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Journal of Virology, November 2008, p. 11437-11445, Vol. 82, No. 22
0022-538X/08/$08.00+0     doi:10.1128/JVI.00779-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Measles Virus Fusion Protein Transmembrane Region Modulates Availability of an Active Glycoprotein Complex and Fusion Efficiency

Michael D. Mühlebach,^1,2 Vincent H. J. Leonard,¹ and Roberto Cattaneo^1*

Department of Molecular Medicine and Virology and Gene Therapy Track, Mayo Graduate School, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905,¹ Division of Medical Biotechnology, Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, D-63225 Langen, Germany²

Received 10 April 2008/ Accepted 5 September 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The glycoprotein complex of paramyxoviruses mediates receptor binding and membrane fusion. In particular, the measles virus (MV) fusion (F) protein executes membrane fusion, after receptor binding by the hemagglutinin (H) protein. Structures and single amino acids influencing fusion function have been identified in the F-protein ectodomain and cytoplasmic tail, but not in its transmembrane (TM) region. Since this region influences function of the envelope proteins of other viruses, we examined its role in the MV F protein. Alanine-scanning mutagenesis revealed that an F protein with a single mutation of a central TM region leucine (L507A) was more fusogenic than the unmodified F protein while retaining similar kinetics of proteolytic processing. In contrast, substitution of residues located near the edges of the lipid bilayer reduced fusion activity. This was true not only when the mutated F proteins were coexpressed with H but also in the context of infections with recombinant viruses. Analysis of the H-F complexes with reduced fusion activities revealed that more precursor (F₀) than activated (F₁₊₂) protein coprecipitated with H. In contrast, in complexes with enhanced fusion activity, including H-F^L507A, the F₀/F₁₊₂ ratio shifted toward F₁₊₂. Thus, fusion activity correlated with an active F-H protein complex, and the MV F protein TM region modulated availability of this complex.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The glycoprotein complex of paramyxoviruses mediates receptor binding and membrane fusion. In particular, the measles virus (MV) fusion (F) protein executes membrane fusion, after receptor binding by the hemagglutinin (H). H is a type II transmembrane (TM) glycoprotein lacking neuraminidase activity, whose head domain was recently crystallized as a dimer (10, 17). The full-length H protein also forms a disulfide-linked dimer (37), which forms complexes with a trimer of the fusion protein (F). F is a type I TM protein synthesized as an inactive precursor (F₀). F₀ trimerizes and is proteolytically cleaved by furin while passing through the trans-Golgi network, producing a large TM fragment, F₁, and a small fragment, F₂ (41). Both fragments are linked by a disulfide bond, and F₀ cleavage is essential for virus-cell and cell-cell membrane fusion (1, 28, 50).

H binds to two receptors, the signaling lymphocytic activation molecule (SLAM; CD150) (20, 46) and the membrane cofactor protein (CD46) (16, 32). Receptor binding triggers conformational changes through the H protein dimer that in turn trigger wide-ranging F-protein trimer conformational changes, ultimately resulting in membrane fusion (33, 34, 52). Fusion activity of the glycoprotein complex is controlled at different levels, including the strength of association of the F-protein trimer with the H-protein dimers (39). Moreover, the membrane-associated viral matrix (M) protein restricts fusion through its interactions with the cytoplasmic tails of F and H, probably by stabilizing the glycoprotein complex (6, 7, 43). In this study, we investigated whether the TM region of the F protein also influences fusion function.

It is known that the TM region of another paramyxovirus F protein modulates inside-out signaling of the cytoplasmic tail (49) and that the TM region of the influenza virus hemagglutinin protein modulates function by influencing its intracellular localization (44). Moreover, the TM region of the vesicular stomatitis virus (VSV) glycoprotein may act as an autonomous domain during late stages of the fusion process (24). Information about the function of the MV F-protein TM region is limited to its palmitoylation, which occurs on at least two of four cysteine residues; mutation of three of these residues to serine reduces or abolishes fusion function (3) (cysteines 503, 515, and 516; numbers used here are according to preferred initiation of F protein translation from the second ATG [5]).

