Correspondence to: Judith M. White, Department of Cell Biology, University of Virginia Health System, School of Medicine, P.O. Box 800732, Charlottesville, VA 22908-0732. Tel:(804) 924-2593 Fax:(804) 982-3912
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Abstract |
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Glycosylphosphatidylinositol-anchored influenza hemagglutinin (GPI-HA) mediates hemifusion, whereas chimeras with foreign transmembrane (TM) domains mediate full fusion. A possible explanation for these observations is that the TM domain must be a critical length in order for HA to promote full fusion. To test this hypothesis, we analyzed biochemical properties and fusion phenotypes of HA with alterations in its 27amino acid TM domain. Our mutants included sequential 2amino acid (2
14) and an 11amino acid deletion from the COOH-terminal end, deletions of 6 or 8 amino acids from the NH2-terminal and middle regions, and a deletion of 12 amino acids from the NH2-terminal end of the TM domain. We also made several point mutations in the TM domain. All of the mutants except
14 were expressed at the cell surface and displayed biochemical properties virtually identical to wild-type HA. All the mutants that were expressed at the cell surface promoted full fusion, with the notable exception of deletions of >10 amino acids. A mutant in which 11 amino acids were deleted was severely impaired in promoting full fusion. Mutants in which 12 amino acids were deleted (from either end) mediated only hemifusion. Hence, a TM domain of 17 amino acids is needed to efficiently promote full fusion. Addition of either the hydrophilic HA cytoplasmic tail sequence or a single arginine to
12 HA, the hemifusion mutant that terminates with 15 (hydrophobic) amino acids of the HA TM domain, restored full fusion activity. Our data support a model in which the TM domain must span the bilayer to promote full fusion.
Key Words: hemagglutinin, hemifusion, transmembrane domain, glycosylphosphatidylinositol anchor, SNARE proteins
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Introduction |
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Influenza virus fusion is mediated by the hemagglutinin (HA)1 trimer (for reviews see -helical propensity, a 27amino acid transmembrane (TM) domain, and a 10amino acid cytoplasmic tail. Studies have demonstrated the importance of the fusion peptide as well as structural changes within the trimeric coiled coil for fusion (
In previous work, we demonstrated that replacing the TM and cytoplasmic tail domains of HA with a glycosylphosphatidylinositol (GPI) anchor generated an HA trimer that could promote only hemifusion (
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Materials and Methods |
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Mutagenesis
HA mutants were generated in HA cDNA (X:31 strain) present in the pTM1 vector using the Quik-Change Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer's instructions. Oligonucleotide primers with stop codons in the TM domain were used to generate cytoplasmic tail- HA (Tail- HA) and then, sequentially, twoamino acid deletions from the COOH-terminal end of the TM domain (2
14; see Fig 1). Oligonucleotide primers were also used to create the following additional HA mutants (in the Tail- HA construct): a deletion of 6 amino acids from the NH2-terminal end of the TM domain (
185190; N
6); a deletion of 6 amino acids from the NH2-terminal end and 2 amino acids from the COOH-terminal end of TM (
185190/
210211; N
6
2); a deletion of 12 amino acids from the NH2-terminal end of TM (
185196; N
12); deletions of 6 or 8 amino acids from the central region of TM (
195200, Mid
6; and 195202, Mid
8); a deletion of 11 amino acids from the COOH-terminal end of TM (
101111;
11); 6 single point mutants (S194L, S194A, G204A, G204L, W185A, and W188A); and two double point mutants (W185A/W188A and S194L/G204L) in the HA TM domain. The point mutants were made in the context of the full-length HA construct (i.e., containing the cytoplasmic tail). We also engineered two HA mutants that contained the TM domain of
12 HA (amino acids 185199), followed by either the entire cytoplasmic tail sequence of HA (
12Tail HA) or a single arginine (
12Arg HA). In this paper we use the term GPI-HA to refer to the construct BHA-PI (K/S) described in
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Expression of Wild-Type HA and Mutant HAs
CV-1 cells (CCL 70; American Type Culture Collection) were maintained in Iscove's modified Dulbecco's medium (IMDM; GIBCO BRL) containing 10% supplemented calf serum (SCS; Hyclone Laboratories, Inc.), 50,000 U penicillin, 50,000 µg streptomycin (GIBCO BRL), and an additional 146 mg glutamine (GIBCO BRL) per 0.5 liter. Wild-type (WT) and mutant HAs were expressed using the vaccinia virus T7 RNA polymerase transient transfection system (10,
12, and N
12, for which we used 7.5 µg cDNA per 6-cm dish. After a 5-h incubation (at 37°C in a 5% CO2 incubator), the DNA/Transit mixture was replaced with IMDM, and the cells were incubated at 31°C for 1520 h.
