A Single Amino Acid Change in the Cytoplasmic Domains of Measles Virus Glycoproteins H and F Alters Targeting, Endocytosis, and Cell Fusion in Polarized Madin-Darby Canine Kidney Cells*

Markus MollDagger , Hans-Dieter KlenkDagger , Georg Herrler§, and Andrea MaisnerDagger

From the Dagger  Institut für Virologie, Philipps-Universität Marburg, D-35037 Marburg, Germany and the § Institut für Virologie, Tierärztliche Hochschule Hannover, D-30559 Hannover, Germany

Received for publication, November 8, 2000, and in revised form, February 26, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

As we have shown previously, release of measles virus (MV) from polarized epithelial cells is not determined by the viral envelope proteins H and F. Although virus budding is restricted to the apical surfaces, both proteins were abundantly expressed on the basolateral surface of Madin-Darby canine kidney cells. In this report, we provide evidence that the basolateral expression of the viral proteins is of biological importance for the MV infection of polarized epithelial cells. We demonstrate that both MV glycoproteins possess a basolateral targeting signal that is dependent upon the unique tyrosine in the cytoplasmic tails. These tyrosines are shown to be also part of an endocytosis signal. In MV-infected cells, internalization of the glycoproteins was not observed, indicating that recognition of the endocytosis signals is disturbed by viral factors. In contrast, basolateral transport was not substantially hindered, resulting in efficient cell-to-cell fusion of polarized Madin-Darby canine kidney cells. Thus, recognition of the signals for endocytosis and polarized transport is differently regulated in infected cells. Mutation of the basolateral sorting signal in one of the MV glycoproteins prevented fusion of polarized cells. These results suggest that basolateral expression of the MV glycoproteins favors virus spread in epithelia.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Epithelial cells play crucial roles in diverse processes such as water and ion balance, secretion, adsorption of nutrients, and signal transduction. The polarized nature of these cells is central to their function. The plasma membrane of these cells is divided into an apical and a basolateral domain that have different lipid and protein compositions (for review, see Ref. 1). Sorting of membrane proteins was shown to occur in the trans-Golgi network and to depend on special targeting signals. The most thoroughly investigated model for epithelial polarity is the Madin-Darby canine kidney (MDCK)1 continuous cell line. Several viral glycoproteins have been shown to be sorted to either the apical or basolateral surfaces (2). Since glycoproteins of enveloped viruses perform critical functions during viral assembly and serve as main targets of humoral immune responses, polarized expression of these proteins affects the viral life cycle. The viral glycoproteins studied in most detail are the vesicular stomatitis virus G protein and the influenza virus hemagglutinin (HA). Vesicular stomatitis virus G protein was found to contain basolateral sorting information that is critically dependent upon the presence of a tyrosine in the cytoplasmic tail (3). The signal responsible for the apical transport of HA is contained within the extracellular or transmembrane portion of the molecule (4-6). Vesicular stomatitis virus was shown to be released mainly from the basolateral side of infected MDCK cells, whereas influenza virus almost exclusively buds from the apical cell surface (7). These data were the basis for the view that budding of enveloped viruses occurs only at the site at which their envelope proteins are mostly concentrated (2, 8, 9).

However, recent data on virus release and glycoprotein sorting indicated that this model is not valid for measles virus (10, 11). Measles virus (MV) is a member of the Paramyxoviridae family and possesses a negative-stranded RNA genome encoding for six structural proteins. The nucleoprotein together with the phosphoprotein, the viral polymerase, and the genomic RNA form the ribonucleoprotein complex that assembles in the cytoplasm of infected cells. The matrix protein (M protein), synthesized in the cytoplasm, mediates the contact of the ribonucleoproteins with viral envelope proteins initiating virus budding at the plasma membrane (12, 13). Hemagglutinin (H protein), the envelope protein responsible for the binding of the virus to the cell surface receptor (14-16), is a type II integral membrane protein with a C-terminal ectodomain. The second envelope protein, the fusion protein (F protein), is a type I glycoprotein possessing an N-terminal ectodomain that has to be cleaved into the F1/F2 subunits to mediate pH-independent fusion of virus with the plasma membrane (17). We have previously shown that both proteins are abundantly expressed on the basolateral side of polarized MDCK cells, although virus budding is restricted to the apical cell surfaces (10). This finding was recently confirmed using another polarized cell line, CaCo2 cells (11). Furthermore, we have demonstrated that upon stable expression in MDCK cells, both H and F proteins are targeted almost completely to the basolateral surface (10). Thus, targeting information resides in the proteins itself. Since MV glycoproteins do not determine the site of virus budding, MV must have developed a maturation strategy different from those of viruses such as influenza virus or vesicular stomatitis virus. Most recent work has shown that MV M protein may specify apical virus release because it is predominantly localized at the apical plasma membrane of polarized MDCK2 and CaCo2 cells (11).

