability to localize the chimeric p14 to DRMs. Chimeric p14 containing the VSV G TMD fails to localize to the plasma membrane. In contrast to the p14TMHA- and p14TMTfR-transfected cells, immunofluorescent cell surface staining using the ectodomain-specific anti-p14 antiserum provided no evidence that p14TMVSV was similarly localized to the plasma membrane in the correct topology (Fig. 6A). This observation was quantitatively confirmed by flow cytometry; the cell surface fluorescence profile of p14TMVSV-transfected cells was identical to that of mock-transfected cells (Fig. 6B). Therefore, while p14TMVSV still incorporates into membranes as an integral membrane protein (Fig. 5), the VSV G TMD is insufficient to direct p14 to the plasma membrane. This result was surprising, since of the three heterologous TMDs, the VSV G TMD most closely approximates the length of the p14 TMD. Furthermore, VSV G and chimeric enveloped virus fusion proteins containing the VSV G TMD all traffic to the cell surface in the correct topology (37). We therefore further explored this unexpected observation. An absence of detectable levels of p14TMVSV on the surfaces of transfected cells could be the result of either (i) an inability to traffic to the plasma membrane or (ii) insertion in the membrane in the incorrect topology, preventing recognition by the p14 ectodomain-specific antibody. To distinguish between defects in trafficking to the plasma membrane versus topogenesis, p14TMVSV-transfected cells were examined by fluorescence microscopy using the polyclonal anti-p14 antiserum that recognizes both the p14 ecto- and endodomains. As shown in Fig. 8, p14TMVSV was clearly detectible in permeabilized cells and displayed the punctate intracellular staining pattern typical of authentic p14 (Fig. 5A). Similar to the ectodomain-specific anti-p14 serum used in Fig. 6, polyclonal anti-p14 serum also detected cell surface-localized authentic p14 but failed to detect p14TMVSV in the plasma membrane (Fig. 8a and b). These results suggested that p14TMVSV is not
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present in the plasma membrane in either the correct Nexo/Ccyt membrane topology or the inverse Ncyt/Cexo topology. To ensure that the polyclonal anti-p14 serum was capable of detecting epitopes in the p14 endodomain that would be exposed on the surface if p14TMVSV were in the wrong orientation, cells were transfected with a plasmid expressing p15endo14, a chimeric p15 FAST protein in which the endodomain was replaced with that of p14. As expected, this antiserum did not detect the p15endo14 protein (which assumes the correct Nexo/Ccyt membrane topology) on the cell surface, but the p14 endodomain in this chimeric p15 protein was easily detectible in the cytosol of transfected cells when the cells were permeabilized with 0.1% TX-100 (Fig. 8c and f). Therefore, while the VSV G TMD efficiently serves as a reverse signal-anchor sequence to direct p14 into the membrane in the correct topology, this chimeric p14 protein fails to accumulate in the plasma membrane and is therefore incapable of inducing cell-cell fusion and syncytium formation. DISCUSSION With the majority of the protein mass comprised of the TMD and endodomain, the mechanism of FAST protein-mediated membrane fusion seems to be particularly focused on the donor membrane. The present results indicate that, aside from their role as reverse signal-anchors, the TMDs of the FAST proteins represent membrane-interactive motifs that play essential roles in the fusion reaction. While TMD substitutions have a diversity of effects on the protein trafficking and subcellular localization of the p14 FAST protein, such effects are independent of the role of the TMD in the fusion reaction. Our results further suggest that structural properties of the FAST protein TMDs may be conserved within this protein family and that these motifs function in a primary sequenceand length-independent manner to directly affect the fusion activities of the FAST proteins. The FAST protein TMDs play a direct role in the membrane fusion reaction, but they are not essential determinants of protein topogenesis and they have variable effects on protein trafficking. Influenza virus HA uses a cleavable N-terminal signal peptide for membrane insertion, with its TMD serving merely as an anchor sequence, while the TfR TMD functions both as a signal sequence and as an anchor to direct TfR into a Cexo/Ncyt topology (45, 59). In spite of these differences, both of these TMDs allowed p14 to assume the correct membrane topology. These results are consistent with the hypothesis that the hydrophobicity of the signal sequence (strongly hydrophobic signals insert in an Nexo/Ccyt topology), the charge ratio flanking the signal sequence (the “positive-inside” rule), and the size and complexity of the ectodomain (small, unfolded N-terminal domains are translocated) are the main topological determinants (25). In this regard, all of the above-mentioned TMDs are highly hydrophobic, all are flanked on the C-terminal side by the conserved polybasic regions present in the endodomains of the FAST proteins, and the N-terminal ectodomains of the FAST proteins are all small and therefore likely to be easily translocated. Although the FAST protein TMDs are not required for membrane insertion, TMDs can influence p14 trafficking. The p14TMTfR protein showed diminished protein surface levels, while the p14TMVSV con-
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FIG. 9. Structural modeling of the FAST protein TMDs. The amino acid sequences of the indicated TMDs were modeled as ␣-helices using Swiss-PDB Viewer v4.0 (http:www.expasy.org/spdbv/), and the output pdb file was manipulated using VMD Viewer v1.8.5 (26) to present the end-on (left) and side (right) views of the modeled helices. Backbone hydrogens and oxygens were omitted for clarity, and the backbone and side chains are colored blue. Green, aromatic side chains; yellow, polyglycine motif in p10; red, oxygens on polar side chains.
