©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a 40-kDa Cell Surface Sialoglycoprotein with the Characteristics of a Major Influenza C Virus Receptor in a Madin-Darby Canine Kidney Cell Line (*)

(Received for publication, April 7, 1995; and in revised form, May 25, 1995)

Gert Zimmer (§) Hans-Dieter Klenk Georg Herrler (¶)

From the Institut fr Virologie, Philipps-Universitt Marburg, Robert-Koch-Stra&cjs1648;e 17, D-35037 Marburg, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Infection of cells by influenza C virus is known to be initiated by virus attachment to cell surface glycoconjugates containing N-acetyl-9-O-acetylneuraminic acid. Using an in vitro virus binding assay, we have detected this carbohydrate on several glycoproteins of Madin-Darby canine kidney cells (type I), a polarized epithelial cell line permissive for infection with influenza C virus. Among these proteins, only one was found to be present to a significant extent on the cell surface. This protein, gp40, was characterized as an O-glycosylated (mucin-type) integral membrane protein of 40 kDa, which was predominantly localized on the apical plasma membrane of filter-grown cells. It is a major cell surface sialoglycoprotein in this cell line and was shown to be subject to constitutive and rapid endocytosis. Thus, this glycoprotein can mediate not only the binding of influenza C virus to the cell surface, but also its delivery to endosomes, where penetration occurs by membrane fusion. Other highly sialylated cell surface glycoproteins were also detected but did not mediate influenza C virus binding to a significant extent, indicating that only gp40 contains 9-O-acetylated sialic acids.


INTRODUCTION

Enveloped viruses enter their host cells by attachment to receptor molecules located in the plasma membrane followed by a fusion process between the viral envelope and the cell membrane, either directly at the cell surface or within endosomes after uptake of the viral receptor-virus complex by endocytosis. As an increasing number of virus receptors has been identified in recent years, it turned out that in many instances viruses selectively recognize specific membrane proteins for attachment to the cell surface, which is considered to be a major determinant for their host range and tissue tropism(1) . On the other hand, it has been known for many years that sialic acid is the receptor determinant for influenza viruses and several other viruses. Since this acidic sugar is conjugated to many cellular glycoproteins and glycolipids, influenza viruses in theory have the choice between multiple receptors. However, sialic acids and sialylated oligosaccharides are very diverse in structure, and it has been shown for influenza A and B viruses that pronounced preferences exist with regard to the sialylated oligosaccharide sequences recognized(2) . Another type of specificity has been observed with influenza C virus. In contrast to influenza A and B viruses, for which N-acetylneuraminic acid (Neu5Ac)()is the primary receptor determinant, influenza C virus exclusively binds to a modified form of this sialic acid which is acetylated at position C-9 (Neu5,9Ac)(3, 4) . The receptor binding (hemagglutinating) activity of influenza C virus is a function of the single viral spike glycoprotein HEF, which is also responsible for the receptor destroying and the fusion activities of this virus(5) . The receptor destroying activity has been identified as an acetylesterase which catalyzes the de-O-acetylation of Neu5,9Ac(3, 6) . The inactivation of the receptor determinant may be important for facilitating the elution of progeny virus from infected cells, for preventing self-aggregation of virions, and/or for inactivating competitive inhibitors. Recent data suggest that the acetylesterase may also be involved in virus entry(7) . However, the role of the receptor-destroying enzyme in this process is not understood because the fusion activity of HEF, which is known to be essential for virus penetration(8) , is not dependent on the inactivation of the receptors (9) . Fusion is believed to be due to a hydrophobic amino acid sequence, which for its activation requires the proteolytic cleavage of the glycoprotein into the subunits HEF and HEF, and the exposure to low pH. Because of the dependence on acidic pH, it is assumed that influenza C virus, like influenza A and B viruses, is internalized by cells via receptor-mediated endocytosis. Acidification of the endosomal compartment by a vacuolar ATPase is believed to induce a conformational change in the viral glycoprotein which results in the fusion of the endosomal and the viral membrane. As receptor binding and receptor inactivation are competitive activities, influenza C virus may bind to certain glycoconjugates of the plasma membrane and, if these molecules are not internalized in time, will elute from them without infecting the cell. Thus, aside from proper glycosylation, a further important criterion for a functional influenza C virus receptor is its rapid internalization.

Madin-Darby canine kidney (MDCK) cells are polarized epithelial cells which are widely used in cell biology for the study of protein transport and cell polarity(10) . A subline of these cells (MDCK I), characterized by a high trans-epithelial electrical resistance, is susceptible to influenza C virus infection. In the present study, we analyzed the glycoproteins of MDCK I cells for their ability to mediate influenza C virus binding as well as for their distribution between the cell surface and intracellular compartments. We describe a cellular glycoprotein which is the major surface protein recognized by influenza C virus and which is subject to rapid endocytosis. This glycoprotein has therefore the characteristics expected from an influenza C virus receptor.