The aim of this study was to analyze the signals responsible for the basolateral sorting of H and F proteins to elucidate the importance of the basolateral glycoprotein expression for the pathogenesis of measles virus infection. We show that polarized targeting of both proteins is dependent upon a tyrosine residue in the cytoplasmic tails. The same tyrosines are shown to be part of an endocytosis signal. Since the signals in the F protein cytoplasmic tail appeared to be more efficiently recognized than those of the H protein, they were transferred to another protein. Chimeric proteins from MV F protein and influenza virus HA revealed that the signals residing in MV F protein can only partly override the targeting signals of an apical protein that is not internalized. To analyze the biological importance of the signals residing in the cytoplasmic tails of the MV envelope proteins, the polarized transport and endocytosis of the glycoproteins in infected cells were monitored. Endocytosis, but not polarized transport, appeared to be inhibited. Fusion of polarized MDCK cells was observed only when basolateral sorting signals of both glycoproteins were intact. This result led us to suggest that endocytosis interferes with the assembly of new viruses at the plasma membrane, whereas basolateral expression of the MV glycoproteins favors virus spread in epithelial cells.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cell Culture-- MDCK cells (strain II) were grown in Eagle's minimal essential medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Life Technologies, Inc.), 100 units/ml penicillin, and 100 µg of streptomycin. For studies of cell polarity, tissue culture-treated 0.4-µm pore size Transwell polycarbonate filters (Costar Corp., Cambridge, MA) were used. Cells were seeded 5 days before experiments (2 × 106 cells/24-mm unit). The polarity was determined by measurement of the transepithelial resistance using a Millipore ERS instrument. MDCK cells formed a tight monolayer with an electrical resistance of 1000-2500 ohms × cm2. The different patterns of surface proteins on the apical and basolateral membranes of the polarized cell line were controlled by surface biotinylation.

Plasmid Constructs and Stable Expression in MDCK Cells-- Cloning of the viral glycoprotein (H and F protein) genes into the expression vector pCG under the control of the cytomegalovirus early promotor has been described previously (18). The glycoprotein mutants were prepared by introduction of site-specific mutations with the complementary primers F549Y/A 1 (5'-GGAACATCAAAATCCGCTGTAAGGTCGCTCTGATCC-3') and F549Y/A 2 (5'-GGATCAGAGCGACCTTACAGCGGATTTTGATGTTCC-3') and H12Y/A 1 (5'-CGGATAAATGCCTTCGCCAAAGATAACCCC-3') and H12Y/A 2 (5'-GGGGTTATCTTTGGCGAAGGCATTTATCCG-3') in the double-stranded pCG plasmids using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The chimeras used in this study are marked by their composition (ectodomain-transmembrane domain-cytoplasmic tail) as H7-H7-F, H7-F-H7, and H7-F-F. H7 represents the respective domain of the influenza virus hemagglutinin, and F represents the respective domain of the measles virus fusion protein. The recombinant proteins H7-H7-F and H7-F-F were generated by a recombinant polymerase chain reaction technique (19) using synthetic oligonucleotides and pSG5new-HAmut7 and pCG-F as templates. The mutant hemagglutinin HAmut7 of the influenza A virus has been described elsewhere (20). The internal sense primers were 5'-CTTGTTTTCATATGTGTGAGGGGGCGTTGTAATAAA-3' (H7-H7-F) and 5'-GAGTAGTGGCTACAAAGATGGTTTATCGAGCACTAGC-3' (H7-F-F), and the internal antisense primers were 5'-TTTATTACAACGCCCCCTCACACATATGAAAACAAG-3' (H7-H7-F) and 5'-GCTAGTGCTCGATAAACCATCTTTGTAGCCACTACTC-3'(H7-F-F). The external primers were 5'-ATACTTATGATCACAGCAAAT-3' (sense) and 5'-GGCGGCGCGCAGATCTTGTGTTTCAAGAGTTGTAGAGG-3' (antisense). pSG5new-H7-F-F and pSG5new-HAmut7 were used to produce H7-F-H7 by the same recombinant polymerase chain reaction technique with the internal primers 5'-GCTTTAATATGTTGCTGCAAGAACGGAAACATGCGG-3' (sense) and 5'-CCGCATGTTTCCGTTCTTGCAGCAACATATTAAAGC-3' (antisense) and the external primers 5'-ATACTTATGATCACAGCAAAT-3' (sense) and 5'-GGCGGCGCGCACTAGTAATAAACAAGTTCTGC-3' (antisense). The sequences of the plasmid constructs were confirmed by dideoxy sequencing. For stable expression, MDCK cells were cotransfected with either of the expression plasmids and the neomycin resistance-conferring plasmid pIGI at a ratio of 1:10 using the cationic lipid transfection reagent LipofectAMINE 2000 (Life Technologies, Inc.) according to the protocol of the manufacturer. Cells were screened for Geneticin resistance by addition of 1.0 mg of Geneticin (Calbiochem)/ml medium. The selected cell clones were screened for expression of foreign proteins by immunofluorescence analysis.

Surface Immunofluorescence Analysis-- MDCK cells stably expressing parental proteins (FEdm and HEdm) or tyrosine mutants (F549Y/A and H12Y/A) were grown on glass coverslips and incubated with the monoclonal antibody (mAb) A504 (directed against MV F protein; kindly provided by J. Schneider-Schaulies) or mAb 8905 (directed against MV H protein; Chemicon, Temecula, CA) for 60 min at 4 °C without prior fixation. The primary antibodies were detected using an FITC-labeled goat anti-mouse IgG (Dako Corp.) for 45 min at 4 °C. The samples were mounted in Mowiol and 10% 1,4-diazabicyclo(2,2,2)octane. The coverslips were viewed and photographed with a Zeiss Axiophot microscope equipped with UV optics.