struct failed to accumulate to detectible levels in the plasma membrane. Several possible explanations for these trafficking defects need to be explored. For example, low surface levels of p14TMTfR might reflect the ability of the TfR TMD to increase the recycling of chimeric surface proteins (58) or the possible presence of ER retention signals in TMDs (56). Similarly, efficient export of the VSV G protein from the ER and trans-Golgi network requires motifs present in its C-terminal endodomain (38, 46). The absence of these export signals might inhibit p14 transport to the plasma membrane, although these export signals normally only slow the exit from the ER of chimeric proteins containing the VSV TMD without affecting overall plasma membrane accumulation of G protein (4, 46). Since the FAST protein TMDs are interchangeable with each other but not with the TMDs of heterologous proteins, we suggest that the FAST proteins may contain family-specific structural features in their TMDs that directly affect fusion activity. The absence of direct linear-sequence conservation in the FAST protein TMDs and the differential effects of glycine substitutions on the p10 and p14 FAST proteins (50) imply that a structural motif conserved within family members and involved in fusion activity is not dependent on primary sequence. Each of the FAST protein TMDs contains four or five aromatic residues (Fig. 3A). Molecular modeling of the TMDs as helices revealed a sided-helix structure in the p10 TMD, with clustering of the bulky aromatic side chains on one face of the helix (Fig. 9), but this periodicity was not conserved in the p14 or p15 TMD. Interestingly, the aromatic residues are clustered in the cytoplasmic side of the p14 and p15 TMDs (Fig. 3A), which imparts a funnel-shaped architecture to these TMDs,
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with the C-terminal side of the TMD displaying a broader cross-sectional area (Fig. 9). The funnel-shaped architecture is not as apparent in the p10 TMD; while 3 of the 4 aromatic residues are contained within the 12 C-terminal residues, the fourth residue resides at the boundary of the TMD and the N-terminal ectodomain (Fig. 9). However, the N-terminal tyrosine is likely to exist in the vicinity of the polar lipid head groups of the outer leaflet, not in the core of the membrane bilayer, and the helix-breaking properties of the triglycine motif may allow this tyrosine to occupy a different position relative to the other aromatic residues. The p10 TMD is also flanked on the C-terminal side by an essential palmitoylated dicysteine motif, which is absent from the p14 and p15 FAST proteins. Insertion of these two fatty acids into the inner leaflet of the bilayer and parallel to the TMD might contribute to the overall architecture of the p10 TMD. One, or a combination, of these properties could contribute to a funnel-shaped architecture for the p10 TMD. As shown for the VSV G TMD (Fig. 9) and the HA and TfR TMDs (data not shown), no such clustering of aromatic residues or funnel-shaped architecture exists in these TMDs, and the TMDs cannot functionally replace the p14 TMD. No clear picture has yet emerged of the role of TMDs in membrane fusion. Generally, replacement of the TMD with a glycosylphosphatidylinositol anchor prevents completion of the full fusion reaction, but mixing of the outer leaflets of the bilayers (i.e., hemifusion) and possibly the formation of small unstable pores still occur, at least to some level (27, 31, 39, 42). There also appears to be a minimal length requirement of ⬃14 to 18 residues for fusion activity, independent of functioning as a membrane anchor, with truncation mutants frequently completing early stages of the fusion reaction but with reduced or eliminated pore formation (1, 10, 47). The inability of the p14TMHA, p14TMTfR, and p14TMVSV constructs to mediate the transfer of a small aqueous dye (Fig. 4C) suggests that the FAST protein TMDs may similarly serve to promote or stabilize pore formation. However, the lack of a quantitative lipid-mixing assay for the FAST proteins means we cannot formally exclude the possibility that the FAST protein TMDs might also influence stages of the fusion reaction prior to pore formation. Several possible mechanisms have been proposed for how TMDs may mediate the transition from hemifusion to pore formation. Based on the essential role of glycine residues, a flexible helix structure of the TMD was proposed as a means to alter TMD-lipid interactions and bilayer structure to destabilize the hemifusion