Preparation of recoated cores.

Recoated cores were prepared from purified cores and insect cell lysates containing T1L μ1 and T1L σ3, with or without T1L or T3D σ1, as previously described (11, 12). Concentrations of the purified particles were determined by densitometry of Coomassie blue-stained gels (11). The recoated cores were biotinylated as described above.

Lectins and antibodies.

The lectins and anticarbohydrate antibodies used in this study and their binding specificities are listed in Table ​Table1.1. Fluorescein isothiocyanate (FITC)-labeled lectins included UEA-I, SNA, EEA, PNA, and MAL-I from Vector Laboratories (Burlingame, Calif.) and AAA and LFA from EY Laboratories (San Mateo, Calif.). Tetramethyl rhodamine isothiocyanate (TRITC)-labeled RCA-I, unconjugated MAL-II, and biotinylated MAL-II were purchased from Vector Laboratories. Unconjugated MAL-II was labeled with TRITC (Pierce) according to the supplier's instructions. Mouse IgG1 and IgM MAbs specific for sialyl Lewis^a and sialyl Lewis^x antigens, respectively, were purchased from Kamiya Biomedical (Thousand Oaks, Calif.). The mouse IgG1 MAb specific for vimentin was purchased from DAKO Corp. (Carpinteria, Calif.). FITC-conjugated goat anti-mouse IgG was obtained from Jackson ImmunoResearch Laboratories (West Grove, Pa.). FITC-conjugated goat anti-mouse IgM was purchased from Kirkegaard and Perry Laboratories (Gaithersburg, Md.). Anti-JAM1 (human) MAb J10.4 was kindly provided by Charles Parkos (Emory University, Atlanta, Ga.).

Binding of biotinylated virions to mouse intestinal M cells in vivo.

Adult female BALB/c mice (Charles River Laboratories, Wilmington, Mass.) were anesthetized with an intraperitoneal injection of Avertin (250 mg/kg of body weight) (Aldrich, Milwaukee, Wis.). A loop of distal small intestine was exposed, a 1- to 2-cm segment including a Peyer's patch was ligated and inoculated with 4 × 10¹¹ biotinylated virus particles, and the intestine was returned to the abdomen for 30 min. Mice were sacrificed by cervical dislocation, and the inoculated Peyer's patch was excised, rinsed in cold PBS, pH 7.3, fixed, and embedded in Epon-Araldite as previously described (26). Trimmed blocks were sectioned at 1 μm and stained with 1% toluidine blue to verify the presence of the follicle-associated epithelium (FAE) and M cells. To visualize bound reovirus particles, Epon was dissolved (41) and sections were treated with 50 mM NH₄Cl in PBS to quench reactive aldehyde groups and with PBS containing 0.2% gelatin (PBS-gelatin) to block nonspecific protein binding sites. To label viral particles and M cells, sections were overlaid with a solution containing 10 μg of FITC-conjugated UEA-I/ml and 0.5 μg of streptavidin-TRITC/ml (Molecular Probes, Eugene, Oreg.) in PBS-gelatin and incubated for 45 min at RT in a humidified chamber. Slides were washed, and coverslips were mounted with Mowiol solution (0.1 g of Mowiol 4-88 [Calbiochem] per ml, 0.5 g of glycerol per ml, and 10 mg of diazabicyclo[2.2.2]octane [Sigma] per ml in 0.1 M Tris-HCl, pH 8.5) as previously described (39). Photography was performed with an Axiophot microscope (Carl Zeiss, Thornwood, N.Y.) equipped for epifluorescence with T-Max 400 or Ektachrome 400 film (Eastman Kodak, Hollywood, Calif.).

Reovirus binding on rabbit Peyer's patch mucosal explants.