Surface Biotinylation and Immunoprecipitation-- Filter-grown MDCK cells were biotinylated essentially as described by Lisanti et al. (21). Cells were washed three times with cold phosphate-buffered saline (PBS) containing 0.1 mM CaCl2 and 1 mM MgCl2. The apical or basolateral cell surface was incubated twice for 20 min at 4 °C with 2 mg/ml sulfo-N-hydroxysuccinimidobiotin (Pierce) by adding 1 ml of the biotinylating reagent to the respective filter chamber. The same volume of PBS containing 0.1 M glycine was placed into the opposite filter chamber. After biotinylation, cells were washed once with cold PBS containing 0.1 M glycine and three times with cold PBS. Cells were lysed in 0.5 ml of radio immunoprecipitation assay buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 10 mM EDTA, 10 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 50 units/ml aprotinin, and 20 mM Tris-HCl, pH 8.5), followed by centrifugation for 20 min at 100,000 × g. The supernatant was immunoprecipitated with mAb A504 (directed against MV F protein), mAb 8905 (directed against MV H protein), or mAb 2A11-H7 (directed against the H7 subtype of the influenza A virus hemagglutinin; kindly provided by W. Garten) at a final dilution of 1:100. After addition of 40 µl of a suspension of protein A-Sepharose CL-4B (Sigma) coated with rabbit anti-mouse IgG (Dako Corp.), immunocomplexes were washed three times with radio immunoprecipitation assay buffer and suspended in reducing (MV H protein) or nonreducing (MV F and recombinant proteins) sample buffer for SDS-polyacrylamide gel electrophoresis. Following separation on a 10% polyacrylamide gel, proteins were blotted onto nitrocellulose by the semidry blot technique. After blocking of nonspecific binding sites by 5% nonfat dry milk in PBS, blots were incubated for 45 min at 4 °C with streptavidin-biotinylated horseradish peroxidase complex (Amersham Pharmacia Biotech, Uppsala, Sweden) diluted 1:2000 in PBS containing 0.1% Tween 20. Biotinylated proteins were detected with the enhanced chemiluminescence system (Amersham Pharmacia Biotech) by exposure to Eastman Kodak XAR autoradiography film.

Antibody Uptake Assay-- MDCK cells were grown on coverslips and transfected with either the parental (pCG-FEdm and pCG-HEdm) or mutant (pCG-F549Y/A and pCG-H12Y/A) protein-expressing plasmids alone or cotransfected with plasmids containing parental proteins using the transfection reagent LipofectAMINE 2000. A total amount of 1 µg of DNA was used. Since infection of confluent MDCK cells has been shown to be very inefficient (10), virus was added to the cells directly after seeding. At 7 h post-infection, the virus-containing growth medium was replaced by fresh minimal essential medium containing 10% fetal calf serum. At 20 h post-transfection or 30 h post-infection, surface-expressed protein was labeled with mAb A504 (directed against MV F protein) or mAb 8905 (directed against MV H protein). After incubation for 60 min on ice, the antibody was removed by washing with PBS, and the cells were incubated with cell culture medium for 15 min at 37 °C to allow endocytosis of the antigen-antibody complexes. Internalization was stopped by rapid cooling on ice. Surface-bound antibodies were detected by incubation with rhodamine-conjugated goat anti-mouse Fab fragments (Dianova) at a dilution of 1:100 in PBS for 60 min on ice. After washing with PBS, the cells were fixed and permeabilized for 5 min at -20 °C with methanol/acetone (1:1). Internalized antibodies were detected with FITC-labeled goat anti-mouse IgG at a dilution of 1:100. The samples were mounted in Mowiol containing 10% 1,4-diazabicyclo(2,2,2)octane and analyzed using a Zeiss Axiophot microscope equipped with UV optics.

Biotin Internalization Assay-- The experiment was mainly performed as described by Graeve et al. (22). MDCK cells stably expressing either one of the parental or one of the mutant proteins were grown on 6-cm plastic dishes and incubated twice for 20 min at 4 °C with a 2 mg/ml concentration of the non-membrane-permeating, cleavable biotin derivative sulfosuccinimidyl 2-(biotinamido)ethyl-1,3'-dithiopropionate (Pierce). Following washing with cold PBS containing 0.1 M glycine and several washings with cold PBS, cells were incubated with cell culture medium for 0, 5, 15, or 30 min at either 4 or 37 °C to allow endocytosis of the labeled proteins. After internalization was stopped by rapid cooling on ice, cells were incubated three times for 20 min at 4 °C with 50 mM 2-mercaptoethanesulfonic acid (Sigma) in 50 mM Tris-HCl, pH 8.7, 100 mM NaCl, and 2.5 mM CaCl2. Biotin exposed at the cell surface was thereby cleaved. After thorough rinsing with PBS containing 20 mM Hepes, cells were lysed in radio immunoprecipitation assay buffer, and proteins were immunoprecipitated as described above. Biotinylated proteins were separated on a 10% SDS-polyacrylamide gel under nonreducing conditions, transferred to nitrocellulose, and detected as described above. The internalization rate was determined by densitometric quantification.

Cell Fusion Assays-- MDCK cells were seeded in 35-mm diameter wells to reach 90-95% confluence 1 day after plating. The MV glycoprotein-expressing plasmids pCG-FEdm and pCG-HEdm were transfected in the absence or presence of either the corresponding parental protein-expressing plasmids (pCG-FEdm and pCG-HEdm) or the mutant protein-expressing plasmids (pCG-F549Y/A and pCG-H12Y/A) using the cationic lipid transfection reagent LipofectAMINE 2000. A total amount of 5 µg of DNA was used. At 20 h post-transfection, the transiently expressing cells were fixed with ethanol and stained with 1:10 diluted Giemsa staining solution (Merck, Darmstadt, Germany).