EXPERIMENTAL PROCEDURES

Materials

The following reagents were obtained from Boehringer Mannheim GmbH (Mannheim, Germany): proteinase K, N-glycosidase F from Flavobacterium meningosepticum, O-glycosidase from Diplococcus pneumoniae, 2,3-dehydro-2-desoxy-N-acetylneuraminic acid, digoxigenin-3-O-succinyl--amidocaproic acid hydrazide hydrochloride, polyclonal sheep anti-digoxigenin Fab fragments conjugated to alkaline phosphatase, nitro blue tetrazolium chloride (NBT), 5-bromo-4-chloro-3-indolyl phosphate 4-toluidine salt (BCIP). The following reagents were purchased from Sigma (Deisenhofen, Germany): wheat germ agglutinin (WGA)-agarose beads, Jacalin agarose beads, concanavalin A-agarose beads, -naphthyl acetate, 4-chloro-2-methylbenzenediazonium salt (Fast Red TR salt), sialidase from Clostridium perfringens (Type X). Sambucus nigra agglutinin (SNA)-agarose beads were from Medac GmbH (Hamburg, Germany). Lens culinaris agglutinin (LCA)-agarose beads were obtained from Pharmacia LKB (Freiburg, Germany). Sulfo-succinimido-biotin (sulfo-NHS-biotin) and streptavidin-agarose beads were supplied by Pierce Chemical Co. ECL detection reagent and prestained protein molecular weight markers were purchased from Amersham Buchler GmbH (Braunschweig, Germany). Vibrio cholerae sialidase was from Behring AG (Marburg, Germany). PI-specific phospholipase C from Bacillus thuringiensis was obtained from Oxford Glycosystems (Oxford, United Kingdom). Pasteurella haemolyticaO-sialoglycoprotease was from Accurate Chemical & Scientific Co. (Westbury, NY). Stock solutions of BCIP (50 mg/ml in dimethylformamide), NBT (75 mg/ml in 70% dimethylformamide), and -naphthyl acetate (10 mg/ml in acetone) were stored at -20 °C.

Virus

Influenza C virus (strain Johannesburg/1/66) was grown in the allantoic cavity of 8-day-old embryonated chicken eggs. After 3 days at 33 °C, the allantoic fluid was harvested and clarified by low speed centrifugation (1000 g, 15 min, 4 °C). Aliquots were stored frozen at -80 °C. Hemagglutination activity of viruses was determined in microtiter plates. Serial 2-fold virus dilutions of 50 µl each were prepared in phosphate-buffered saline (PBS) and 50 µl of a 0.5% suspension of chicken erythrocytes were added to each dilution. After 60 min at 4 °C, the hemagglutination activity (HA-units/ml) was determined as the reciprocal value of the highest dilution causing complete agglutination.

Cell Culture

MDCK cells (type I) were grown in Eagle`s minimal essential medium supplemented with 10% fetal calf serum. For studies of cell polarity, 0.4-µm pore size polycarbonate Transwell filters (Costar, Cambridge, MA) were used. On the fourth day of culture, the polarization of the monolayer was assessed by measuring the electrical resistance between the apical and the basolateral compartments of the filter chamber using a Millicell-ERS instrument (Millipore). Only filter cultures with an electrical resistance of at least 2000 cm were used for experiments.

Cell Surface Labeling

MDCK I cells were grown to confluence in 94-mm Petri dishes (about 7 10 cells), rinsed with ice-cold phosphate-buffered saline, pH 7.4, containing 1 mM Ca and 1 mM Mg (PBS+), and cooled in this buffer for 10 min at 4 °C. Cell surface proteins were labeled by incubating the cells with sulfo-NHS-biotin (0.5 mg/ml in PBS+, 3 ml/dish) for 30 min at 4 °C with gentle agitation. The monolayers were washed once with ice-cold PBS+ containing 0.1 M glycine and incubated in the same buffer for 15 min at 4 °C. For selective labeling of the apical or basolateral surface of filter-grown cells, the biotinylation reagents were added either to the apical (1.5 ml) or basolateral (2.5 ml) compartment of the filter chamber, while the opposite compartment was incubated with PBS+/0.1 M glycine.

Streptavidin and Lectin Precipitation

Cell monolayers derivatized with biotin were scraped from the Petri dishes into ice-cold PBS, pH 7.4, using a rubber policeman. The cells were pelleted by centrifugation (600 g, 10 min, 4 °C), resuspended in 500 µl of 20 mM Tris-HCl, pH 7.5, and lysed by addition of 500 µl of 2 concentrated RIPA-buffer (2% Triton X-100, 2% deoxycholate, 0.2% SDS, 400 mM NaCl, 40 mM Tris-HCl, pH 7.5, 20 mM iodoacetamide, 2 mM PMSF, 100 units/ml aprotinin). Following incubation on ice for 60 min, insoluble material was removed by centrifugation (105,000 g, 60 min, 4 °C). The cell lysate (1 ml) received 100 µl of a 50% slurry of streptavidin agarose (prewashed three times with RIPA buffer) and was incubated overnight at 4 °C while rotating head over tail. The streptavidin agarose was pelleted by centrifugation (14,000 g, 2 min, 4 °C), washed three times with RIPA buffer and once with 20 mM Tris-HCl, pH 6.8. Precipitated proteins were eluted by heating the streptavidin-agarose in 50 µl of 2 concentrated reducing SDS sample buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 0.02% bromphenol blue, 200 mM dithiothreitol) at 95 °C for 10 min with constant agitation. Isolation of cellular glycoproteins by immobilized lectins was performed in principle like the streptavidin precipitation differing only in the lysis buffer used (final concentration: 1% Triton X-100, 200 mM NaCl, 20 mM Tris-HCl, pH 7.5, 2 µg/ml leupeptin, 2 µg/ml pepstatin). In addition, divalent cations Ca, Mg, and Mn (1 mM each) were added to the lysates prior to overnight incubation with immobilized lectins at 4 °C. Triton lysis buffer without divalent cations was used for washing the beads.

In Vitro Virus Binding Assay

Cell surface proteins isolated by streptavidin precipitation or cellular glycoproteins isolated by lectin precipitation were separated by 10% SDS-PAGE under reducing conditions (11) and transferred to nitrocellulose membranes by electroblotting(12) . To prevent loss of O-acetyl groups, electroblotting was modified by lowering the pH values of the two buffers at the anode side from 10.4 to 9.0 (facing the anode) and 7.4 (facing the nitrocellulose), respectively. Nonspecific binding sites were blocked by incubating the nitrocellulose with PBS containing 1% BSA for 60 min at room temperature. The blots were washed with PBS for 5 min. After removal of the buffer, some drops of virus suspension with a hemagglutinating activity of at least 512 HAU/ml were added and spread over the whole blot by covering it with a piece of parafilm. Virus was allowed to bind for 60 min at 4 °C. The nitrocellulose was washed three times with ice-cold PBS containing 0.05% Tween 20, 5 min each, and incubated with a filtered solution of PBS containing 1 mM -naphthyl acetate and 0.1% (mass/volume) 4-chloro-2-methylbenzenediazonium salt. The esterase reaction was allowed to take place at room temperature and was stopped by rinsing the blots with water. For alkaline hydrolysis of O-acetyl esters, the nitrocellulose membranes were incubated directly after electroblotting with 0.1 M NaOH for 30 min at 4 °C and washed three times with PBS, 5 min each. For detection of glycoproteins the blots were probed with different digoxigenin-labeled lectins as described previously(13) .