diaphragm (10). While this model could apply to p10, the fusion activity of the p14 TMD is not dependent on glycine residues. An alternate view is that glycines contribute to a flexible helix as a means to generate a funnelshaped structure in the p10 TMD. A second, “elastic-coupling” model proposes that the TMD translates the ectodomain conformational changes that result in hairpin formation and tilting into local bilayer stress, which is relieved by fusion pore formation (35). A variation of the elastic-coupling model envisions the hairpin structure present in the postfusion state of the ectodomain bringing the N-terminal fusion peptide and Cterminal TMD into close proximity. In the case of influenza virus HA, a glycine ridge in the fusion peptide may allow the fusion peptide to interact with the TMD during membrane
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merger, thereby leading to pore formation (52). We feel it is unlikely that either of these versions of the elastic-coupling model will apply to the FAST proteins. With ectodomains of only 20 to 40 residues that may be mostly disordered (15), it seems unreasonable to expect the FAST proteins to exert the same types of mechanical stresses that drive membrane fusion by the more complex enveloped virus fusion proteins. Moreover, there is no glycine ridge in the p10 and p14 ectodomain fusion peptides, p15 lacks a fusion peptide motif in its ectodomain, and there is no sequence conservation in the FAST protein fusion peptides or TMDs. These considerations make it improbable that the different FAST protein TMDs would specifically interact with the different fusion peptide motifs present in these proteins. The predicted funnel morphology of the FAST protein TMDs can be integrated into two other models of TMD function. In the first model, TMD-TMD interactions, either within or between multimeric fusion proteins, induce the lipid rearrangements required for pore formation (33, 34, 53). The multimerization status of the FAST proteins is presently unclear (5, 50), although it is probable that multiple FAST proteins function in a cooperative manner to mediate membrane fusion. The FAST protein TMDs might therefore stabilize multimers or interact transiently during the fusion reaction. It is noteworthy that, with the funnel-shaped structure of the TMDs, the geometry of a multimeric FAST protein TMD complex would increase the curvature of the membrane toward the target membrane, the same curvature change required for transition from a hemifusion diaphragm to pore formation (7, 9). Similar curvature changes could occur via interactions of the funnel-shaped FAST protein TMDs with membrane lipids. The concept that TMDs might alter membrane curvature by exerting a direct effect on their lipid environment was previously suggested (10). One interesting possibility is that the bulky conical shape of the C-proximal end of the TMD, which would reside almost entirely within the inner leaflet of the bilayer, might induce positive curvature in this leaflet. In this regard, the FAST protein TMDs would function in a manner similar to that of chlorpromazine, an amphipathic weak base that preferentially accumulates in the inner leaflets of cell membranes, promoting positive curvature and rupture of the hemifusion diaphragm (6, 32). Both structurally and functionally, the FAST proteins are an exception to the paradigm that viral fusion proteins have evolved to mediate virus-cell fusion and that the fusion process is dependent on dramatic structural remodeling of complex, multimeric ectodomains. In contrast, the FAST proteins have evolved specifically to induce cell-cell fusion, an evolutionary imperative that has no doubt contributed to the unusual structural features that define this viral protein family. The FAST proteins may well adhere to the dominant fusion-throughhemifusion model of membrane fusion and therefore would need to promote the same types of membrane curvature events captured within this model. Their unique biological and structural features, however, suggest they have devised a different means to accomplish this objective. The present results suggest that, in the absence of complex ectodomain conformational changes that exert stress on the membrane, the FAST protein TMDs may have evolved to utilize a family-specific TMD structure to remodel lipid interactions and promote the