To analyze the biological activity of the MV glycoproteins in polarized MDCK cells, MDCK cells stably expressing either MV FEdm or MV F549Y/A were seeded on coverslips at high density. At 24 h post-seeding, cells were transfected with pCG-HEdm or pCG-H12Y/A, respectively. For comparison, MDCK cells were infected as described above. At 7 h post-transfection or post-infection, transfection complexes or virus-containing growth medium was removed, and cells were incubated for 24 h in Dulbecco's modified essential medium either with or without calcium. Since the maintenance of tight junctions requires calcium, the lack of calcium abolishes cell polarity. To visualize syncytium formation in polarized or non-polarized cells, immunostaining was performed. After fixation and permeabilization at -20 °C with methanol/acetone (1:1), cells were incubated with monoclonal antibody 8905 (directed against MV H protein) and an FITC-labeled goat anti-rabbit IgG (Dako Corp.). The samples were mounted and analyzed as described above.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Both H12Y/A and F549Y/A Mutant Proteins Are Transported to the Cell Surface and Are Biologically Active-- Cytoplasmic tyrosines have been identified as important constituents of many, but not all, basolateral sorting signals. The cytoplasmic tails of both MV glycoproteins contain a single tyrosine at position 12 in the H protein and at position 549 in the F protein. To determine if these tyrosines residues contribute to a basolateral targeting signal, mutants were constructed in which amino acid 12 of the H protein and amino acid 549 of the F protein were changed to an alanine. The sequences of parental proteins and mutants H12Y/A and F549Y/A (tyrosine mutants) are shown in Fig. 1A. Successful use of site-specific mutagenesis to identify amino acids serving as recognition signals during intracellular transport requires that local changes do not disturb other, perhaps distant, parts of the protein, resulting in inefficient transport or loss of function. Without prior fixation, surfaces of transiently expressing MDCK cells were incubated with anti-H protein (mAb 8905) or anti-F protein (A504) antibodies and an FITC-conjugated secondary antibody. The surface immunofluorescence in Fig. 1B shows that the exchange of the tyrosine did not prevent surface transport. A common assay for testing the biological function of H and F proteins is to analyze cells coexpressing H and F proteins for their ability to induce syncytium formation. Only fusion-competent F proteins in combination with H proteins that bind to MV receptors and that can interact with the F protein support virus-to-cell or cell-to-cell fusion (23). To analyze the mutants for syncytium formation, subconfluent MDCK cells coexpressing either parental HEdm or H12Y/A in combination with parental FEdm or F549Y/A were fixed at 20 h post-transfection and stained with Giemsa staining solution. As shown in Fig. 1C, H or F proteins expressed alone did not induce fusion, whereas syncytium formation was observed in cells expressing different combinations of parental and mutant proteins. This result indicates that tyrosine mutants were not affected in their ability to mediate fusion of non-polarized cells.


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Fig. 1.   A, sequences of the cytoplasmic domains of parental and mutant H and F proteins. Protein sequences are shown in single-letter code. The vertical lines separate the transmembrane sequences from those predicted to be in the cytoplasm. TM, transmembrane domain; CD, cytoplasmic domain. B, surface expression of parental and mutant glycoproteins. MDCK cells transiently expressing either the parental (HEdm and FEdm) or mutant (H12Y/A and F549Y/A) proteins were incubated with either anti-H or anti-F protein monoclonal antibodies at 4 °C without prior fixation. Surface-bound antibodies were detected with FITC-conjugated anti-mouse immunoglobulins. C, fusion activity of parental and mutant proteins. HEdm and FEdm were transiently expressed in the absence or presence of either the corresponding parental protein (FEdm and HEdm) or mutant protein (F549Y/A and H12Y/A). At 20 h post-transfection, cells were fixed and stained with Giemsa staining solution.

Both Tyrosine Mutants Are Re-targeted in Polarized Epithelial Cells-- To analyze the polarized transport of membrane proteins, stably expressing cell lines have to be established. For this purpose, plasmids expressing either the parental HEdm or FEdm protein or tyrosine mutants were transfected into MDCK cells, and neomycin-resistant transfectants were selected using G418. To monitor the targeting of the expressed proteins, the cells were cultured on permeable filter supports, where they form polarized monolayers. Cells grown on filters were cooled to 4 °C, and either the apical or basolateral surface proteins were labeled by adding the non-membrane-permeating reagent sulfo-N-hydroxysuccinimidobiotin to the respective filter chamber. The cells were lysed, and H or F proteins were immunoprecipitated by specific antibodies (mAb 8905 or A504). After separation on a 10% SDS-polyacrylamide gel (reducing conditions for H proteins and nonreducing conditions for F proteins), proteins were transferred to nitrocellulose. Biotin-labeled proteins were detected with peroxidase-conjugated streptavidin. As shown in Fig. 2 (lanes b), efficient biotinylation of both parental proteins was obtained only after labeling the cells from the basolateral side (HEdm, 95%; and FEdm, >99%). This confirms our previous results that both MV glycoproteins are predominantly expressed on the basolateral surface of polarized MDCK cells. In contrast, strong labeling of both tyrosine mutants was detected only after apical surface biotinylation (F549Y/A and H12Y/A panels, lanes a). Only a minor band was found after basolateral labeling (lanes b). The protein distribution was further confirmed by surface immunofluorescence of filter-grown cells using a confocal laser scanning microscope (data not shown). The redirection of the mutant proteins to the apical cell membrane indicates that both MV glycoproteins contain a tyrosine-dependent basolateral sorting signal.


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Fig. 2.   Surface distribution of parental and mutant proteins. MDCK cells stably expressing HEdm, H12Y/A, FEdm, or F549Y/A were grown on filters and surface-biotinylated from either the apical (lanes a) or basolateral (lanes b) side. After cell lysis, proteins were immunoprecipitated with an H or F protein-specific monoclonal antibody. Precipitates were analyzed by SDS gel electrophoresis, transferred to nitrocellulose, and probed with peroxidase-conjugated streptavidin.