Peyer's patch tissue was obtained from female New Zealand White rabbits weighing 1.4 to 3.8 kg (Charles River Laboratories) as previously described (39). Mucosal tissue samples were washed with Dulbecco's minimal essential medium (DMEM), covered with DMEM containing biotinylated virions or ISVPs (2 × 10¹¹ to 3 × 10¹¹ particles) in 200 μl of DMEM containing 10% fetal calf serum and protease inhibitors [aprotinin (1 μg/ml; Sigma), leupeptin (5 μg/ml; Sigma), 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF; 48 μg/ml; Calbiochem), and bestatin (1 μg/ml; Sigma)], and incubated for 40 min at RT. For experiments using fixed tissue, freshly excised Peyer's patch mucosa was fixed overnight at 4°C in 4% paraformaldehyde (PFA) in PBS. Tissue was cut into 2- by 2-mm blocks, washed in PBS, quenched in 0.1 M glycine in PBS, blocked with PBS-gelatin, and incubated with biotinylated virions or ISVPs as described above. All tissues were washed and fixed as described previously (39). Tissues were then frozen, and cryostat sections were stained with streptavidin-TRITC and mounted with Mowiol. To assess the effect of MAL-II lectin on reovirus binding to rabbit Peyer's patch explants, live and fixed tissue blocks were pretreated with 100 μg of MAL-II/ml for 20 min at RT prior to the addition of biotinylated reovirus particles. Control tissue was incubated with DMEM only. All tissues were then washed in DMEM and further processed as described above.

Reovirus overlay assay on paraffin sections of rabbit Peyer's patches.

Peyer's patch mucosa was fixed as described above and embedded in paraffin. Deparaffinized sections were quenched with 50 mM NH₄Cl, blocked with PBS-gelatin, overlaid with 200 μl of PBS-gelatin containing biotinylated virions, ISVPs, or ISVP-like particles derived from recoated cores (3 × 10¹¹ to 4 × 10¹¹ particles/ml), and incubated in a humidified chamber for 45 min at RT. After being washed in PBS, sections were fixed for 15 min at RT with 4% PFA, quenched with NH₄Cl, and incubated with 0.5 μg of streptavidin-TRITC/ml for 45 min at RT. M cells were identified by using a mouse monoclonal IgG1 specific for vimentin (DAKO Corp.) followed by secondary FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories).

Some deparaffinized sections were treated with periodate to disrupt carbohydrate epitopes (75). Briefly, sections were incubated with 10 mM sodium metaperiodate (Sigma) diluted in 50 mM sodium acetate buffer (pH 4.5) followed by 50 mM sodium borohydride in PBS (pH 7.6). Control sections were treated with acetate buffer alone. To verify disruption of carbohydrate residues, periodate-treated and control sections were incubated with the lectins UEA-I, SNA, and RCA-I (10 μg/ml) for 45 min at RT. Periodate-treated sections showed loss of lectin labeling (data not shown). Periodate-treated and control sections were quenched with NH₄Cl, blocked with PBS-gelatin, and overlaid with biotinylated virions or ISVPs. To test the capacities of different monosaccharides to inhibit binding of virus particles in the overlay assay, aliquots of biotinylated virions and ISVPs were diluted in PBS-gelatin containing 2% (wt/vol) l-(−)-Fuc, d-(+)-galactose (Gal), NeuAc, or N-acetyl-d-galactosamine (GalNAc) (Sigma), overlaid onto tissue sections for 45 min at RT, and further processed as described above.

Inhibition of reovirus binding by lectins and anticarbohydrate antibodies was tested by using the lectins SNA, MAL-I, and MAL-II and MAbs anti-sialyl Lewis^a and anti-sialyl Lewis^x. For inhibition studies, lectins and antibodies were used at concentrations ranging from 10 to 200 μg/ml. Deparaffinized sections were quenched, blocked, overlaid with lectin or antibody solutions, and incubated for 45 min at RT. The slides were washed twice in PBS and exposed to biotinylated virus particles and further processed as described above.

Reovirus binding and inhibition assays using Caco-2_BBe cell monolayers.

The cloned Caco-2_BBe cell line (54) was obtained from Mark Mooseker (Yale University, New Haven, Conn.) and from the American Type Culture Collection (Manassas, Va.). Cells were grown as described previously (20) and were seeded directly onto 12-mm-diameter glass coverslips (Bellco, Vineland, N.J.) precoated with rat tail collagen, in 24-well plates. Monolayers were confluent at 2 days after seeding. Binding of reovirus particles to monolayers at 2, 6, and 12 days postconfluence (dpc) was quantitated as the percentage of virus-positive cells in randomly chosen high-power fields containing an average of 275 total cells. Monolayers at 2 dpc were used for most binding and inhibition experiments.