We performed systematic alanine-scanning mutagenesis of the MV F-protein TM segment and tested its function. Initially, blocks of three or four residues were mutated, and then a relevant block was analyzed in detail. Cell-to-cell fusion activity of an MV F-protein mutant in which a central leucine was mutated to alanine (L507A) was enhanced. In contrast, alanine substitution of certain residues located near the edges of the lipid bilayer led to reduced cell-to-cell fusion activity. We show that fusion activity correlates with the availability of an active F-H protein complex.

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Cells. Vero (African green monkey kidney; ATCC CCL-81) cells were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS). B95a (a marmoset B-cell line kindly provided by D. Gerlier) and 293T (human embryonic kidney; ATCC CRL-11268) cells were maintained in DMEM and 10% FBS. The rescue helper cell line 293-3-46 (40) was grown in DMEM with 10% FBS and 1.2 mg of G418/ml. Vero/hSLAM (Vero cells stably expressing human SLAMs; kindly provided by Y. Yanagi) were maintained in DMEM supplemented with 10% FBS and 0.5 mg of G418/ml.

Plasmid construction. pCG-MVvac-F and pCG-MVvac-H expressing the F and H genes of the molecular clone of the vaccine strain Moraten were cloned from pBR-MVvac (15) into the pCG-F or pCG-H (5) expression plasmid using the unique restriction sites PacI/NarI or PacI/SpeI, respectively. Numbering of amino acid residues of the F protein was done according to preferred initiation of F translation from the second ATG, as described previously (5). The different alanine-scanning mutants were produced with a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer's protocol. Mutated genes were fully sequenced between the PacI and NarI restriction sites, and plasmids without secondary mutations were used.

Transfection of mammalian cells. Cells were seeded on 12-well tissue culture plates and allowed to reach 80% confluence prior to transfection. Cells were transfected with equal amounts (1 µg) of pCG-MVvac-H and one pCG-MVvac-F mutant using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Briefly, 5 µl of Lipofectamine 2000 was diluted in 100 µl of OptiMEM (Invitrogen), mixed, and incubated at room temperature for 5 min. Meanwhile, plasmid DNAs were diluted in 100 µl of OptiMEM. The two solutions were combined, mixed, and incubated for 20 min before being added to the cells.

Immunoblotting. Cells were transfected with F and H expression plasmids or infected with the indicated virus at a multiplicity of infection (MOI) of 0.1. At the appropriate time, cells were lysed by the addition of lysis buffer (50 mM Tris [pH 8.0], 62.5 mM EDTA, 1% Igepal CA-630 [formerly NP-40], 0.4% deoxycholate; Sigma, St. Louis, MO) supplemented with Complete protease inhibitor (Roche Biochemicals, Indianapolis, IN). The lysates were clarified by centrifugation at 12,000 x g for 15 min at 4°C. The cell extract protein concentration was determined with a DC protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of proteins from cell extracts were denatured for 10 min at 95°C in 2x urea sample buffer (5% sodium dodecyl sulfate [SDS], 8 M urea, 200 mM Tris-HCl, 0.1 mM EDTA, 0.03% bromphenol blue, 2.5% dithiothreitol; pH 8.0), fractionated by SDS-polyacrylamide gel electrophoresis, and blotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were blocked with 10% skim milk powder in Tris-buffered saline plus 0.1% Tween 20 for 0.5 h at room temperature. The membranes were incubated with rabbit anti-F or rabbit anti-H cytoplasmic tail serum (anti-Fcyt and anti-Hcyt, respectively) (8) diluted 1:10,000. After incubation with peroxidase-conjugated donkey anti-rabbit immunoglobulin G (Jackson Immunoresearch Laboratories, West Grove, PA) for 1 h at room temperature, proteins were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). For densitometric analysis, the immunoblots were scanned with a transilluminating scanner (UMAX; Biostep GmbH, Jahnsdorf, Germany) and saved as TIFF files. These were analyzed with the TotalLab analytic software package (Phoretix, Newcastle upon Tyne, United Kingdom).