The Tyrosine Responsible for Basolateral Transport of H and F Proteins Also Mediates Endocytosis-- In certain cases, the cytoplasmic signal responsible for efficient localization to coated pits may be very similar to that responsible for basolateral targeting (24-26). To determine if MV glycoproteins also possess an endocytosis signal, we performed an antibody uptake experiment. Without prior fixation, H or F proteins on the surface of stably expressing cells were labeled with the respective antibodies at 4 °C and warmed to 37 °C for 15 min to allow endocytosis to occur. Surface-bound antibodies were detected by incubation of the living cells with a rhodamine-conjugated second antibody at 4 °C. After permeabilization of the cells, internalized H or F protein-antibody complexes were detected with FITC-conjugated second antibody. In Fig. 3A, the result of the double immunofluorescence (surface and intracellular) is shown. Cells expressing parental HEdm and FEdm proteins showed both surface fluorescence and numerous small fluorescent intracellular vesicles distributed throughout the cell. Both MV glycoproteins were internalized during 15 min at 37 °C, indicating that both proteins possess an endocytosis signal. Since the antibody uptake experiment is only a qualitative test, we performed a biotin internalization assay (see "Experimental Procedures"). With this assay, it can be determined at what rate MV proteins are internalized and whether mutation of tyrosine 12 in the H protein and tyrosine 549 in the F protein affects endocytosis. In Fig. 3C, quantitation of the blots shown in Fig. 3B revealed that the FEdm protein was endocytosed at a higher rate than the HEdm protein (3 versus 2.5%/min). Since the antibody uptake experiment is only a qualitative assay, this difference could not be detected. The amount of intracellular F protein increased with increasing incubation at 37 °C. In contrast, the amount of intracellular H protein decreased after 15 min of endocytosis. The same reduction was also observed when the total amount (surface and intracellular H proteins) was determined (data not shown). Thus, the decrease was due to intracellular degradation. The endocytosis rate of the tyrosine mutants was significantly lower than that of parental proteins, indicating that the tyrosines responsible for the basolateral transport are also responsible for endocytosis of H and F proteins. Tyrosine mutants are internalized at a slow rate (<1%/min) that is more characteristic of bulk uptake during membrane turnover than of active clustering into endocytotic vesicles.


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Fig. 3.   A, internalization of antibodies bound to MV glycoproteins. Living MDCK cells expressing either HEdm or FEdm were incubated with an H or F protein-specific antibody at 4 °C. After 60 min, cells were warmed to 37 °C to allow endocytosis to occur. After 15 min, surface-bound antibodies were detected by incubation at 4 °C with a rhodamine-conjugated anti-mouse serum (surface panels). After permeabilization of the cells, internalized antibodies were stained with an FITC-conjugated anti-mouse serum (intracellular panels). B, endocytosis of parental and mutant proteins. MDCK cells stably expressing HEdm, H12Y/A, FEdm, or F549Y/A were surface-labeled with sulfosuccinimidyl 2-(biotinamido)ethyl-1,3'-dithiopropionate at 4 °C. Cells were shifted to 37 °C for the times indicated to allow endocytosis to occur. Subsequently, cell-surface proteins were either reduced with 2-mercaptoethanesulfonic acid (MESNA) at 4 °C (+) or left untreated (-). After cell lysis, proteins were immunoprecipitated with an H or F protein-specific antibody. The precipitates were separated by SDS gel electrophoresis under nonreducing conditions and transferred to nitrocellulose. Biotinylated proteins were detected with peroxidase-conjugated streptavidin. C, rate of internalization of parental and mutant proteins. The percentage of internalized protein measured in the experiment shown in Fig. 3B is plotted as a function of the time that cells were incubated at 37 °C before reduction with 2-mercaptoethanesulfonic acid in the cold. black-diamond , FEdm; black-square, F549Y/A; black-triangle, HEdm; , H12Y/A.