Metabolic Labeling
CV-1 cells expressing WT and mutant HAs were metabolically labeled with 35S-Translabel (ICN Biomedicals) essentially as described previously (
Cell Surface Biotinylation, Immunoprecipitations, and Western Blot Analyses
Biotinylation of cell surface proteins was performed as described previously (
Sucrose Gradient Analysis
CV-1 cells expressing WT and mutant HAs were treated with trypsin, then STI, and lysed as described above. Cell lysates were layered on continuous 330% sucrose (wt/vol) gradients. After centrifugation, 12 395-µl fractions were collected and prepared as described previously (
C-HA1 Conformational Change Assay
Transfected CV-1 cells were metabolically labeled overnight as described above. After treatment with trypsin and STI (see above), the cells were incubated at 37°C for 10 min in fusion buffer (100 mM NaCl, 10 mM Hepes, 10 mM MES, 10 mM succinate, and 2 mg/ml glucose) adjusted to the indicated pH. After reneutralization with pH 7.0 fusion buffer, the cells were lysed and immunoprecipitated with the C-HA1 antibody as described previously (
RBC Labeling, Binding, and Lipid and Content Mixing Assays
Freshly collected human RBCs were either colabeled with octadecylrhodamine B chloride (R18) and carboxyfluorescein (CF; Molecular Probes, Inc.) or labeled with CF only (
Preparation of Microsomal Membranes
Transfected CV-1 cells were biotinylated and treated with trypsin and STI as described above. The cells were then released from their dishes by incubation for 10 min at RT in 1.0 ml PEEG (PBS containing 0.5 mM EDTA, 0.5 mM EGTA, and 10 mM glucose), transferred to a 1.5-ml Eppendorf tube, pelleted at 325 g for 2 min at 4°C, resuspended in 0.8 ml DHB buffer (10 mM Tris-HCl, pH 7.5, 1 mM MgCl2), and incubated for 5 min on ice (to induce cell swelling). The cells were then passed 10 times through a 25-gauge needle. Sucrose was added to bring the solution to a final 1.18 M (wt/wt) concentration by the addition of 2.0 M sucrose (in DHB), and the suspension was overlaid with 3.0 ml of 0.25 M sucrose (in DHB). The nuclei were pelleted by centrifugation in an SW55 rotor at 192,000 g for 90 min at 4°C. The interface containing the microsomal membrane fraction was collected, transferred to a tube containing 4.0 ml of 0.25 M sucrose (in DHB), and centrifuged as before. The pellet containing the microsomal membranes was collected.
Carbonate Extraction
The pellet containing the microsomal membranes was resuspended in 0.3 ml 50 mM TEA, pH 7.5 (triethanolamine, pH adjusted with acetic acid). The pH was adjusted to 11.0 by the addition of 0.1 volume 1 M Na2CO3 (pH 11.0), and the suspension was incubated on ice for 20 min. Membranes were layered on top of a 0.68-ml sucrose cushion (0.2 M sucrose, 20 mM Hepes-NaOH (pH 11.0), 150 mM potassium acetate, 2.5 mM magnesium acetate) and centrifuged at 135,000 g in a TLS-55 rotor for 20 min at 4°C. The supernatant was collected and reneutralized by the addition of 30 mM HCl and designated the "supernatant fraction." The pellet was lysed in 0.5 ml lysis buffer containing protease inhibitors, incubated on ice for 20 min, centrifuged to clear debris at 16,000 g for 10 min at 4°C, and transferred to a fresh 1.5-ml Eppendorf tube and designated "pellet fraction." HA from the supernatant and pellet fractions was immunoprecipitated using the Site A mAb as described above, resolved by SDS-PAGE on a reducing 10% gel, transferred to nitrocellulose, and probed with streptavidin-HRP as described above.