Radioimmunoprecipitation and pulse-chase analysis. For each construct and time point, one six-well plate of semiconfluent Vero cells was transfected with a plasmid encoding one F mutant and pCG-IC₃₂₃-H (26) using Lipofectamine 2000. Sixteen hours after transfection, the cells were washed, and the medium was replaced by DMEM without glutamine, methionine, or cysteine. After incubation at 37°C for 1.5 h, the medium was exchanged for 1 ml of DMEM without glutamine, methionine, or cysteine, supplemented with 100 µCi of [³⁵S]methionine (Amersham Pharmacia Biotech).

For pulse-chase experiments, cells were labeled for 30 min, washed three times with phosphate-buffered saline (PBS), and incubated with DMEM supplemented with 10% FBS. The cells were lysed 0, 0.5, 1.5, 3, and 6 h after the pulse with 500 µl of radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1.0% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl [pH 8.0]) with protease inhibitors (Complete; Roche Biochemicals) for 20 min at 4°C. The lysate was cleared by centrifugation at 5,000 x g for 15 min at 4°C, and the supernatant was added to 50 µl protein G agarose beads (Roche Biochemicals) with the anti-Fcyt antibody. After incubation at 4°C for 3 h, the beads were washed in RIPA buffer and denatured in urea sample buffer, and the samples were subjected to SDS-polyacrylamide gel electrophoresis. The gels were dried for 2 h at 70°C and analyzed with a Fuji BAS 1000 phosphorimager (Fujifilm Europe GmbH, Düsseldorf, Germany) after 2-h to overnight exposure of the imager plates. Autoradiograms were subjected to densitometric analysis with AIDA Image Analyzer v4.10 software (Raytest, Straubenhardt, Germany).

Fusion assays. To assess fusion activity of different F-protein mutants, Vero cells plated in 12-well plates were cotransfected with pCG-MVvac-H and a plasmid encoding one F mutant. After overnight incubation at 37°C, the numbers of nuclei per syncytium were determined for 40 syncytia for each mutant. To assess the fusion activity of viral envelope protein complexes in the context of an ongoing viral infection, Vero cells were infected at an MOI of 0.001 and incubated at 37°C. After 48 h, infected cells were subjected to microscopic analysis, and the numbers of nuclei per syncytium were determined for 25 independent syncytia for each virus.

Quantitative fusion assays based on the luciferase gene as the reporter gene (35) were performed by transfection of Vero cells in 24-well plates with a plasmid encoding one F mutant together with pCG-MVvac-H and pTM1-luc at a molar ratio of 1:1:0.7 with Lipofectamine 2000. For each transfected well, a similar amount of B95a cells had been infected with modified vaccinia virus Ankara expressing the bacteriophage T7 polymerase (MVA-T7) (42) at an MOI of 0.5 the day before. Four hours posttransfection, the B95a cells were washed, detached with Versene (Invitrogen), resuspended in 1 ml of DMEM plus 5% FBS, and transferred onto the transfected Vero cells. Luciferase activity was determined with the Steady-Glo luciferase assay system (Promega, Madison, WI) and a luminometer that can read a 96-well plate (Topcount-NXT; Packard, Los Angeles, CA).

Coimmunoprecipitation. Cells in six-well plates were cotransfected with 2.5 µg each of plasmids encoding MVvac-H and one F mutant. After a washing with PBS, cells were scraped in coimmunoprecipitation buffer (10 mM HEPES [pH 7.4], 50 mM sodium pyrophosphate, 50 mM sodium fluoride, 50 mM sodium chloride, 5 mM EDTA, 5 mM EGTA, 100 µM sodium vanadate, 1% Triton X-100) containing protease inhibitors (Complete; Roche Biochemicals) and 1 mM phenylmethylsulfonyl fluoride. Lysates were cleared by centrifugation for 25 min at 20,000 x g and 4°C, and 300 µg of total protein was incubated with antibodies directed against MV H (MAB8905, Chemicon, Temecula, CA) for 90 min at 4°C. Immune complexes were adsorbed to protein G-agarose (Roche) for 90 min at 4°C, washed in buffer 1 (100 mM Tris [pH 7.6], 500 mM lithium chloride, 0.1% Triton X-100, 1 mM dithiothreitol) and then buffer 2 (20 mM HEPES [pH 7.2], 2 mM EGTA, 10 mM magnesium chloride, 0.1% Triton X-100, 1 mM dithiothreitol), incubated in urea buffer for 25 min at 50°C, and subjected to Western analysis using antibodies specific for the MV-F or MV-H cytoplasmic tail. As an internal standard to assess protein expression in transfected cells, 5 µg of total protein denatured in urea sample buffer was used.