Construction and Analysis of Chimeric Proteins from MV F Protein and the H7 Protein of Influenza Virus-- In infected MDCK cells, the F protein was found to be targeted to the basolateral surface, as found in cells singly expressing FEdm (10). Basolateral transport of the H protein was found to be less stringent in infected cells. Furthermore, endocytosis of MV F protein was more efficient than was internalization of the H protein. Taken together, the F protein cytoplasmic tail appeared to have stronger transport signals. To know whether this signal functions as an autonomous sorting sequence, we wanted to determine whether it is able to redirect an apical membrane protein to the basolateral cell surface. For this purpose, we chose the hemagglutinin (H7) protein of fowl plaque virus, an avian influenza A virus. The apical localization of this protein is well documented (7, 27). Furthermore, it was shown that it is excluded from coated pits (28). Therefore, foreign sequences introduced into the H7 protein can be analyzed not only for basolateral targeting information, but also for their ability to mediate endocytosis through coated pits. For the construction of chimeras, an H7 mutant was used in which the recognition site for proteolytic cleavage by furin-like enzymes was destroyed. As we described previously (29), establishment of cells stably expressing the H7 protein is facilitated when the protein is not cleaved intracellularly and thus is unable to mediate cell-to-cell fusion. The chimeric proteins used in this study are shown in Fig. 4A. In the H7-H7-F chimera, the 11-amino acid tail of the H7 protein was replaced with the 33 amino acids of the cytoplasmic portion of MV F protein. In the H7-F-F chimera, the transmembrane and cytoplasmic domains of the H7 protein were exchanged with the respective domains of MV F protein. In the H7-F-H7 chimera, only the transmembrane domain of the H7 protein was replaced with the corresponding portion of MV F protein. For all constructs, stably expressing MDCK cell lines were established. The surface-expressed chimeric proteins were analyzed whether they were present on the apical or basolateral domain of the plasma membrane. For this purpose, cells were cultured on filters and subjected to domain-specific biotinylation as described above for the tyrosine mutants. Following immunoprecipitation with mAb 2A11-H7, the proteins were analyzed by SDS-polyacrylamide gel electrophoresis, and biotinylated proteins were detected with peroxidase-conjugated streptavidin. As shown in Fig. 4B, the H7 wild-type protein (>99%) as well as H7-H7-F (98%) were predominantly expressed on the apical cell surface of MDCK cells. H7-F-H7 was found almost equally distributed on both cell surfaces (60% apical and 40% basolateral). Only when both the cytoplasmic tail and the transmembrane domain of MV F protein were transferred to the luminal domain of the H7 protein (H7-F-F) the protein was re-targeted to the basolateral side (>95%). This result indicates that the cytoplasmic tail containing the tyrosine critical for the basolateral transport of MV F protein is not sufficient to redirect the apical H7 protein. The additional replacement of the transmembrane domain appeared to be required either for the recognition of the basolateral targeting signal in the F protein cytoplasmic tail or to provide a second basolateral targeting signal. To assay how far the endocytosis signal can be transferred from the F to H7 protein, cells stably expressing chimeric molecules were analyzed by a biotin internalization assay in which endocytosis was allowed to occur for 5-25 min. After immunoprecipitation using an H7 protein-specific antibody, the samples were separated by SDS gel electrophoresis, and endocytosed biotinylated protein was detected after blotting onto nitrocellulose with peroxidase-conjugated streptavidin. In Fig. 4C, quantification of the endocytosis is shown. As described by Lazarovits et al. (28) for the H2 hemagglutinin, the H7 protein was not internalized during 25 min at 37 °C. In contrast, endocytosis of all chimeras could be measured; but none of the proteins was internalized at a rate significantly higher than that of F549Y/A or the bulk of the plasma membrane. Uptake of the H7 chimera containing both the MV F transmembrane and cytoplasmic domains (H7-F-F) was found to be only slightly enhanced in comparison with H7-F-H7 and H7-H7-F. This result indicates that the endocytosis signal in the F protein cytoplasmic tail is not recognized in the H7 protein context even if the transmembrane domain of the F protein is present at the same time.


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Fig. 4.   A, diagram of the chimeric and parental MV F and influenza H7 proteins. MV F protein is shown in gray boxes, and the H7 protein is shown in white boxes. The luminal (LD), transmembrane (TM), and cytoplasmic (CD) domains are not drawn to scale. B, apical/basolateral distribution of the chimeric proteins on the surface of MDCK cells. MDCK cells stably expressing H7, H7-H7-F, H7-F-H7, or H7-F-F were grown on filters and surface-biotinylated from either the apical (lanes a) or basolateral (lanes b) side. Cells were lysed, and proteins were immunoprecipitated with an H7 protein-specific antibody. Precipitates were separated on a 10% SDS gel, blotted onto nitrocellulose, and probed with peroxidase-conjugated streptavidin. C, internalization rate of chimeric proteins. Stably expressing MDCK cells were surface-biotinylated. The internalization assay was performed as described in the legend to Fig. 3. The percentages of endocytosed H7 protein (black-diamond ), H7-H7-F (black-square), H7-F-H7 (), and H7-F-F (black-triangle) are depicted.

Apical Expression of the MV Glycoproteins Prevents Syncytium Formation in Epithelial Cells-- To elucidate the importance of the basolateral sorting of glycoproteins H and F for MV spread, we analyzed whether inactivation of the targeting signal affects the biological activity of MV glycoproteins in polarized MDCK cells. In polarized cell monolayers, only lateral cell membranes can fuse. Therefore, fusogenic proteins must be expressed on the basolateral cell surface to induce fusion (30). Apically expressed proteins are unable to mediate fusion. Thus, we examined whether replacement of the tyrosines in the cytoplasmic tails of H and F proteins affects the syncytium formation in polarized cell monolayers. For this purpose, cells stably expressing either FEdm or F549Y/A were grown to over-confluency. The cells that had already established a polarized phenotype were then transfected either with the parental HEdm protein or with the mutant H12Y/A protein. As a control, MDCK cells were infected with MV. At 7 h post-infection or post-transfection, the culture medium was removed, and cells were grown for 24 h either in normal growth medium or in calcium-depleted medium. In the absence of calcium, tight junctions could not be maintained or formed, resulting in non-polarized cells in which apical and basolateral surfaces were no longer separated. At 30 h post-transfection or post-infection, cells were fixed and stained with an H protein-specific antibody and an FITC-conjugated secondary antibody. Immunofluorescence was used to monitor cell-to-cell fusion since syncytium formation in over-confluent MDCK cell monolayers is difficult to detect by staining with Giemsa staining solution. As expected, all samples demonstrated large syncytia in the absence of calcium (Fig. 5, -Ca panels). In contrast, fusion of polarized cells cultured in normal growth medium (+Ca panels) was observed only when parental HEdm and FEdm proteins were coexpressed either in transfected or infected cells (MV and FEdm+HEdm panels). In polarized MDCK cells expressing a tyrosine mutant in combination with a parental protein (F549Y/A+HEdm and FEdm+H12Y/A panels), fusion was largely prevented. This result indicates that the presence of a basolateral targeting signal in the cytoplasmic tails of H and F proteins has significant consequences for cell-to-cell fusion in polarized cells and thus for the phenotype of MV-infected cells.