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formation of the nonbilayer structures required for pore formation and membrane merger. ACKNOWLEDGMENTS We thank Jingyun Shou for excellent technical assistance. This research was supported by grants from the Canadian Institutes of Health Research (CIHR). E.K.C. was funded by scholarships from the Nova Scotia Health Research Foundation (NSHRF) and the Cancer Research Training Program (CRTP). R.D. was the recipient of a CIHR-RPP Investigators Award. REFERENCES 1. Armstrong, R. T., A. S. Kushnir, and J. M. White. 2000. The transmembrane domain of influenza hemagglutinin exhibits a stringent length requirement to support the hemifusion to fusion transition. J. Cell Biol. 151:425–437. 2. Arnaoutova, I., A. M. Smith, L. C. Coates, J. C. Sharpe, S. Dhanvantari, C. R. Snell, N. P. Birch, and Y. P. Loh. 2003. The prohormone processing enzyme PC3 is a lipid raft-associated transmembrane protein. Biochemistry 42:10445–10455. 3. Brosig, B., and D. Langosch. 1998. The dimerization motif of the glycophorin A transmembrane segment in membranes: importance of glycine residues. Protein Sci. 7:1052–1056. 4. Brown, E. L., and D. S. Lyles. 2003. Organization of the vesicular stomatitis virus glycoprotein into membrane microdomains occurs independently of intracellular viral components. J. Virol. 77:3985–3992. 5. Cheng, L. T., R. K. Plemper, and R. W. Compans. 2005. Atypical fusion peptide of Nelson Bay virus fusion-associated small transmembrane protein. J. Virol. 79:1853–1860. 6. Chernomordik, L. V., V. A. Frolov, E. Leikina, P. Bronk, and J. Zimmerberg. 1998. The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation. J. Cell Biol. 140:1369–1382. 7. Chernomordik, L. V., and M. M. Kozlov. 2008. Mechanics of membrane fusion. Nat. Struct. Mol. Biol. 15:675–683. 8. Chernomordik, L. V., and M. M. Kozlov. 2003. Protein-lipid interplay in fusion and fission of biological membranes. Annu. Rev. Biochem. 72:175– 207. 9. Chernomordik, L. V., J. Zimmerberg, and M. M. Kozlov. 2006. Membranes of the world unite! J. Cell Biol. 175:201–207. 10. Cleverley, D. Z., and J. Lenard. 1998. The transmembrane domain in viral fusion: essential role for a conserved glycine residue in vesicular stomatitis virus G protein. Proc. Natl. Acad. Sci. USA 95:3425–3430. 11. Coffin, W. F., III, T. R. Geiger, and J. M. Martin. 2003. Transmembrane domains 1 and 2 of the latent membrane protein 1 of Epstein-Barr virus contain a lipid raft targeting signal and play a critical role in cytostasis. J. Virol. 77:3749–3758. 12. Cohen, F. S., and G. B. Melikyan. 2004. The energetics of membrane fusion from binding, through hemifusion, pore formation, and pore enlargement. J. Membr. Biol. 199:1–14. 13. Corcoran, J. A., and R. Duncan. 2004. Reptilian reovirus utilizes a small type III protein with an external myristylated amino terminus to mediate cell-cell fusion. J. Virol. 78:4342–4351. 14. Corcoran, J. A., J. Salsman, R. de Antueno, A. Touhami, M. H. Jericho, E. K. Clancy, and R. Duncan. 2006. The p14 fusion-associated small transmembrane (FAST) protein effects membrane fusion from a subset of membrane microdomains. J. Biol. Chem. 281:31778–31789. 15. Corcoran, J. A., R. Syvitski, D. Top, R. M. Epand, R. F. Epand, D. Jakeman, and R. Duncan. 2004. Myristoylation, a protruding loop, and structural plasticity are essential features of a nonenveloped virus fusion peptide motif. J. Biol. Chem. 279:51386–51394. 16. Danieli, T., S. L. Pelletier, Y. I. Henis, and J. M. White. 1996. Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers. J. Cell Biol. 133:559–569. 17. Dawe, S., J. A. Corcoran, E. K. Clancy, J. Salsman, and R. Duncan. 2005. Unusual topological arrangement of structural motifs in the baboon reovirus fusion-associated small transmembrane protein. J. Virol. 79:6216–6226. 18. Dawe, S., and R. Duncan. 2002. The S4 genome segment of baboon reovirus is bicistronic and encodes a novel fusion-associated small transmembrane protein. J. Virol. 76:2131–2140. 19. Duncan, R., Z. Chen, S. Walsh, and S. Wu. 1996. Avian reovirus-induced syncytium formation is independent of infectious progeny virus production and enhances the rate, but is not essential, for virus-induced cytopathology and virus egress. Virology 224:453–464. 20. Duncan, R., J. Corcoran, J. Shou, and D. Stoltz. 2004. Reptilian reovirus: a new fusogenic orthoreovirus species. Virology 319:131–140. 21. Duncan, R., and K. Sullivan. 1998. Characterization of two avian reoviruses that exhibit strain-specific quantitative differences in their syncytium-inducing and pathogenic capabilities. Virology 250:263–272. 22. Earp, L. J., S. E. Delos, H. E. Park, and J. M. White. 2005. The many
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