Surface biotinylation. Vero cells seeded in six-well plates were cotransfected with 3 µg each of plasmids encoding MVvac-H and one F mutant. After overnight incubation, cells were washed in cold PBS, incubated in PBS containing 0.5 mg of succinimidyl 2-(biotinamido)-ethyl-1,3'-dithiopropionate-biotin/ml (Pierce) for 20 min at 4°C. One ml of 0.5 M glycine-PBS was then added to quench the excess biotin, and cells were incubated for 20 min at 4°C. Cells were scraped into coimmunoprecipitation buffer containing protease inhibitors (Complete; Roche Biochemicals) and 1 mM phenylmethylsulfonyl fluoride, and lysates were cleared by centrifugation for 20 min at 20,000 x g and 4°C. As an internal standard, 30 µl of total protein was mixed with urea buffer. Biotinylated proteins were adsorbed to Sepharose-coupled streptavidin (Amersham Pharmacia Biotech) for 90 min at 4°C, washed first in buffer 1 and then in buffer 2, incubated in urea buffer for 25 min at 50°C, and subjected to Western blot analysis using antibodies specific for the MV-F cytoplasmic tail.

Generation of recombinant viruses. The F-mutant open reading frames were transferred into the infectious MV genome by cloning the PacI/NarI fragment containing the F open reading frame from pCG-MVvac-F into PacI/NarI-digested pBR-MVvac-GFP(N) (15). All engineered MV genomes were verified by sequencing and were hexameric, to conform to the "rule of six" (4, 31). The Moraten-based parental MV strain (15) and all its recombinant derivatives were rescued as described previously (40). Briefly, the helper cell line 293-3-46 stably expressing MV nucleoprotein, phosphoprotein, and the T7 polymerase was transfected by calcium phosphate precipitation (ProFection kit; Promega) with one plasmid coding for the relevant MV genome and one coding for MV polymerase (pEMC.La [40]). Three days after transfection, the helper cells were overlaid on Vero cells, and the appearance of infectious centers was monitored. Single syncytia were picked and propagated on Vero cells. To prepare virus stocks, Vero or Vero/hSLAM cells were infected at an MOI of 0.01 and incubated at 37°C. At the peak of viral production, cells were scraped in Opti-MEM and freeze-thawed once. Titers were determined by 50% tissue culture infective dose (TCID₅₀) titration by the method of Kärber (21) on Vero cells.

Virus growth kinetics. Vero cells were infected at an MOI of 0.03 TCID₅₀/cell. At various time points, supernatants were clarified by centrifugation, and cells were scraped into Opti-MEM and subjected to freeze-thaw cycles. Released and cell-associated viral titers were determined by TCID₅₀ titration.

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Alanine-scanning mutants of the F-protein TM region are efficiently transported to the cell surface. To assess the role of the F protein TM region in the fusion process, we constructed six mutants of the vaccine strain (Moraten) protein in which blocks of three or four residues are replaced by alanines (Fig. 1A, F ^A0 to F^A5). Secondary-structure analysis of the TM region of the mutant proteins in silico using NNPREDICT (23) and JPRED3 (12) predicted a higher local content of alpha-helical structures for all initial mutated F-protein mutants, with no major effects on other domains' secondary structure.

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FIG. 1. Analysis of the processing of F proteins with mutated TM regions. (A) (Top) schematic drawing of MV F protein mutants. The N and C termini, the F1 and F2 subunits, and the TM region are indicated. (Bottom) Amino acid sequences of the TM regions of vaccine strain (Moraten) F protein and nine mutants. Numbers indicate the positions of the amino acids in the F protein. (B) Expression and processing of the F-protein mutants coexpressed with MV H in Vero cells and analyzed by immunoblotting. (Top) Detection of F0 and F1 using an antibody directed against the cytoplasmic tail. F1 to F0 ratios were determined by densitometry. (Bottom) Detection of H using an antibody directed against its cytoplasmic tail. NC, negative control.