Detection of Sialic Acid and Biotin Residues

For selective oxidation of sialic acids, proteins immobilized on nitrocellulose were incubated in the dark for 20 min at 0 °C with 1 mM sodium metaperiodate in 100 mM acetate buffer, pH 5.5. The membranes were washed as above, and oxidized sialic acids were labeled by incubation with digoxigenin-succinyl--amidocaproic acid (1:5000) in 100 mM acetate buffer, pH 5.5, for 60 min at room temperature. The blots were rinsed twice in PBS and washed three times, 10 min each with PBS before incubation with 1% BSA in PBS overnight at 4 °C. After washing the membrane as above, digoxigenin-labeled sialoglycoproteins were detected by incubating the blots with anti-digoxigenin Fab fragments conjugated to alkaline phosphatase (1:1000 in PBS) for 60 min at room temperature. The blots were washed and immersed at room temperature in a solution of 37.5 µl of BCIP and 50 µl of NBT diluted in 10 ml of 100 mM Tris-HCl, pH 9.5, 50 mM MgCl, 100 mM NaCl. The reaction was stopped by rinsing the blots with water. For detection of biotinylated proteins, blots were blocked with 1% BSA in PBS for 60 min at room temperature or overnight at 4 °C. After washing the nitrocellulose three times, 5 min each with PBS, some drops of streptavidin-horseradish peroxidase (1:1000) in PBS were added, the blots covered with a piece of parafilm, and the streptavidin-horseradish peroxidase allowed to bind for 60 min at 4 °C. The blots were rinsed twice with PBS containing 0.1% Tween 20 and washed with this buffer three times for 10 min each. A chemoluminescent substrate (ECL detection reagent, Amersham, Braunschweig) was used for detection of streptavidin-horseradish peroxidase by short exposure to an autoradiography film.

Triton X-114 Phase Separation and Enzyme Treatments

Cell surface biotinylated MDCK I cells (7 10 cells) were extracted with the detergent Triton X-114 followed by temperature-induced phase separation of the extracts(14) . Reextracted aqueous and detergent phases were diluted to 1 ml with Tris-buffered saline (TBS) and subjected to WGA precipitation or streptavidin precipitation. Precipitates were probed with influenza C virus in a ligand blot assay (see above). Phosphatidylinositol-specific phospholipase C treatment of detergent phases with subsequent temperature-induced phase separation was performed as described(14) . For treatment with glycosidases, the biotin-labeled proteins were precipitated from the detergent phase with immobilized streptavidin and were digested with one of the following glycosidases while still bound to the streptavidin-agarose: 100 milliunits of sialidase from V. cholerae (50 mM acetate buffer, pH 5.5, 154 mM NaCl, 9 mM CaCl), 2 units of N-glycosidase F (50 mM phosphate buffer, pH 7.0, 1% Triton X-100, 1 mM 2,3-dehydro-2-deoxy-N-acetylneuraminic acid), 5.7 milliunits of O-glycosidase (cf. buffer for N-glycosidase F). Glycosidases (diluted in 100 µl of buffer) were added to the streptavidin-agarose and incubated in a thermomixer 5436 (Eppendorf) at 37 °C with agitation for 60 min (sialidase) or overnight (N-glycosidase F, O-glycosidase). Of note, all glycosidase treatments were performed in the presence of leupeptin (1 µg/ml), pepstatin (1 µg/ml), and PMSF (1 mM). The streptavidin-agarose was washed and the biotinylated proteins eluted as described above (see the previous section on streptavidin and lectin precipitation). The eluted proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose, and probed with influenza C virus or streptavidin-horseradish peroxidase (see previous sections). Polyacrylamide gels were silver stained according to the methods of Merril et al.(15, 16) using two commercially available kits (Silver Stain Kit, Silver Stain Plus Kit, Bio-Rad, Mnchen). Desialylation of glycoproteins transferred to nitrocellulose was performed after blocking the membrane with 1% BSA in PBS (60 min, room temperature) by adding some drops of V. cholerae sialidase (1 units/ml, buffer as above) to the blot and covering it with a piece of parafilm. Sialidase was allowed to act for 2 h at 37 °C in a humid atmosphere followed by three wash steps with PBS, 5 min each. For P. haemolyticaO-sialoglycoprotease treatment, biotinylated MDCK I monolayers (4 10 cells) were incubated for 60 min at 37 °C with 1 ml of PBS+ in the presence or absence of the enzyme (25 µl). After washing the cells three times with ice-cold PBS+, they were lysed and subjected to streptavidin precipitation (see above). The precipitated cell surface proteins were analyzed by the in vitro virus binding assay and streptavidin-horseradish peroxidase binding (see above).

Endocytosis Assay

Surface-labeled cells were incubated with PBS+ at 37 °C. At different times, cells were rinsed with ice-cold PBS, and cell surface proteins were digested with 1 mg/ml proteinase K in PBS containing 5 mM EDTA at 4 °C for 45 min. The digestion was stopped by addition of 2 mM PMSF. Protease-resistant, biotinylated proteins were precipitated from cell lysates with immobilized streptavidin and internalized influenza C virus receptors were detected by the in vitro virus binding assay described above. Quantitation was performed by densitometric scanning of the blots.