FACS® Analysis
Transfected CV-1 cells (6-cm dishes) were released from the dish with PEEG (as described above) and transferred to a 1.5-ml Eppendorf tube. The cells were then washed twice with cold PBS+ containing 0.02% azide (PBSA) and centrifuged at 325 g for 2 min at 4°C. The cells were resuspended in 0.2 ml cold PBSA containing 2% SCS and incubated for 30 min on ice with 1.7 µl of 1.0 mg/ml Site A mAb. The cells were then washed twice with PBSA as described above, resuspended in PBSA containing 2% SCS, and incubated with 1.0 µl FITC-conjugated goat antimouse IgG for 30 min on ice. The cells were then washed twice with cold PBSA, resuspended in PBS containing 2% paraformaldehyde, and analyzed by FACS® at the University of Virginia Core Facility, using a FACScanTM flow cytometer (Becton Dickinson).
Endo F Treatment
Transfected CV-1 cells (6-cm dishes) were metabolically labeled and treated with trypsin and STI as described above. The cells were released from the dish by a brief treatment with PEEG (as described above) and transferred to a 1.5-ml Eppendorf tube. Lysates were prepared and HA was immunoprecipitated with the Site A mAb as described above. After the immunoprecipitation, 50 µl N-Glycosidase F buffer (1% octylglucoside, 0.2% SDS, 40 mM Tris, pH 8.0, 5 mM EDTA, and 1% ß-mercaptoethanol) was added to the protein AAgarose (PAA) beads. The beads were then treated at 95°C for 3 min, followed by centrifugation for 2 min at 16,000 g. The supernatant was then transferred to a new 1.5-ml Eppendorf tube and treated with 1.0 U N-Glycosidase F (Roche) for 2 h at 37°C. SDS gel loading buffer containing 0.14 M ß-mercaptoethanol was added, the samples were reboiled, and the proteins were resolved by SDS-PAGE on a 15% gel. The gel was then fixed in a solution of 40% methanol/10% acetic acid for 20 min at RT, followed by incubation in 1 M salicylic acid for 20 min at RT. The gel was then dried and exposed to film.
Cholesterol Depletion and Triton X-100 Extraction
For fusion experiments, transfected CV-1 cells were treated with trypsin and STI as described above, and depleted of cholesterol by a 30-min treatment with 20 mM methyl ß-cyclodextrin (MßCD; Sigma-Aldrich) in PBS+ at 37°C before RBC binding and fusion (see above). For Triton X-100 extraction experiments, transfected CV-1 cells were biotinylated, trypsin treated, cholesterol depleted with MßCD as described above, and lifted off the dish by treatment with PEEG for 10 min at 4°C. The cells were pelleted in a refrigerated microfuge chilled to 2°C for 2 min at 325 g. After centrifugation, the cells were placed on ice in a 4°C coldroom, resuspended in 500 µl cold lysis buffer (50 mM Tris, pH 8.0, 1% Triton X-100, and protease inhibitors), and incubated on ice for 20 min. The insoluble fraction was removed by centrifugation for 15 min at 16,000 g and 4°C, again in a microfuge chilled to 2°C. The supernatant was designated the "soluble fraction." The insoluble (pellet) fraction was resuspended in 500 µl cold lysis buffer containing 0.1% SDS and protease inhibitors, incubated for 1 h at RT with occasional vortexing, centrifuged to clear debris at 16,000 g for 10 min at 4°C, transferred to a fresh 1.5-ml Eppendorf tube, and designated "pellet fraction." HA from the soluble fraction and the pellet fraction was immunoprecipitated with the Site A mAb, detected as described above with streptavidin-HRP, and quantitated by PhosphorImager® analysis.