Expression levels, stability, and processing of the six block mutants and of three double or single mutant proteins were verified after cotransfection of the vaccine strain F and H expression plasmids in Vero cells, followed by immunoblotting. All F-protein mutants were expressed and processed. Processing ratios between the F₁ product and the precursor (F₀) are presented in Fig. 1B and ranged between 0.4 and 1.1. As controls, an F protein with a trypsin instead of a furin cleavage site (F_trypsin) (28) and an F protein bearing an endoplasmic reticulum retention signal (F_ER) were used (38). As expected, F_ER remained exclusively in the F₀ form. Low levels of F_trypsin processing were detected, but the F₁-like form migrated as a doublet in contrast to the other F₁ proteins, suggesting different sites of cleavage.

The kinetics of F cleavage was characterized by pulse-chase analysis followed by immunoprecipitation. F mutants were coexpressed with the H protein of the wild-type MV recombinant strain IC-323 (45) in order to limit fusion followed by proteolytic degradation of the F protein. With the exception of the mutant F^A1, the processing kinetics of mutant F proteins maintained characteristics similar to those of the unmodified protein (Fig. 2A) as quantified by densitometry (Fig. 2B). The half-lives of the F₀ form of the unmodified protein and most mutants were about 4.5 h (Fig. 2B). Only F^A1 was processed with a slower kinetics, with about 40% of the F₀ form being cleaved after a 6-h chase (Fig. 2B). Coexpression and precipitation of the F-protein mutants with H of the vaccine strain yielded similar results, but the most fusogenic F mutants caused early cell death and proteolytic degradation following extensive fusion (data not shown).

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FIG. 2. F-protein mutants' processing kinetics. (A) Pulse-chase analysis of the F TM region mutants after coexpression with wild-type H protein in Vero cells. After pulsing for 30 min and chasing for the indicated times (in hours), the F proteins were immunoprecipitated using an antibody recognizing their cytoplasmic tail. NC, negative control. (B) Schematics of the quantitation of the pulse-chase autoradiograms. Values are percentages of F1 protein relative to unprocessed F0.

Since preliminary immunofluorescence analyses indicated that all F proteins except F_ER were transported to the cell surface (data not shown), surface biotinylation experiments were planned to determine the amount and forms of F proteins expressed. Figure 3 shows that similar quantities of the processed F₁ subunit were detected at the cell surface for all variants, whereas only trace amounts (<1%) of F₀ were present. As expected, low levels of F_trypsin were detected in the inactive F₀ form, and F_ER did not reach the cell surface (Fig. 3). Thus, the F mutants do not vary significantly in terms of their transport to the cell surface.

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FIG. 3. Surface transport of the F-protein mutants. Vero cells expressing the F TM region mutants and H were biotinylated and lysed. Lysates were subjected to precipitation with streptavidin-coated agarose beads, and precipitated surface proteins were subjected to immunoblot analysis using antibodies directed against the cytoplasmic tail of F or H. Molecular mass markers (kDa) are on the right.

Mutation of a central residue in the F-protein TM region enhances fusion activity. To identify F-protein TM region residues important for the fusion process, we counted the nuclei in syncytia produced after coexpression of H and the F mutants in Vero cells. This assay revealed that five mutants (F^A0, F^A1, F^A2, F^A4, and F^A5) had impaired fusion activity (three to five nuclei per syncytium) compared to unmodified F and mutant F^A3 protein (12 nuclei per syncytium) (Fig. 4A).