Kinetics of Virus Internalization

MDCK I cell monolayers (1-2 10 cells) were washed twice with ice-cold PBS+ and influenza C virus (about 5 HAU/ml in PBS+) was allowed to bind to the monolayers for 60 min at 4 °C. Virus not adsorbed to the cells was washed away with ice-cold PBS+. The monolayers were incubated with medium at 37 °C for different time periods after which the cells were rinsed with ice-cold PBS+. Virus that had not been internalized was neutralized at the cell surface by a polyclonal antiserum (1:100 in PBS+) for 60 min at 4 °C. The monolayers were washed three times with ice-cold PBS+ and incubated with 2 ml of medium without fetal calf serum at 33 °C. After 24 and 48 h, 100 µl of medium was removed and virus released in the supernatant titrated by a hemagglutination assay (see above).


RESULTS

The Receptor Determinant Neu5,9Ac Is Present on Several MDCK I Glycoproteins

We analyzed the glycoproteins of MDCK I cells for their ability to mediate influenza C virus attachment. Glycoproteins were precipitated from cell lysates using immobilized lectins of different specificity. ConA preferentially interacts with N-linked oligosaccharides of the high mannose and hybrid type, weakly with bi-antennary complex structures, and not with tri- and tetra-antennary complex oligosaccharides(17, 18) . Recognition by lentil lectin (LCA) requires both the presence of two -linked mannose residues and the presence of fucose attached to the Asn-linked GlcNAc. Exposure of terminal GlcNAc residues enhances the binding(19) . Jacalin recognizes O-glycans of the sequence Gal1-3GalNAc-Ser/Thr. This sequence with an additional sialic acid residue 2,3-linked to Gal is also recognized(20) . WGA shows affinity for GlcNAc and sialic acid residues(21) , and SNA specifically binds to sialic acids 2,6-linked to Gal(22) . The precipitated glycoproteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with influenza C virus. The viral acetylesterase activity was used to directly visualize receptor-bound virions with a chromogenic esterase substrate. As shown in Fig. 1, influenza C virus recognized a different number of glycoproteins depending on the lectin used for precipitation. A strong binding was mediated by a cluster of glycoproteins in the 38-55 kDa range precipitated by both WGA and Jacalin. Glycoproteins precipitated by the other lectins were only poorly recognized by influenza C virus with the exception of a 50-55-kDa glycoprotein precipitated by LCA.


Figure 1: Binding of influenza C virus to MDCK I glycoproteins. Glycoproteins were precipitated from MDCK I cell lysates by the immobilized lectins indicated on top of the lanes. The glycoproteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with influenza C virus. Bound virus was detected using a chromogenic esterase substrate, which was cleaved by the viral O-acetylesterase.



Because of its ability to precipitate a great number of glycoproteins which are recognized by influenza C virus, we used WGA to further study the specificity of the viral interaction with cellular proteins. Fig. 2A shows that influenza C virus binding to these glycoproteins was abolished when the blots were pretreated with sialidase (lane c) or with sodium hydroxide (lane d). The latter treatment hydrolyzes ester linkages such as the 9-O-acetyl ester of sialic acids. As a further control, we also tested glycoproteins from influenza C virus-infected cells. The viral glycoprotein is synthesized at the rough endoplasmatic reticulum and is transported to the plasma membrane via the constitutive secretory pathway. On the way to the cell surface, it passes the Golgi compartment where the cellular O-acetyltransferase is thought to be located(23, 24) . Therefore, HEF has the chance to de-O-acetylate both intracellular and surface sialoglycoproteins with the consequence that influenza C virus cannot bind to them in the ligand blot assay (lane b). For detection of sialoglycoproteins, a parallel blot was subjected to mild metaperiodate oxidation. The oxidized sialic acids were labeled with digoxigenin and detected by anti-digoxigenin Fab fragments conjugated to alkaline phosphatase (Fig. 2B, lane a). De-O-acetylation of sialic acids either by acetylesterase activity during influenza C virus infection (lane b) or by alkaline treatment of the blots (lane d) did not impair the reaction, whereas sialidase treatment abolished the detection of glycoproteins (lane c). These findings demonstrate that influenza C virus binds to MDCK I glycoproteins via Neu5,9Ac residues.


Figure 2: Analysis of the influenza C virus binding specificity. MDCK I cells were used either as non-infected cells (lanes a, c, and d) or 20 h after infection with influenza C virus (lanes b). Glycoproteins were precipitated from cell lysates by immobilized WGA, electrophoresed on polyacrylamide gels, and blotted to nitrocellulose membranes. The nitrocellulose was cut into strips that were incubated with PBS (lanes a and b), sialidase (lanes c), or 0.1 M NaOH (lanes d) as described under ``Experimental Procedures.'' The nitrocellulose strips were probed with influenza C virus (A) or subjected to mild metaperiodate oxidation (B). Oxidized sialic acid residues were labeled with digoxigenin and detected using anti-digoxigenin Fab fragments conjugated to alkaline phosphatase.



A 40-kDa Glycoprotein Is the Major Cell Surface Protein Recognized by Influenza C Virus

An important criterion for a functional virus receptor is its location on the cell surface. Therefore, we analyzed the distribution of O-acetylated sialoglycoproteins between the plasma membrane and intracellular compartments by determining their sensitivity to cell surface digestion by sialidase at 37 °C (Fig. 3). Comparison with the untreated control (lane a) indicates that many glycoproteins were protected from desialylation (lane b). Binding of influenza C virus to only a few glycoproteins was diminished by the sialidase treatment, the major band being a protein of 40 kDa. To confirm this result, we labeled cell surface proteins at 4 °C using a water-soluble, membrane-impermeable sulfonated N-hydroxysuccinimide ester of biotin. The biotinylated proteins were specifically precipitated from cell lysates by immobilized streptavidin. Among the O-acetylated sialoglycoproteins only the 40-kDa glycoprotein was recovered by this procedure to a significant extent (lane c). When cell surface labeling was followed by sialidase treatment, the recognition of this protein by influenza C virus was totally abolished (lane d). We repeated the experiment using proteinase K at 4 °C instead of sialidase at 37 °C to allow effective digestion of cell surface proteins at a temperature, at which endocytosis is arrested. The result obtained with this approach (not shown) was the same as that shown in Fig. 3. The most prominent glycoprotein recognized by influenza C virus was a 40-kDa glycoprotein which mainly resided at the plasma membrane, whereas many other O-acetylated sialoglycoproteins were predominantly found in intracellular compartments. The 40-kDa cell surface sialoglycoprotein was designated gp40.