Videomicroscopy Lipid Mixing Assay
Transfected CV-1 cells expressing either WT HA or 10 HA were processed for fusion as described above. RBCs (0.05%) labeled with R18 as described above were bound to the CV-1 cells. Fusion was triggered at 37°C with fusion buffer adjusted to the indicated pH. The cells were maintained at 37°C on a warm stage and monitored by videomicroscopy for 5 min using the software package Openlab (Improvision). Fusion was quantified using Scion Image (National Institutes of Health, Bethesda, MD). Care was taken such that one to three RBCs were bound per cell. Each field contained 40 RBCs, and the amount of R18 fluorescence per field was quantified. The values from three to four fields per time point were averaged, and the data were plotted as a function of time. From these plots, values for the lag time, initial rate, and final extent of lipid mixing were calculated.
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Results |
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Biochemical Properties of HA TM Truncation Mutants D2D14
We initially made a set of mutants in which we sequentially deleted 2, 4, 6, 8, 10, 12, and 14 amino acids from the COOH-terminal end of the HA TM domain in the context of a Tail- HA construct (Fig 1). We first asked whether these mutant HAs could be expressed at the cell surface in a fusion-permissive form (i.e., cleaved from HA0 to HA1-S-S-HA2). We also examined them for a shift in the migration of their HA2 subunits. With the exception of 14 (data not shown), all of the mutants were expressed at the cell surface as HA0 and were efficiently cleaved to HA1 and HA2 by the addition of trypsin (Fig 2 A).
2
8 HA exhibited the expected shift in the mobility of the HA2 subunits (Fig 2 B, left).
10 and
12 HA exhibited an increased mobility compared with WT HA (and Tail- HA; data not shown), but these mutants migrated more slowly than
8 HA (Fig 2 B, right). The fact that
12 was, but that
14 was not, expressed at the cell surface is consistent with previous observations that a mutant HA with a 13amino acid truncation of the TM domain was not transported beyond the cis-Golgi compartment (
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We next asked if the mutant HAs form trimers. Processed forms of 2
12 HA migrated to a similar position on sucrose gradients as WT HA (Fig 3 A, arrows). The higher molecular weight band seen in some of the gradients corresponds to intracellular HA0.
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To address whether the mutant HAs change conformation at the same pH as WT HA, HA-expressing cells were briefly incubated at the indicated pH, lysates were prepared, and HA was immunoprecipitated using C-HA1, a conformation-specific antibody (2
12 HA changed conformation with a pH dependence similar to that of WT HA.
Fusion Activity of 2
12
We evaluated the fusion activity of 2,
4,
6,
8,
10, and
12 HA using a dye transfer assay. RBCs colabeled with a lipid dye (R18) and a soluble content dye (CF) were bound to HA-expressing cells, and fusion was induced as described in Materials and Methods. After 5 min at 37°C and pH 5, the cells were returned to neutral pH medium and examined with a fluorescence microscope. As seen in Fig 4, both dyes transferred efficiently to cells expressing
2,
4,
6,
8, and
10 HA. A different phenotype was seen for cells expressing
12 HA: whereas we observed efficient lipid dye transfer (97%), content dye transfer was severely restricted (<10 vs. 97% for WT HA). As expected, we did not observe transfer of R18 or CF by WT or mutant HA-expressing cells at neutral pH or at low pH if the cells had not been pretreated with trypsin to process HA0 (data not shown).
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Given the striking observation that 10 HA mediated robust lipid and content mixing whereas
12 HA mediated robust lipid mixing with minimal content mixing, we also tested a mutant lacking the 11 COOH-terminal residues of the TM domain (
11 HA).
11 HA exhibited biochemical properties similar to WT HA (Table 1). Whereas
11 HA mediated efficient lipid mixing (Fig 4), it was significantly impaired in its ability to mediate content dye transfer (Fig 4).