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FIG. 4. Fusion activity of the F-protein mutants. (A and D) Average numbers of nuclei per syncytium after coexpression with H protein in Vero cells. The cells were incubated overnight at 37°C, fixed, and stained with crystal violet, and numbers of nuclei per syncytium were determined. (B and E) Fusion activity assessed by luciferase expression levels. Luciferase activity of Vero cells cotransfected with vaccine strain H protein, mutant F protein, and T7 promoter-dependent luciferase expression constructs was determined after overlay with B95a cells infected with MVA-T7. After 6 h, cultures were lysed, and activity was determined with a luminometer. RLU, relative light units; NC, negative control (without F). (C) Thermodynamic analysis of F-mediated fusion. After expression of different F-protein mutants and H protein in Vero cells and overnight incubation at 37°C in the presence of fusion-inhibiting peptide, cells were incubated without fusion-inhibiting peptide for 2 h at the temperatures indicated. The cells were fixed and stained with crystal violet, and numbers of nuclei per syncytium were determined. (F) Amino acid sequences of the TM regions of the vaccine strain (Moraten) F protein and the mutants FA3.B, FI508A, and FG509A. Numbers indicate the positions of the amino acids in the F protein. Amino acids in the TM region are boxed.

We then repeated these measurements using an assay based upon fusion-induced luciferase expression (35). This quantitative assay confirmed the data obtained by counting the nuclei in syncytia: the fusion activity of F^A0, F^A1, F^A2, F^A4, and F^A5 was two to three times lower than that of the standard F protein, whereas F^A3 had slightly higher activity in this assay. Therefore, the role of the amino acids mutated in variant F^A3 was further examined. Mutation of two residues (F^A3.A) enhanced fusogenic activity to an average of 35 nuclei per syncytium and nearly tripled luciferase activity (Fig. 4A and B). This effect was attributed to the mutation L507A by further mapping. F^L507A had about three-times-higher fusion activity in both assays, similar to F^A3.A (Fig. 4A and B).

To complete this analysis, we also mutated amino acids 508 and 509 to alanines separately and in combination (Fig. 4F). The F^I508A mutant and the double mutant F^A3.B had reduced fusion activity (10 nuclei per syncytium) compared to unmodified F (18 nuclei per syncytium), while the single mutant F^G509A had levels of fusion similar to those of unmodified F (17 nuclei per syncytium) (Fig. 4D). These measurements were confirmed by the luciferase assay, revealing 1.5-fold reduction of fusion activity of F^A3.B or F^I508A compared to that of unmodified F protein (Fig. 4E). Thus, I508A counteracts the effect of L507A, which is consistent with the nearly normal levels of fusion of F^A3.

Next, we measured fusion activity at 32°C and 42°C, to assess whether this effect occurs under different thermodynamic conditions (Fig. 4C). The fusion activity of each mutant protein at 42°C was about double that at 32°C. Similar ratios of fusion between mutants were observed as at 37°C (data not shown). Altogether, these results indicate that a central residue of the F-protein TM region has a strong influence on the efficiency of membrane fusion.

Fusion directly correlates with availability of an active F-H protein complex. We then hypothesized that the mutations in the TM region of the F protein may alter the formation or stability of the active F₁-H complex, thereby also modulating fusion activity. To assess the availability of these complexes, we performed coimmunoprecipitation assays on cells coexpressing H and the F mutants (Fig. 5). The F proteins were coimmunoprecipitated by an anti-H antibody (Fig. 5A, top). For the standard F protein, F₀ was coimmunoprecipitated in complex with H more efficiently than F₁, as previously described (38).

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FIG. 5. Analysis of F-H protein hetero-oligomers. Vero cells coexpressing F and H proteins were lysed, and proteins were analyzed by immunoblotting after (A) or before (B) coimmunoprecipitation with an antibody directed against native H. Antibodies used for protein detection were directed against cytoplasmic tails of F (top panels) or H (bottom panels). Numbers below the top panels are ratios of F1 to F0 for each mutant as determined by densitometry. Fusion activity is indicated in panel A below the blot for coprecipitated F.

The amount of inactive F₀ protein coimmunoprecipitated with H was inversely correlated to the fusion activity, as indicated in Fig. 5A. Small amounts of F₀ protein coprecipitated for the hyperfusogenic mutants F^A3.A and F^L507A, large amounts for the hypofusogenic mutants F^A0, F^A1, F^A2 and F^A4, and intermediate amounts for unmodified F, F^A3, F^A5, and F^G506A (Fig. 5A). The antibody directed against the H ectodomain precipitated similar amounts of H in all transfected cells (Fig. 5A), revealing little bias in the experiment due to differential binding of the complexes to the precipitation antibody. An additional control documented that similar amounts of F and H protein were expressed (Fig. 5B).