Figure 3: Cellular distribution of 9-O-acetylated sialoglycoproteins in MDCK I cells. Confluent MDCK I monolayers were surface biotinylated at 4 °C. A, the monolayers were either incubated with MES puffer (lanes a and c) or treated with C. perfringens sialidase (300 milliunits/ml in MES puffer) for 60 min at 37 °C (lanes b and d). After cell lysis, glycoproteins were precipitated either by immobilized WGA (lanes a and b) or by immobilized streptavidin (lanes c and d). After SDS-PAGE and transfer to nitrocellulose, the precipitated proteins were probed with influenza C virus. The position of gp40, the major cell surface glycoprotein recognized by influenza C virus is indicated.



For detection of major cell surface sialoglycoproteins irrespective of O-acetylation, Western blots of biotin-labeled and streptavidin-precipitated cell surface proteins were subjected to mild metaperiodate oxidation followed by labeling of the oxidized sialic acids with digoxigenin. The reaction with an antibody directed to digoxigenin revealed the presence of several cell surface sialoglycoproteins including gp40 (Fig. 4A, lane a). Only the latter was recognized to a significant extent when the Western blots were probed with influenza C virus (lane b). De-O-acetylation of sialic acids by NaOH pretreatment abolished the virus binding but did not alter the antibody reaction (not shown; see also Fig. 2B for comparison). The lectin from Arachis hypogea (PNA) exhibits a stringent specificity for the sequence Gal1-3GalNAc on O-glycans and does not tolerate substitution of Gal or GalNAc by sialic acid(25) . Fig. 4B shows that PNA did not react with any cell surface glycoprotein (lane a) unless they were pretreated with sialidase (lane b). Desialylation did not markedly change the mobility of gp40 on SDS-PAGE as verified by staining with streptavidin-horseradish peroxidase (cf.Fig. 7). We found that among several O-glycosylated cell surface proteins only gp40 efficiently mediated influenza C virus binding (lane c).


Figure 4: Detection of major MDCK I cell surface sialoglycoproteins. Confluent MDCK I monolayers were surface biotinylated, and the labeled proteins were precipitated from cell lysates by immobilized streptavidin. A, the precipitated proteins were separated by SDS-PAGE, blotted to nitrocellulose, and either subjected to mild metaperiodate oxidation with subsequent labeling and detection of sialic acid residues via an enzyme-linked immunoassay (lane a) or probed with influenza C virus (lane b). B, Precipitated proteins were either incubated with acetate buffer (lanes a and c) or treated with V. cholerae sialidase (lanes b and d) as described under ``Experimental Procedures.'' Western blots of the precipitates were either probed with PNA (lanes a and b) or influenza C virus (lanes c and d) as described under ``Experimental Procedures.'' The position of gp40 is indicated.




Figure 7: Effect of glycosidases on gp40. Confluent MDCK I monolayers were surface biotinylated and extracted with the detergent Triton X-114. After temperature-induced phase separation, the labeled proteins were precipitated from the detergent phase with immobilized streptavidin. The precipitates were treated with buffer (lanes a), V. cholerae sialidase (lanes b), N-glycosidase F (lanes c), sialidase + N-glycosidase F (lanes d), O-glycosidase (lanes e), sialidase + O-glycosidase (lanes f) as described under ``Experimental Procedures.'' The digested samples were separated by SDS-PAGE and transferred to nitrocellulose. A, the blots were probed with influenza C virus. B, biotinylated proteins were visualized by strepavidin-horseradish peroxidase. The arrows mark the undigested and digested forms of gp40. The 35 kDa band in lanes a, c, and e is not related to gp40 because its molecular mass was reduced after sialidase treatment and it was not recognized by influenza C virus.



Surface Distribution of gp40 on Polarized Cells

MDCK cells have provided a well characterized in vitro model system for the study of cell polarity(10) . Polarized cells are characterized by two distinct plasma membrane domains facing different physiological compartments. The apical and basolateral membrane are separated by junctional complexes and differ from each other in their protein and lipid composition. The epithelial organization found in vivo can be mimicked in culture when the cells are grown on permeable polycarbonate filters. Using this system, influenza C virus was able to infect the cells from either membrane domain, but infection was more efficient when virus was applied to the apical compartment (Table 1). Progeny virus, titrated by its hemagglutination activity, was only detected in the apical medium indicating that maturation and budding of influenza C virus is a polar process as has been reported(26) . To determine the distribution of gp40 between the apical and basolateral plasma membrane, filter-grown cells were selectively biotinylated at either domain. The labeled proteins were recovered by streptavidin precipitation, and gp40 was detected by the virus binding assay (Fig. 5). Using this approach, gp40 was labeled at either domain, but a larger amount was recovered from the apical (lane c) than from the basolateral (lane d) plasma membrane. As a control for the equal labeling efficiency on both plasma membrane domains, the biotinylated apical and basolateral proteins were stained in parallel with streptavidin-horseradish peroxidase complex (lanes a and b). The domain-specific distribution of many other proteins demonstrates the high polarity of filter-grown MDCK I cells.