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Fig. 5.   Syncytium formation in polarized MDCK cells. Transient coexpression of glycoproteins H and F was achieved either after infection with MV or after transfection. For transfection, confluent MDCK cells stably expressing FEdm were transfected with a plasmid encoding for either the parental HEdm protein (FEdm+HEdm panels) or the mutant H12Y/A protein (FEdm+H12Y/A panels). Cells stably expressing mutant F549Y/A were transfected with DNA encoding for HEdm (F549Y/A+HEdm panels). At 7 h post-transfection or post-infection, culture media were exchanged with either calcium-containing (+Ca panels) or calcium-deficient (-Ca panels) medium. After incubation for 24 h, syncytia were visualized by indirect immunofluorescence using an anti-H protein monoclonal antibody and an FITC-conjugated second antibody.

Endocytosis of the MV Glycoproteins Is Inhibited in Virus-infected Cells-- The basolateral sorting signals were shown to be functional in virus-infected MDCK cells (10). To analyze whether endocytosis signals function also in infected cells, we analyzed the endocytosis of the MV glycoproteins by an antibody uptake experiment as described above. None of the glycoproteins was found to be endocytosed to a detectable amount (Fig. 6A). This observation indicates that internalization of H and F proteins is prevented in infected cells. In contrast, cells cotransfected with plasmids containing HEdm and FEdm showed internalization of both glycoproteins (Fig. 6B). Since internalization was also observed in coexpressing cells having already formed syncytia, lack of endocytosis in infected cells cannot be explained by general cell damage induced by cell fusion. This is further supported by the observation that neither endocytosis of a fluid-phase marker (FITC-dextran) nor endocytosis of a constitutive cellular membrane protein (CD4613F/Y) (25) is affected in virus-infected cells (data not shown).


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Fig. 6.   Endocytosis of MV glycoproteins in infected or coexpressing cells. At 30 h post-infection, MV-infected cells (A) or at 20 h post-transfection, cells transfected with pCG-HEdm and pCG-FEdm (B) were incubated with an H protein-specific (HEdm panels) or an F protein-specific (FEdm panels) antibody at 4 °C. After 60 min, endocytosis was allowed to proceed for 15 min at 37 °C. Surface-bound antibodies were detected by incubation at 4 °C with a rhodamine-conjugated anti-mouse serum (surface panels). Internalized antibodies were stained after permeabilization of the cells with an FITC-conjugated anti-mouse serum (intracellular panels). Arrows indicate syncytia.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have shown here that both MV envelope proteins possess targeting signals that critically depend upon a tyrosine residue in the cytoplasmic tails. Stable expression of the MV glycoproteins in MDCK cells showed that the tyrosine residues are involved both in basolateral transport and in endocytosis. In MV-infected cells, recognition of the sorting signals of F and H proteins is differently regulated. Whereas endocytosis appeared to be abolished, basolateral transport was not substantially prevented; and thus, efficient fusion of polarized epithelia was observed. This suggests that the basolateral signals have biological importance for virus replication in epithelia.

Both MV Glycoproteins Possess Overlapping Signals for Endocytosis and Basolateral Transport-- Most cellular transmembrane proteins are internalized together with the bulk of the plasma membrane; others are destined to be rapidly endocytosed and are concentrated in clathrin-coated pits (31-33). Rapid internalization is known to be mediated by the cytoplasmic domain containing a structural motif that includes one or more aromatic residues, generally a tyrosine. In polarized cells, proteins containing an endocytosis signal are generally transported to the basolateral membrane. Motifs responsible for internalization and polarized targeting appear to share common beta -turn structures with consensus sequences involving a 4-amino acid motif: either YXXØ (where X is any amino acid and Ø is an amino acid with a bulky hydrophobic side chain) or NPXY (for review, see Ref. 26). It has been shown that interaction with specific recognition molecules, the adaptor complexes, leads to a selective recruitment of cargo proteins into either endosomes or lysosomes or to the basolateral cell membrane (Refs. 26 and references therein). Even when basolateral sorting signals that depend on a tyrosine overlap or are collinear with internalization signals, the two sorting processes are sensitive to different characteristics of the sequence (34, 35). Furthermore, the position of the signal in the cytosolic domain and the presence of other signals in this domain may contribute to differential interactions with the adaptor complexes (29). Both MV glycoproteins contain a tyrosine in the cytoplasmic tail. Since the sequences around do not properly fit the YXXØ or NPXY motif, they possess only "degenerated" signals. Recognition of such signals cannot be predicted with confidence. Examples are known of basolateral proteins that appeared to possess a "tyrosine" internalization signal, but that have been shown to function poorly or not at all (3, 34, 36, 37). Thus, signals for polarized transport and endocytosis do not necessarily overlap. Here, we showed that in the MV glycoproteins, they do. Mutation of the critical tyrosines abolished basolateral transport and rapid internalization, suggesting that the sequences around the tyrosines are involved in forming a beta -turn.