To test viral binding to live Caco-2_BBe cells, monolayers were washed with gel-saline solution (137 mM NaCl, 270 μM CaCl₂, 840 μM MgCl₂, 19 mM H₃BO₃, 130 μM Na₂B₄O₇, 0.3% gelatin [pH 7.4]) and incubated with virus at 4°C for 60 min by inversion of coverslips onto 50-μl droplets of gel-saline containing biotinylated virions or ISVPs (3 × 10¹¹ to 4 × 10¹¹ particles/ml). After incubation, coverslips were washed twice with gel-saline and fixed in 3% PFA for 30 min at RT. For experiments using fixed cells, coverslips were immersed in 1 ml of 3% PFA for 30 min at RT, washed with gel-saline, inverted onto virus-containing droplets as described above, washed, and fixed again. Reactive aldehydes were quenched on all coverslips with 50 mM NH₄Cl, and nonspecific protein binding sites were blocked in gel-saline. Coverslips were then inverted onto 50-μl droplets of gel-saline containing 0.5 μg of streptavidin-TRITC or streptavidin-Cy5 (Zymed, South San Francisco, Calif.)/ml and incubated for 45 min in a humidified chamber at RT. They were then washed, mounted on slides with Mowiol, and examined and photographed as described above.

To test inhibition of viral binding, fixed Caco-2_BBe cell monolayers were incubated with biotinylated virions, ISVPs, or ISVP-like particles derived from recoated viral cores as described above, washed twice with gel-saline, and inverted onto 50-μl droplets of gel-saline containing 0.5 μg of streptavidin-TRITC/ml along with lectins or antibodies for 45 min. Lectins tested included FITC-conjugated UEA-I, SNA, PNA, and MAL-I and TRITC-conjugated RCA-I and MAL-II (all at 10 μg/ml). Antibodies included anti-JAM MAb J10.4 and anti-sialyl Lewis^x and anti-sialyl Lewis^a MAbs (all at 10 μg/ml). The anti-JAM MAb was detected with TRITC-conjugated goat anti-mouse IgG, anti-sialyl Lewis^x labeling was detected with 50 μg of FITC-conjugated goat anti-mouse IgM (Kirkegaard and Perry Laboratories)/ml, and anti-sialyl Lewis^a labeling was detected with 5 μg of FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories)/ml. Coverslips were washed, mounted, and examined as described above. Inhibition of virus binding to Caco-2_BBe cells by the lectins MAL-I, MAL-II, and SNA and by the anti-JAM MAb J10.4 was tested by using Caco-2_BBe cell monolayers fixed at 2 and 6 dpc. Monolayers were washed, quenched, and blocked as described above and incubated with unlabeled lectins at 10, 50, or 200 μg/ml or with MAb J10.4 at 10 μg/ml in gel-saline for 1 h at RT. The coverslips were washed and then incubated with biotinylated virions or ISVPs as described above, and the bound viral particles were detected by labeling with streptavidin-FITC.

To remove NeuAc residues from 2-dpc Caco-2_BBe cell surfaces, monolayers were briefly fixed in PFA as described above, washed once with sodium acetate buffer (pH 5.5) containing 0.1% (wt/vol) bovine serum albumin, inverted onto 50-μl droplets of sodium acetate buffer containing 0.25 U of Vibrio cholerae neuraminidase (Calbiochem)/ml, 0.08% bovine serum albumin, and 1 mM CaCl₂, and incubated for 90 min at 37°C. Control monolayers were incubated with the same solution without neuraminidase. Following incubation, monolayers were washed twice in PBS and exposed to biotinylated virus particles and further processed as described above. To assess the degree of desialylation, some neuraminidase-treated and control monolayers were labeled with FITC-conjugated SNA to detect residual NeuAc or FITC-conjugated PNA to monitor exposure of terminal Galβ1-3GalNAc residues.