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The density of HA at the cell surface can influence the fusion phenotype (12 HA (7.5 µg cDNA) mediates efficient lipid transfer but very poor content dye transfer. The fusion phenotype of cells expressing
12 HA was thus similar to that seen for cells expressing GPI-HA (Fig 4). Representative micrographs showing the fusion patterns of WT HA, GPI-HA,
10 HA, and
12 HA are shown in Fig 5.
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The fusion data presented in Fig 4 and Fig 5 suggested that there is a stringent length requirement for the HA TM domain to be able to mediate both lipid and content mixing. To further test this possibility, we generated a second 12amino acid truncation in the HA TM domain, but in this case we deleted 12 amino acids from the NH2-terminal end of the TM domain (Fig 1). This mutant, referred to as N12 HA, was then examined for biochemical properties. Like all of the other truncation mutants, N
12 HA was expressed at the cell surface, was processed by trypsin into HA1 and HA2, and exhibited a faster migrating HA2 subunit than tail_HA (Table 1, and data not shown). By all of these criteria, N
12 HA resembled
12 HA. However, it was not as well expressed at the cell surface as
12 HA, as determined by FACS® analysis and RBC binding (
80% compared with
12 HA; Fig 4 and Table 1). In terms of fusion with RBCs, N
12 HA mediated significant lipid mixing, albeit less than seen with
12 HA (63 vs. 97%). With respect to content mixing, N
12 HA mediated <5% dye transfer similar to the behavior of
12 HA and GPI-HA (Fig 4 and Fig 5). Neither
12 HA nor N
12 HA promoted significant content mixing (>10%) even after 60 min of incubation at 37°C at either pH 4.8 or 5.0 (data not shown).
Comparison of the Lipid and Content Mixing Ability of D10 HA and WT HA
Given the dramatic decrease in content mixing ability between 10 HA and
12 HA (and
11 HA), we examined the fusion activity of
10 HA in more detail. For this purpose, we compared the lag times, initial rate, and final extent of lipid mixing with WT HA and
10 HA at different pH values. At all pH values tested, the lag time before the onset of dye transfer was equivalent for
10 HA and WT HA (Table 2). There was no difference in either the initial rate or the final extent of lipid mixing for
10 HA and WT HA at pH 5.0 and 5.25. At pH 5.5, the latter parameter was somewhat lower for
10 HA. Hence, the lipid mixing properties of
10 HA were very similar to those of WT HA at all pH values tested. In addition, when incubated at the suboptimal pH of 5.25 for 2 min at 37°C,
10 HA meditated content mixing to the same extent as WT HA (data not shown).
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Effect of CPZ on Content Mixing
Previous work has shown that treatment with 0.1 mM CPZ, a membrane-permeable amphipathic reagent that partitions preferentially into the inner leaflet of the plasma membrane, efficiently induces full fusion in cases of "stunted fusion" caused by performing fusion experiments under suboptimal conditions (12 HA, N
12 HA, and GPI-HA to promote content transfer. After binding double-labeled RBCs (R18 and CF) to HA-expressing cells, fusion was triggered by lowering the pH to 5.0 for 5 min at 37°C. The medium was reneutralized, and CPZ was added to the cells at neutral pH. After a 1-min incubation at RT, the CPZ solution was replaced with PBS+. The percentage of R18-stained HA-expressing cells that became labeled with CF was then determined. In the absence of CPZ,
1, 7, and 3% of cells expressing N
12 HA,
12 HA, and GPI-HA, respectively, received aqueous dye (Fig 6). The addition of 0.1 mM CPZ increased content dye transfer to
5, 8, and 4%, respectively. Addition of 0.5 mM CPZ induced a greater extent of CF transfer:
20, 27, and 22%, respectively. Representative images of aqueous dye transfer before and after the addition of either 0.1 or 0.5 mM CPZ are shown in Fig 7. Hence, cells expressing N
12 HA and
12 HA respond similarly to CPZ as do cells expressing GPI-HA in terms of their ability to promote aqueous dye transfer. The presence of R18 in the RBC membrane augments the transfer of aqueous contents to GPI-HAexpressing cells (
12 HA and N
12 HA still responded similarly to CPZ as did GPI-HA: a brief treatment with 0.5 but not 0.1 mM CPZ increased content dye transfer (data not shown).