Figure 5: Distribution of gp40 between the apical and basolateral plasma membrane of MDCK I cells. Filter-grown MDCK I monolayers were surface labeled by addition of sulfo-NHS-biotin to either the apical (lanes a and c) or basolateral (lanes b and d) compartment of the filter chamber. The biotinylated proteins were recovered from cell lysates by streptavidin precipitation, run on SDS-PAGE, and blotted to nitrocellulose. Biotinylated proteins were detected with streptavidin-horseradish peroxidase (lanes a and b). After incubation of the blots with influenza C virus, bound virions were visualized by esterase activity (lanes c and d). The position of gp40 is indicated.



Gp40 Is an Integral Membrane Protein

Triton X-114 phase separation (27) was used to study the association of gp40 with the plasma membrane. In Fig. 6the binding of influenza C virus to sialoglycoproteins precipitated from the resulting detergent and aqueous phases with immobilized WGA is shown. The majority of proteins recognized by influenza C virus was recovered from the aqueous phase (lane b) with the exception of a 40-kDa protein which was enriched in the detergent phase (lane a). Treatment of the detergent phase with phosphatidylinositol-specific phospholipase C with subsequent phase separation did not release this glycoprotein into the aqueous phase (not shown) indicating that it is not attached to the plasma membrane via a glycosylphosphatidylinositol anchor. The 40-kDa protein could be biotinylated at the cell surface and precipitated from the detergent phase with immobilized streptavidin (lane c) confirming its identity with gp40. Influenza C virus bound also to some cell surface proteins that were precipitated from the aqueous phase but to a much lesser extent (lane d). The hydrophobic nature of gp40 suggests that this cell surface protein contains one or more membrane-spanning segments.


Figure 6: Analysis of the membrane association of 9-O-acetylated sialoglycoproteins. Confluent MDCK I monolayers were surface biotinylated and extracted with the detergent Triton X-114. After temperature-induced phase separation the resulting detergent phases (lanes a and c) and aqueous phases (lanes b and d) were either subjected to precipitation with immobilized WGA (lanes a and b) or immobilized streptavidin (lanes c and d). The precipitates were run on 8% polyacrylamide gels, transferred to nitrocellulose, and probed with influenza C virus. The position of gp40 is indicated.



Gp40 Is a Mucin-type Glycoprotein

We have shown above that gp40 is a major cell surface sialoglycoprotein of MDCK I cells, which is bound by Jacalin and PNA, lectins specific for O-glycans. Despite its sialylation, gp40 was not recognized by Maackia amurensis-agglutinin or SNA when Western blots of cell surface proteins were probed with these two sialic acid-specific lectins (not shown). To understand this reactivity and to gain a more detailed insight into the glycosylation of this protein, we analyzed the susceptibility of gp40 to different exo- and endoglycosidases. Biotin-labeled cell surface proteins were precipitated with immobilized streptavidin and subjected to digestion by sialidase and two endoglycosidases. Gp40 was detected on Western blots either by virus binding (Fig. 7A) or by streptavidin-horseradish peroxidase complex (Fig. 7B). Comparison with the untreated control (lanes a) shows that desialylated gp40 (lanes b) was not recognized by influenza C virus though it did not show a markedly altered mobility on SDS-PAGE. The endoglycosidases used were N-glycosidase F, which cleaves all classes of N-linked carbohydrate chains at the peptide-carbohydrate linkage(28) , and O-glycosidase, which exhibits a stringent specificity for the disaccharide core structure Gal1-3GalNAc cleaving the linkage between GalNAc and Ser or Thr (29) . These enzymes had no effect both on virus binding to gp40 and on the electrophoretic mobility (lanes c and e). As O-glycosidase is known not to tolerate any substitution on Gal or on GalNAc, gp40 was desialylated prior to incubation with O-glycosidase treatment. After this treatment, gp40 migrated as two bands of about 35 and 37 kDa (Fig. 7B, lane f). Only the 37 kDa band was observed when gp40 was treated with O-glycosidase alone, without addition of a sialidase inhibitor (not shown; the O-glycosidase preparation contained a low sialidase activity, which could be inhibited by addition of 2,3-dehydro-2-desoxy-N-acetylneuraminic acid). The 37 kDa band is, therefore, thought to be a partly desialylated form of gp40 that was not completely digested by the O-glycosidase. Desialylation of gp40 did not improve its susceptibility to N-glycosidase F (Fig. 7B, lane d). This result indicates the presence of sialylated O-linked oligosaccharides of the sequence Gal1-3GalNAc. Gp40 did not react with silver stains of the diamine type(16) , but intensely stained yellow to brown when a silver-staining procedure adapted from photographic chemical development processes was used(15) , which is known to stain highly O-glycosylated sialoglycoproteins(30) . After treatment of gp40 with sialidase and O-glycosidase, the resulting 35 kDa band was still visualized by this silver-staining procedure, indicating that it still contained O-glycan sequences (not shown).

The O-sialoglycoprotease from Pasteurella haemolytica has been shown to specifically cleave mucin-type sialoglycoproteins(31, 32) . As shown in Fig. 8, treatment of biotinylated MDCK I cells with this enzyme resulted in a drastically reduced binding of influenza C virus to gp40 (compare lane c with d). Parallel detection of the biotin groups by streptavidin-horseradish peroxidase shows that only gp40 but not other cell surface proteins had been proteolytically cleaved (lanes a and b, longer exposure time; lanes a` and b`, shorter exposure time). Two bands in the range of 22 kDa became visible following O-sialoglycoprotease treatment (lane b) and are thought to represent proteolytic fragments of gp40. Part of gp40 escaped digestion by O-sialoglycoprotease possibly due to internalization (see below). Taken together, these results indicate that gp40 has characteristics typical for mucin-type glycoproteins.


Figure 8: Effect of P. haemolytica O-sialoglycoprotease on gp40. Surface biotinylated, confluent MDCK I monolayers were treated with either PBS+ (lanes a and c) or O-sialoglycoprotease in PBS+ (lanes b and d) for 60 min at 37 °C. The biotinylated proteins were precipitated from the cell lysates with immobilized streptavidin. Western blots of the precipitates were probed with either streptavidin-horseradish peroxidase (lanes a and b) or influenza C virus (lanes c and d). The exposure times for chemoluminescent detection were 30 s for lanes a and b and 15 s for lanes a` and b`.