The ratio of coprecipitated F₁ to F₀ changed depending on the mutation, as indicated in Fig. 5A. The hypofusogenic mutants F^A0, F^A1, F^A2, and F^A4 coprecipitated mainly in the uncleaved F₀ form. In contrast, the hyperfusogenic mutants coprecipitated mainly in the cleaved form F₁ (Fig. 5A). The highest F₁/F₀ ratio (2.2) was documented for F^L507A, whereas for the hypofusogenic mutant F^A5 a ratio of less than 0.01 was documented. In Western blot controls of tissue lysates, the ratio of F₁/F₀ varied by a factor of 6, at most, being 3.5 for unmodified F and 0.6 for F^A1, and did not correlate well with the fusion efficiencies (Fig. 5B). Thus, fusion activity correlates with the availability of the complex of activated F with H.

F-protein TM region mutation confers enhanced fusogenicity on recombinant virus. To assess the effect of the mutations in the context of viral infection, we cloned the open reading frames of the nine F mutants into the infectious cDNA of the MV Moraten strain (15). Viruses were rescued for each infectious cDNA, and multistep growth kinetics were determined on Vero cells. These analyses did not reveal significant differences (Fig. 6A).

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FIG. 6. Growth characteristics and fusion efficiency of recombinant MV expressing different F protein variants. (A) Multistep growth curves. Vero cells were infected at an MOI of 0.03, and titers of cell-associated and released viruses were measured at the time points indicated. (B) Fluorescence microscopy of representative single syncytia 48 h after infection of Vero cells with the indicated virus strains. All these viruses express the reporter protein green fluorescent protein. (C) Number of nuclei per syncytium 48 h after infection of Vero cells. Averages and standard deviations are indicated.

Syncytium formation induced by the recombinant MV infection in Vero cells was documented by microscopy (Fig. 6B) and quantified by counting the nuclei per syncytium 48 h postinfection (Fig. 6C). These data show that the fusogenicity of the different MV strains correlated with the fusogenicity of the respective F-protein TM mutants expressed transiently (Fig. 4).

The only exception was the recombinant virus carrying the F^A0 mutation, which produced larger syncytia than unmodified recombinant Moraten MV (Fig. 6C). In contrast, the F^A0 protein coexpressed with H was less fusogenic than the standard F protein (Fig. 4A and B). The fusogenic activity of recombinant viruses harboring membrane-proximal amino acid substitutions in the respective F protein (MV-F^A1, MV-F^A2, MV-F^A4, and MV-F^A5) was reduced about twofold (9 to 12 nuclei per syncytium) compared to unmodified recombinant virus (19 nuclei per syncytium on average). Fusogenic activity of the variants with central mutations MV-F^A3 and MV-F^G506A was in the same range (21 and 22 nuclei per syncytium, respectively), whereas the variants MV-F^A3.A and MV-F^L507A exhibited the highest fusion activity (52 to 62 nuclei per syncytium). Thus, with one exception, the fusion activity of the F-TM mutants correlates directly with the cell-to-cell fusion activity of the respective virus.

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Mutation of leucine 507, located near the center of the MV F-protein TM region, enhanced membrane fusion efficiency, while mutation of other residues in this TM region reduced it. More activated F₁₊₂-H hetero-oligomers of the F^L507A protein were available than F₀-H hetero-oligomers. On the other hand, all TM region mutations that reduced membrane fusion had a lower ratio of active F₁₊₂-H hetero-oligomers to inactive F₀-H hetero-oligomers. These data are consistent with a mechanism of cell fusion in which the availability of active F₁₊₂-H hetero-oligomers at the cell surface determines the efficiency of the process, as proposed for other paramyxoviruses (2, 36, 51).

Mutations in the TM region of F may influence the activity of the glycoprotein complex by changing the F-protein routing through the secretory pathway, possibly altering its folding process. Small hydrophobic patches left by imperfect folding of the F trimer may not interfere with transport to the cell surface but may lower the activity of the complex, possibly by modifying the lateral interactions with the H-protein dimer.