Sorting Signals in the F Protein Cytoplasmic Tail Lack Functionality in HA-F Protein Chimeras-- The properties of influenza virus HA completely differ from those of the MV glycoproteins. HA is preferentially expressed on the apical surface of epithelial cells and is internalized 40 times more slowly than is the bulk of the plasma membrane (33). A mutant that is rapidly endocytosed and targeted to the basolateral cell surface could be generated by replacement of cysteine 543 with a tyrosine residue in the short cytoplasmic tail of HA (HA-Y543) (38, 39). Just as the MV glycoprotein sorting signals, the YRIC motif from the HA-Y543 mutant does not follow the YXXØ or NPXY pattern. MV F protein and HA-Y543 have an additional feature in common. In both proteins, the tyrosine responsible for endocytosis and basolateral transport is located at position -5 from the cytoplasmic end of the proteins. Nevertheless, transfer of the F protein cytoplasmic tail with a tyrosine at position -5 to the HA protein (H7-H7-F) resulted neither in efficient endocytosis nor in re-targeting to the basolateral membrane. The lack of functionality cannot be explained by a more degenerated signal (YXXS in H7-H7-F versus YXXC in HA-Y543) since an HA mutant (HA-Y543/S546) with a serine instead of cysteine (YXXS) was endocytosed efficiently (40). This indicates that other information than only a tyrosine in the correct position is required to override the intrinsic properties of HA. HA was only re-targeted by MV F protein sequences when both the transmembrane and cytosolic portions of the F protein were transferred to the luminal portion of HA (H7-F-F). From this it follows that the tyrosine-dependent targeting signal in the F protein cytoplasmic tail either is overridden by a counteracting apical signal in the HA transmembrane domain or requires an additional supporting signal in the transmembrane region of the F protein. This view is supported by the observation that the F protein transmembrane domain alone (H7-F-H7) could not redirect HA to the basolateral surface, although it abolished the strict apical localization of the chimera. In contrast to basolateral transport, the F protein cytosolic portion was unable to mediate efficient endocytosis even in the presence of the F protein transmembrane domain. Thus, we conclude that the internalization signal in the F protein cytoplasmic tail functions only in the context of the authentic protein.

Endocytosis, but Not Basolateral Targeting, of MV Glycoproteins Is Prevented in Virus-infected Cells-- Given the importance of cell-associated viral glycoproteins in mediating cytopathic effects and as targets of host immune responses, endocytosis of MV envelope proteins may be a regulatory mechanism to prevent extensive expression of viral antigen on the cell surface. A likely explanation for the lack of endocytosis in MV-infected cells is that interaction of the H and F protein cytoplasmic tails with the viral matrix protein initiating assembly of new virions (13, 41) prevents the interaction with cellular adaptor complexes. We propose that MV has developed a system that removes those H and F proteins from the cell surface that are not associated with the M protein and are therefore not destined for incorporation into virions. This view is supported by our observation3 that both MV glycoproteins were efficiently endocytosed in cells infected with recombinant MV lacking the M protein (MVDelta M) (12). Similar mechanisms to regulate the expression of viral envelope proteins by interaction with viral core proteins have been proposed for Sendai virus and the human immunodeficiency virus (42, 43). In contrast, glycoproteins from the simian immunodeficiency virus and simian virus 5 have been found to be internalized even in the presence of viral core proteins (44, 45). This points out that the biological relevance of endocytosis signals in viral envelope proteins cannot be predicted, but has to be analyzed in the context of virus infection.

In contrast to endocytosis, we found the basolateral transport of MV glycoproteins not to be severely impaired in infected cells. We have reported previously that both glycoproteins are expressed on the basolateral surface of infected MDCK cells (10). Here we show that cell-to-cell fusion of polarized MDCK cells occurs both in virus-infected cells and in cells coexpressing H and F proteins in the absence of other viral proteins. The ability to mediate fusion critically depends upon intact targeting signals in both proteins. We propose that the presence of H and F proteins on the basolateral surface promotes spread of virus infection from cell to cell. Although it has been recently reported that the M protein can partially re-target the glycoproteins to the apical membrane of polarized CaCo2 cells (11), a sufficient amount of glycoprotein appeared to reach the basolateral membrane to induce fusion.

In summary, MV regulates the recognition of its signals for endocytosis and basolateral targeting by differential interaction with other viral components. In this way, MV glycoproteins can have a tyrosine-dependent endocytosis and sorting signal that permits polarized transport, but prevents internalization of glycoproteins during infection of epithelial cells. As virus release was shown to occur only at the apical cell surface, basolateral glycoprotein expression may be required for MV to spread from the primarily infected epithelia to underlying tissues by direct cell-to-cell fusion. In the absence of viral core proteins, glycoprotein expression on the cell surface is down-regulated by endocytosis, thereby preventing the exposure of viral antigens on the cell surface. Although just one component of the complex biology of the virus, it is clear from the experiments described here that the tyrosine-dependent signals contribute to the cytopathic properties of MV in culture and may also be important for the pathogenesis in vivo.

    ACKNOWLEDGEMENTS

We thank J. and S. Schneider-Schaulies, W. Garten, and R. Cattaneo for kindly providing monoclonal antibodies and cloned MV genes.

    FOOTNOTES

* This work was supported by a grant from the Deutsche Forschungsgemeinschaft (to A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Inst. für Virologie, Robert-Koch-Str. 17, D-35037 Marburg, Germany. Tel.: 49-6421-2865146; Fax: 49-6421-2868962; E-mail: maisner@mailer.uni-marburg.de.

Published, JBC Papers in Press, February 28, 2001, DOI 10.1074/jbc.M010183200

2 P. Riedl, M. Moll, H.-D. Klenk, and A. Maisner, submitted for publication.

3 M. Moll, and A. Maisner, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: MDCK, Madin-Darby canine kidney; HA, influenza virus hemagglutinin; MV, measles virus; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline.

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RESULTS
DISCUSSION
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