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Membrane Association of 12 HA
Given the striking phenotype of HA lacking 12 amino acids in the TM domain (lipid, but not content, mixing), we explored how 12 HA is anchored in the membrane. Like WT HA,
12 HA (as well as GPI-HA) was resistant to carbonate extraction (Fig 8 A). Given that some GPI-anchored proteins associate with cholesterol and sphingomyelin-rich detergent-insoluble membrane fractions (DIGs;
12 HA and N
12 HA in Triton X-100 at 4°C before and after treating cells with methyl ß-cyclodextrin to remove cholesterol (
12 HA and N
12 HA (data not shown) were readily solubilized by Triton X-100 at 4°C, suggesting that they do not associate with DIGs. In contrast, both WT HA and GPI-HA were partially insoluble in Triton X-100 at 4°C, and depletion of cholesterol appeared to increase their solubility (Fig 8 B).
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Effect of MßCD on Fusion by 12 HA and N
12 HA
Because of the general interest in glycoprotein localization to plasma membrane microdomains (12 HA, or N
12 HA with 20 mM MßCD influenced their fusion activity. Treatment of WT HA, GPI-HA,
12 HA, and N
12 HA-expressing cells with MßCD did not affect the fusion phenotype. WT HA still mediated efficient lipid and content dye transfer, whereas the mutant HAs still demonstrated significant lipid mixing but little or no content mixing (Fig 8). Similar results were obtained using WT HA of the Japan strain (H2N2;
Why 12 HA May Cause Lipid, but Not Content, Mixing
We have considered two general models for why 12 HA mediates only lipid mixing whereas
10 HA mediates content mixing as well. In the first model (Fig 9 A, 1), we consider that
12 HA has recruited specific (e.g., shorter fatty acyl chain) lipids around it such that it spans a thinned bilayer. The lipids in such a thinned bilayer may not be competent to promote the hemifusion to fusion transition. In the second model, we consider that the TM domain of
12 HA is simply too short to span a bilayer; it may be anchored either perpendicularly (Fig 9 A, 2a), obliquely (Fig 9 A, 2b), or parallel (Fig 9 A, 2c) to the membrane normal. To test between these models, we analyzed a mutant HA in which we added back the hydrophilic cytoplasmic tail (10 amino acids) to the end of
12 HA. We reasoned that if
12 HA spans a thinned bilayer (model 1), addition of the cytoplasmic tail should not affect its fusion phenotype. If, however,
12 HA does not span a bilayer (model 2), then addition of the cytoplasmic tail may force
12 HA to span a bilayer and it may therefore be able to support full fusion. As seen in Fig 10 A, the mutant
12Tail HA clearly promotes full fusion. Next, we tested whether the first residue of the cytoplasmic tail, an arginine, added to the end of the
12 HA TM domain, was sufficient to restore full fusion activity. As seen in Fig 10 B,
12Arg HA clearly promotes full fusion.