Gp40 Is Subject to Rapid and Constitutive Endocytosis

Influenza C virus as well as other influenza viruses require the exposure to low pH to display fusion activity(33, 34, 35) . As this requirement is met by the acidic milieu of secondary endosomes, influenza viruses are thought to enter host cells by receptor-mediated endocytosis. To understand the dynamics of this process, we first allowed influenza C virus to bind to MDCK I monolayers for 1 h at 4 °C, a temperature at which endocytosis is arrested. The cells were then incubated at 37 °C to allow endocytosis to take place. After various times the process was stopped by putting the cells on ice. Virus which had not been internalized was inactivated by a neutralizing antiserum. Internalized virus could escape neutralization and initiate productive infection. Progeny virus released into the medium was titrated 24 and 48 h post-infection using a hemagglutination assay (Fig. 9). Some internalization of infectious virus already occurred during the first 10 min after raising the temperature to 37 °C and increased as a function of time. We assume that internalization of influenza C virus is mediated by a receptor which follows a similar endocytosis kinetics. To test whether gp40 meets this criterion, MDCK I monolayers were surface labeled with biotin at 4 °C. After an incubation at 33 or 37 °C for 45 min, the cells were rapidly cooled, and gp40 still exposed at the surface was digested with proteinase K at 4 °C. The virus binding assay of Fig. 10A shows that gp40, that had been internalized during the 33 or 37 °C chase and thereby had acquired resistance to exogenous protease treatment, could be recovered by streptavidin precipitation, whereas surface-labeled gp40 was completely digested, when the cells were kept at 4 °C. The kinetics of gp40 internalization at 37 °C (Fig. 10B) shows that uptake of gp40 could already be detected 10 min after raising the temperature and reached a maximum at 40 min. With a longer incubation time, a decrease of protected gp40 was observed which may be the result of intracellular degradation or of a recycling process back to the plasma membrane.


Figure 9: Kinetics of influenza C virus internalization. Influenza C virus was allowed to bind to MDCK I monolayers (10 cells) for 60 min at 4 °C. The cells were incubated at 37 °C for the indicated time periods after which the cells were rapidly chilled and treated with a neutralizing antiserum directed against influenza C virus for 60 min at 4 °C. After 24 h () and 48 h () at 33 °C, the virus yield was titrated by measuring the hemagglutination activity of the cell supernatant.




Figure 10: Endocytosis of surface-labeled gp40. A, confluent MDCK I monolayers were labeled at 4 °C with sulfo-NHS-biotin and incubated for 45 min at the indicated temperatures. After cell surface digestion with proteinase K at 4 °C, protease-resistant biotinylated proteins were precipitated from cell lysates with immobilized streptavidin, electrophoresed on polyacrylamide gels, transferred to nitrocellulose, and probed with influenza C virus. Control cells (first lane) were kept at 4 °C and were not digested with protease. B, time course of gp40 internalization. The endocytosis assays was performed at 37 °C for the time intervals indicated and quantitated by densitometric scanning of the Western blots.




DISCUSSION

A great number of viruses including influenza viruses, paramyxoviruses, coronaviruses, polyomaviruses, rotaviruses, and reoviruses are known to require sialic acid as a crucial part of cellular receptors for attachment to cell surfaces(1) . For some influenza viruses, the type of sialic acid and the structure of the oligosaccharide backbone required for optimal binding has been elucidated(2, 4, 36) . On the other hand, the glycoconjugates on the surface of permissive cell lines that mediate virus attachment and virus infection have not been identified. We have previously shown that MDCK cells (type I) are highly susceptible to infection by influenza C virus but are rendered resistant to infection by pretreatment with sialidase(37) . The susceptibility to infection is entirely restored after resialylation of the cells with Neu5,9Ac, whereas other types of sialic acids transferred are ineffective in this respect (38, 39) . These results demonstrate that influenza C virus uses Neu5,9Ac as a receptor determinant to initiate the infection of MDCK I cells.

In this study, we demonstrated the direct binding of influenza C virus to MDCK I glycoproteins by probing Western blots with intact virions. Lectin precipitation resulted in specific enrichment of cellular glycoproteins that facilitated the detection of virus binding. Among the various glycoproteins recognized by influenza C virus one glycoprotein was remarkable for two properties not shared by the others. It was the major protein expressed at the cell surface, and it showed the characteristics of an integral membrane protein. Because of its apparent molecular mass of 40 kDa, this glycoprotein has been designated gp40. It is present at both plasma membrane domains of filter grown cells with a stronger apical expression. Our finding that gp40 is subject to rapid endocytosis is most important because it shows that gp40 is able to exhibit a dual receptor function. It can bring about the binding of influenza C virus to the cell surface and mediate its delivery to the endosomal compartment, which provides the acidic milieu required for the fusion of the viral envelope with the cellular membrane. A critical point in this context is the kinetics of internalization because infection of a cell by influenza viruses will only be successful, if virus internalization proceeds more rapidly than the elution of the virus from the receptor caused by the receptor-destroying enzyme. Endocytosis of gp40 has been shown to be a very rapid process with a kinetics similar to that of virus internalization. The rapid endocytosis of various known plasma membrane receptors, for example those for transferrin, low density lipoprotein, and asialoglycoproteins, is based on a common mechanism(40, 41, 42, 43) : adaptor proteins (adaptins) recognize a short sequence in the cytoplasmic tail of these receptors and induce the formation of a clathrin coat. In this way, the receptors are preferentially concentrated in coated pits, which readily invaginate and pinch off the membrane, while other plasma membrane proteins lacking an endocytosis signal are excluded from the clathrin-coated pits and will internalize only slowly(44, 45) . Further studies have to show if this mechanism is also responsible for the rapid endocytosis of gp40.