HA assay.

Serial twofold dilutions of purified T1L virions or ISVPs (1 × 10¹¹ to 1.6 × 10⁹) or serial threefold dilutions of unconjugated lectins MAL-I, MAL-II, and SNA (200 to 0.3 μg/ml) in 50 μl of cold PBS were placed in 96-well round-bottom microtiter plates (Costar). Bovine erythrocytes or human type A erythrocytes were washed twice in cold PBS, and 50-μl aliquots were added to the wells containing virus or lectin to a final concentration of 0.4% (vol/vol) erythrocytes. The plates were briefly agitated and maintained at 4°C overnight. The lowest concentration of virus or lectin that caused agglutination was recorded.

L929 cell adherence and lectin inhibition assay.

Mouse L929 cells were seeded onto 12-mm-diameter round glass coverslips (Bellco) in 24-well plates at approximately 5 × 10⁴ cells/well. After overnight culture, the cells were fixed, rinsed, and treated with lectins at 1, 50, 100, or 250 μg/ml and/or biotinylated virions or ISVPs at the concentrations and conditions described above for Caco-2_BBe cells. Biotinylated virions and ISVPs were visualized with fluorophore-conjugated streptavidin, and the presence or absence of bound virus was evaluated by fluorescence microscopy.

Establishment of a reovirus binding assay with rabbit Peyer's patch tissue sections.

We next sought to develop an in vitro assay in which lectins and monosaccharides could be screened for inhibition of viral binding. Rabbit Peyer's patches were tested for this purpose because their M cells are numerous and have broad apical surfaces that are readily visualized by light microscopy (42). Although rabbits are susceptible to reovirus infection (66), it was not known whether reovirus binds preferentially to rabbit Peyer's patch M cells. To test this, mucosal explants of a rabbit Peyer's patch were incubated with biotinylated T1L virions or ISVPs, washed, and stained with streptavidin-TRITC. We tested both live and fixed explants because fixation can alter the availability of cell surface binding sites (39) but on the other hand lectins can damage enterocyte brush borders in live intestinal tissue (71). In both live and fixed explants, T1L virions and ISVPs adhered selectively to M cells (data not shown).

We subsequently tested whether T1L would show preferential binding to M cells when overlaid onto tissue sections of a rabbit Peyer's patch. The overlay approach has been used by others to identify cell-specific receptors for bacterial adhesins (19). Sections of rabbit Peyer's patch were deparaffinized, incubated with biotinylated T1L virions or ISVPs, washed, and stained with streptavidin-TRITC. Both virions (data not shown) and ISVPs (Fig. ​(Fig.1A)1A) bound to M cell apical surfaces in the sections, consistent with the observed M cell-selective binding on explanted rabbit mucosa. M cells were identified in this experiment by costaining with a MAb specific for vimentin, an intermediate filament protein expressed by M cells but not by other epithelial cells in rabbits (23). Vimentin filaments are concentrated around the nuclei of M cells and serve to mark the position of these cells in the epithelium (Fig. ​(Fig.1B).1B). The findings confirmed that both virions and ISVPs of reovirus T1L bind preferentially to M cells in the overlay assay.

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FIG. 1.

Reovirus T1L adhered to M cell apical surfaces in an overlay assay on sections of a rabbit Peyer's patch. Deparaffinized 5-μm sections of Peyer's patch mucosa were dual labeled with biotinylated T1L ISVPs and an anti-vimentin MAb. Bound ISVPs were visualized with streptavidin-TRITC, and vimentin was visualized with an FITC-conjugated secondary antibody. Sections were viewed by fluorescence (A and B) and phase-contrast (C) microscopy. ISVPs (A) (arrow) adhered to apical surfaces of most (but not all) of the epithelial cells that were identified as M cells by dual labeling with the MAb specific for vimentin, an intermediate filament protein that is concentrated around the nuclei of M cells (B). A phase-contrast image of the same section (C) confirmed that the virus-positive cell shows M cell features. Bar, 25 μm.