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Additional HA TM Domain Mutants
To ascertain whether we could detect any specific TM domain sequences needed for HA to promote full fusion, we constructed additional point and deletion mutations within the HA TM domain (Fig 11). We mutated the tryptophans (to alanines) within the highly conserved WILW sequence at the beginning of the HA TM domain. We mutated a serine at position 194, since this residue is the analogue of a glycine implicated as being important for the fusion activity of Japan HA (
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Discussion |
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Cells expressing the ectodomain of HA linked to the membrane via a GPI anchor (GPI-HA) promote lipid, but not content, mixing (14, which did not reach the cell surface (data not shown), all of the mutant HAs possessed biochemical properties similar to WT HA (Table 1 and Table 3). In terms of membrane fusion, all HAs studied with
17 amino acids in their TM domains were able to efficiently mediate full fusion (both lipid and content mixing). In contrast, HAs with 15 or 16amino acid TM domains (and no cytoplasmic tail) mediated robust lipid mixing, but were either severely impaired (16amino acid TM domain) or virtually unable (15amino acid TM domain) to mediate content mixing. All of the point mutants examined efficiently promoted full fusion. Our findings suggest that there is a stringent length requirement, 17 amino acids, for the HA TM domain to be able to support the hemifusion to fusion transition. Additional experiments (see below) suggested that the HA TM domain must span its bilayer to properly execute the fusion reaction.
12 HA and N
12 HA Mediate Hemifusion
Hemifusion is functionally defined as the merger of the outer, but not the inner, leaflets of the fusing bilayers, such that aqueous continuity is not established. Many investigators have proposed that biological fusion events proceed through a hemifusion intermediate (
Studies with GPI-HA indicate that progression to a fusion pore, as monitored by the transfer of small content dyes, does not occur when GPI-HAexpressing cells are induced to fuse with RBCs (12 HA and N
12 HA, are severely restricted in their ability to mediate mixing of a 376mol wt content dye, CF. Since the level of CF mixing seen with
12 HA and N
12 HA is similar to that seen with GPI-HA (Fig 6 B), it is likely that
12 HA and N
12 HA are unable to efficiently promote the hemifusion to fusion transition and are unable to support pore enlargement. Additional evidence that
12 HA and N
12 HA are blocked at the stage of hemifusion, and not at stunted fusion, is that 0.5 mM CPZ is required to induce appreciable content dye transfer (Fig 6), as has been seen with GPI-HA (
12 HA and N
12 HA are protein mimetics of GPI-HA.
Length Requirement of the HA TM Domain
We have uncovered a surprisingly stringent length requirement for the HA TM domain to be able to (efficiently) promote the hemifusion to fusion transition. HAs harboring a 17amino acid (predicted) TM domain promote full fusion, whereas an HA with a 16amino acid (predicted) TM domain is severely impaired in promoting full fusion and HAs with 15amino acid (predicted) TM domains appear to arrest at hemifusion.
The finding that there is a stringent length requirement of 17 amino acids for the HA TM domain to efficiently promote the hemifusion to fusion transition suggests that HAs with TM domains 17 amino acids are anchored differently in the bilayer than fusion-impaired HAs that have shorter TM domains (
16 amino acids). Using a synthetic peptide representing the transmembrane segment of X:31 HA,
-helix that aligns roughly perpendicular to the bilayer normal. As discussed in Results (with reference to Fig 9 A), we have considered two general models for how the fusion-defective TM domain mutants (with TM domains
16 amino acids) are anchored in the bilayer. The first (Fig 9 A, 1) envisions that the short (
16 amino acids) TM domains are aligned like the WT HA TM domain (as a perpendicular
-helix), but to span the bilayer, they have had to recruit specific lipids (e.g., with short fatty acyl tails) around them. Such lipids may not be able to adopt the necessary curvature to allow fusion to progress beyond hemifusion (
16 amino acids) cannot span the bilayer (Fig 9 A, 2a, 2b, and 2c). Our finding that addition of the hydrophilic 10amino acid cytoplasmic tail sequence to a TM domain of 15 hydrophobic amino acids generated an HA that efficiently promotes full fusion (Fig 10 A) supports the latter model (Fig 9 A, 2), as it suggests that the addition of these hydrophilic residues has forced the 15amino acid TM domain to span the bilayer. Even more striking is the finding that addition of a single arginine to a 15 amino acid TM domain generated an HA that promotes full fusion (Fig 10 B). We propose that addition of one or more hydrophilic residues forced the 15 amino acid TM domain to span the bilayer. The need for a hydrophilic residue following a 15 amino acid TM domain is underscored by comparing
11 HA and
12Arg HA (see Fig 4 and Fig 10, legend). Collectively, these findings support a model in which the HA TM domain must span a bilayer to efficiently promote full fusion.