A surprising finding of our work was that influenza C virus preferentially binds to a single cell surface glycoprotein. The reason for this might be that gp40 is expressed in larger amounts or that it is sialylated to a larger extent than are other cell surface glycoproteins. We have found that gp40 is a major sialoglycoprotein of the MDCK I plasma membrane. However, we detected also other prominent sialoglycoproteins that did not mediate virus binding. Thus, the quantitative aspect alone is not sufficient to explain the selectivity of virus binding. In spite of its sialylation, removal of sialic acids did not result in a shift to a lower molecular weight. This unusual behavior is typically seen in heavily O-glycosylated sialoglycoproteins(46) . In agreement with these observations, gp40 was found to be susceptible to P. haemolyticaO-sialoglycoprotease, a neutral metalloprotease that has been shown to specifically cleave mucin-type sialoglycoproteins like glycophorin, sialophorin (CD43), or CD34(31, 32) . In addition, the lectins M. amurensis-agglutinin and SNA that recognize sialic acids on N-glycans but not on O-glycans(13, 47) , failed to bind to gp40. Other characteristics of mucin-type sialoglycoproteins, like the predominance of O-glycosylation compared to N-glycosylation and the weak reactivity with protein stains were also found with gp40 indicating that this protein is a mucin-like glycoprotein. Secreted mucins, e.g. ovomucin, have been known for a long time as potent inhibitors of hemagglutination by influenza viruses(48, 49) . The macromolecular structure of these compounds is crucial since the tryptic glycopeptides of mucins or small sialyloligosaccharides had no inhibitory effect(49) . The binding of influenza viruses to erythrocytes is mediated by glycophorin, another mucin-type glycoprotein. In contrast to most soluble mucins which have molecular masses of often several hundred kDa, glycophorin resembles gp40 in being a small integral glycoprotein of about 31 kDa. Typical for mucins is the high level of O-glycosylation at clusters of serine- and threonine-rich sequences(50) . The clustering of the O-linked carbohydrates may be favorable for a polyvalent interaction and thus enhance the binding avidity of influenza C virus. Interestingly, the preferential binding of simian rotaviruses to O-linked sialoglycoconjugates and mucins is also thought to be due to clustering of sialic acids(51) .

O-Acetylated sialic acids have been detected in quite diverse types of glycoconjugates including gangliosides, mucins, serum glycoproteins, and membrane proteins(52) . For example, rat serum glycoproteins and bovine submandibular mucin, which both have a high content of Neu5,9Ac, very efficiently inhibit the hemagglutination activity of influenza C virus(3) . This suggests that the structural features of the oligosaccharide backbone may only be of minor importance for the recognition of Neu5,9Ac by influenza C virus. This conclusion is supported by the finding that asialo-erythrocytes which have been resialylated to contain Neu5,9Ac in three different defined oligosaccharide sequences were agglutinated equally well by influenza C virus(4) . On the other hand, 9-O-acetylation of sialic acid shows remarkably tissue-specific and developmentally regulated expression in a variety of systems(53) . In addition, certain types of sialoglycoconjugates have been found to be predominantly O-acetylated in a given cell type(54, 55) . Therefore, it is likely that the sialic acids of gp40 are selectively O-acetylated rendering this glycoprotein a preferred ligand for influenza C virus. From the observation that gp40 was easily oxidized by metaperiodate, we conclude that only part of its sialic acids are 9-O-acetylated because this substitution is known to render sialic acid rather resistant to periodate oxidation(52) . This agrees with the finding that de-O-acetylation prior to oxidation did not markedly improve the reaction as would be expected if most sialic acids were 9-O-acetylated. Perhaps 9-O-acetylation is restricted to certain sialic acid residues of gp40. Interestingly, structural studies of different O-acetylated gangliosides have shown that in almost all cases the 9-O-acetyl group is located on a specific terminal 2,8-linked sialic acid(53) . Future work has to show what factors are responsible for the preferential O-acetylation of gp40.

We have previously shown that the lack of appropriate receptors on the cell surface is a major reason for the restricted cell tropism of influenza C virus(37) . However, it is not understood whether this phenomenon is due to a general low level of 9-O-acetylation in these cells or due to lack of an appropriate cell surface molecule capable of mediating both efficient binding and internalization of influenza C virus. To our knowledge, gp40 is the first defined physiological receptor glycoprotein of an influenza virus. Correlating cell type-specific expression of gp40 with the ability of cells to support influenza C virus infection should help to evaluate the role of gp40 and of O-acetyltransferase activity in the cell tropism of this virus. It would be interesting to see if recombinant expression of gp40 in a non-permissive cell line will be sufficient to render these cells permissive. As various coronaviruses have been shown to use 9-O-acetylated sialic acids for attachment to cell surfaces (56, 57) , gp40 may be a promising receptor candidate also for these viruses.


FOOTNOTES

*
This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 286. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
This work was conducted in partial fulfillment of the requirements for the Dr. rer. nat. degree from FB17, Philipps-Universitt Marburg.

To whom correspondence should be addressed: Institut fr Virologie, Philipps-Universitt Marburg, Robert-Koch-Str. 17, D-35037 Marburg, Germany. Tel.: 06421-285360; Fax: 06421-285482.

The abbreviations used are: Neu5Ac, N-acetylneuraminic acid; BCIP, 5-bromo-4-chloro-3-indolyl phosphate 4-toluidine salt; BSA, bovine serum albumine; HAU, hemagglutinating units; NBT, nitro blue tetrazolium chloride; Neu5,9Ac, N-acetyl-9-O-acetylneuraminic acid; LCA, Lens culinaris agglutinin; MDCK, Madin-Darby canine kidney; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonylfluoride; PNA, peanut agglutinin; SNA, Sambucus nigra agglutinin; WGA, wheat germ agglutinin.


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