The F^L507A protein is the most interesting mutant, because it has enhanced fusion activity, and it seems counterintuitive that gain of function may occur through a folding change. Rather, we think that the physicochemical properties of the mutated TM segment of F^L507A change the association of the complex with the lipid bilayer, which may include lipid rafts (44, 48). Lattices of F and H proteins may be organized differently, or grow larger (27), in cells expressing F^L507A than in cells expressing standard F, enhancing fusion efficiency.

MV envelope glycoprotein complex formation differs from that of other paramyxoviruses because hetero-oligomerization of MV F trimers and H dimers is already substantial in the endoplasmic reticulum, before proteolytic activation of the F₀ protein precursor (38). On the other hand, glycoproteins of the other paramyxoviruses, like SV5, hPIV3 (36), or Sendai virus (47), associate only at the cell surface. It has been suggested that paramyxoviruses recognizing the abundant sialic acids as receptors have to prevent formation of an active complex to avoid premature membrane fusion in the producing cells, whereas the risk of receptor encounter for the MV glycoprotein complexes could be relatively low (11). It is possible that the fusion processes are fundamentally different, but our experiments, while confirming early association of the F and H oligomers, do not give insights into the fusion mechanism.

Viruses with F-protein TM regions conferring higher or lower fusion capacity have replication kinetics similar to that of standard MV. This implies that membrane fusion is not a limiting factor in this experimental system. Syncytium formation is limited after experimental infection of monkeys (14, 26, 29), and extensive fusion can be detrimental because it amplifies the type I interferon response (18). Thus, wild-type MV may be under selective pressure to limit fusion; from this perspective, the generation of F-protein TM mutants with enhanced fusion function is more likely than if wild-type MV were highly fusogenic.

Interestingly, whereas fusion efficiencies of eight F TM mutants were similar in the H coexpression and virus infection assays, the F^A0 mutation had opposite effects in the two assays. The main difference between the assays is the availability of the viral ribonucleoprotein and of the M protein, the membrane-associated assembly organizer, only in the virus infection assay. These components can influence fusion, and the M protein does this by interacting with the F- and H-protein cytoplasmic tails (6, 7, 39, 43). Remarkably, the F^A0 mutation is located on the outer leaflet of the membrane, opposite to the site of association of the M protein. Thus, if the interaction between M and the outer segment of the F TM domain is direct, it may occur when the fusion pore opens.

A direct effect of modifications of the TM region on F-protein function is also possible. Mutations in the TM region or replacements by a glycosylphosphatidylinositol anchor in viral class I fusion proteins like influenza HA and the VSV glycoprotein reduced or abolished fusion activity (9, 22, 30). For the VSV glycoprotein, the TM region has been postulated to act as an autonomous domain during late stages of the fusion process (24). Interestingly, the fusion activity of model peptides mimicking a TM region was enhanced by decreasing the leucine content, possibly because of stabilization of the -helical structure (19). On the other hand, enhanced structural flexibility of TM regions has been correlated with enhanced fusion activity for model peptides mimicking the TM regions of VSV G protein (13, 24) or cellular SNARE proteins (25). Thus, replacement of L507 in the center of the MV F protein TM region may enhance the structural flexibility of F^L507A, resulting in higher fusion activity.

 
We thank Sompong Vongpunsawad for outstanding technical support, Christoph Springfeld for helpful discussions, and Christian J. Buchholz for continuous support.

This work was supported by research scholarship grant Mu 2327/1-1 from the Deutsche Forschungsgemeinschaft (M. D. Mühlebach) and by NIH grant R01 CA90636.

 
* Corresponding author. Mailing address: Mayo Clinic, Department of Molecular Medicine, 200 First Street SW, Rochester, MN 55905. Phone: (507) 538-1188. Fax: (507) 266-2122. E-mail: Cattaneo.Roberto{at}mayo.edu

Published ahead of print on 10 September 2008.

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Journal of Virology, November 2008, p. 11437-11445, Vol. 82, No. 22
0022-538X/08/$08.00+0     doi:10.1128/JVI.00779-08
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