Why might it be necessary for the HA TM domain to span its bilayer to efficiently promote fusion? One possibility is based on the concept that the TM domain of HA plays an important role in disrupting the lipid bilayer during fusion (-helical structures that may be required for specific proteinlipid or proteinprotein (e.g., TM domainTM domain or TM domainfusion peptide) interactions that are required for progression to full fusion. Future biophysical experiments will address how the short fusion-incompetent TM domains are oriented in a bilayer.
Sequence Requirements of the HA TM Domain
We also asked whether we could identify any specific residues within the HA TM domain that are required to promote the hemifusion to fusion transition. During analysis of four additional truncation mutants (of six or eight amino acids) and eight point mutants at different locations in the TM domain (Table 3), we were unable to uncover any specific sequence requirement for fusion. In particular, two highly conserved tryptophan residues within the WILW motif at the NH2-terminal end of the TM domain, which appear to be important for targeting HA to the apical surface of epithelial cells (
If one models the WT HA TM domain as an -helix, there is a short face of four polar residues (two cysteines and two serines). However, several of our mutant HAs disrupt this motif (i.e., leave only two polar residues), but do not impair fusion. Hence, although we cannot exclude the possibility that there may be a sequence motif that is important for the X:31 HA TM domain to promote the hemifusion to fusion transition, we have not found such a motif. The differences we observe in the sequence requirements for fusion with X:31 HA (H3N2 subtype) and that reported for Japan HA (H2N2 subtype) may be due to differences in the subtype of HA or the techniques used.
Possible Parallel Roles for the Fusion Peptide and the TM Domain in the Hemifusion to Fusion Transition
Four indirect lines of evidence suggest that although they have different net hydrophobicities, the HA fusion peptide and the HA TM domain may play parallel roles in the hemifusion to fusion transition. First, we have recently demonstrated that replacement of the glycine at the first position of the HA fusion peptide with a serine (Ser HA) arrests HA fusion at the hemifusion stage (17 amino acids) to efficiently promote full fusion.
Sequence Requirements of the TM Domains of Other Viral Fusion Proteins
We have not observed any specific sequence requirements within the X:31 HA TM domain for full fusion. However, other investigators have suggested that there are specific sequence requirements for fusion within the TM domain of other viral fusion proteins. As discussed above, specific TM domain sequence requirements have been suggested for the VSV G glycoprotein (
Possible Relevance to SNARE-mediated Fusion
Recent structural and biochemical data indicate that the SNARE (soluble N-ethylmaleimidesensitive factor [NSF] attachment protein receptor) complexes, key players in intracellular fusion events, share similarities with many viral fusion proteins (
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Footnotes |
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1 Abbreviations used in this paper: CF, carboxyfluorescein; CPZ, chlorpromazine; GPI, glycosylphosphatidylinositol; HA, hemagglutinin; MßCD, methyl ß-cyclodextrin; RT, room temperature; SCS, supplemented calf serum; SNARE, soluble N-ethylmaleimidesensitive factor attachment protein receptor; STI, soybean trypsin inhibitor; TM, transmembrane; VSV G, vesicular stomatitis virus envelope glycoprotein; WT, wild-type.
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Acknowledgements |
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We are grateful to Drs. F. Cohen, G. Melikyan, D. Castle, L. Tamm, and S. Green for thoughtful discussions and critical reading of the manuscript. The authors also thank Jennifer Gruenke for help preparing Fig 9 and for invaluable assistance with videomicroscopy image capture and quantification.
This work was supported by National Institutes of Health grant AI22470 to J.M. White.
Submitted: 2 May 2000
Revised: 2 August 2000
Accepted: 22 August 2000
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References |
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