Reovirus T1L binding to rabbit M cells involves NeuAcα2-3-containing glycoconjugates.

To determine if carbohydrate structures are important for reovirus T1L binding to M cells, deparaffinized sections of the rabbit Peyer's patch were treated with periodate at low pH, an oxidation procedure that destroys most carbohydrate epitopes by disrupting monosaccharides that contain vicinal hydroxyl groups, including NeuAc (75). Control sections were treated with low-pH buffer alone. Following treatment, the sections were incubated with biotinylated T1L virions or ISVPs, washed, and stained with streptavidin-TRITC. Both virions (data not shown) and ISVPs (Fig. ​(Fig.2A)2A) bound to M cells in control sections, but there was no binding in the periodate-treated sections (Fig. ​(Fig.2B2B and data not shown). This suggested that reovirus binding to M cells requires a carbohydrate component. In an effort to identify key monosaccharides involved in this binding, we overlaid deparaffinized sections with biotinylated T1L virions or ISVPs that had been preincubated with 2% (wt/vol) Fuc, Gal, or GalNAc. We observed no effect on the binding of either particle type (data not shown). Preincubation of the particles with 2% (wt/vol) NeuAc, however, abolished the binding of ISVPs (Fig. 2C and D) but not virions (data not shown).

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FIG. 2.

Periodate oxidation and excess NeuAc inhibited reovirus T1L adherence to rabbit Peyer's patch sections. (A and B) Deparaffinized 5-μm Peyer's patch sections were pretreated with periodate at pH 4.5 before being overlaid with biotinylated T1L ISVPs. ISVPs (arrows) visualized with streptavidin-TRITC bound to control sections treated with buffer (pH 4.5) alone (A) but not to adjacent sections pretreated with periodate (B). (C and D) Biotinylated T1L ISVPs were incubated with NeuAc and then applied to sections of a rabbit Peyer's patch as described above. Untreated ISVPs bound to M cells (C), but adherence was abolished in the presence of NeuAc (D). Bar, 25 μm.

We then tested whether antibodies or lectins specific for defined NeuAc-containing epitopes could inhibit reovirus binding to rabbit M cells. These included two MAbs specific for tetrasaccharides, one for sialyl Lewis^a and one for sialyl Lewis^x, and three lectins (SNA, MAL-I, and MAL-II) that recognize distinct carbohydrate structures containing NeuAcα2-3(Table 1). Deparaffinized rabbit Peyer's patch sections were preincubated with the antibody or lectin, washed, and then incubated with biotinylated T1L virions or ISVPs, followed by staining with streptavidin-TRITC. In these studies it was not possible to distinguish or count individual viral particles clustered on M cells. In addition, M cell numbers varied widely among tissue samples. Viral binding was therefore scored as “inhibited” only when M cell binding was completely abolished. The lectins MAL-I and MAL-II, both of which recognize epitopes containing NeuAcα2-3Gal (32, 36, 37, 77), inhibited binding of both virions (data not shown) and ISVPs (Fig. ​(Fig.3)3) at the lowest lectin concentration tested (10 μg/ml). In contrast, the MAbs against sialyl Lewis^a and sialyl Lewis^x antigens, which recognize epitopes that differ from the MAL-I and -II epitopes by the presence of a Fuc side chain (30, 60), did not inhibit the binding of either particle type (data not shown). The lectin SNA, which recognizes epitopes containing NeuAcα2-6 (59), also failed to inhibit the binding of either virions or ISVPs, even when applied at 200 μg/ml (data not shown).

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FIG. 3.

The lectin MAL-II inhibited M cell-selective reovirus T1L binding to rabbit Peyer's patch sections. Deparaffinized 5-μm Peyer's patch sections were incubated with MAL-II lectin, washed, and overlaid with biotinylated T1L ISVPs, which were then visualized with streptavidin-TRITC. (A) On control sections, ISVPs adhered to M cell apical surfaces (arrows). (B) Adjacent sections of Peyer's patch mucosa preincubated with MAL-II did not bind ISVPs. Bar, 25 μm.

Reovirus T1L binding to polarized Caco-2_BBe cells also involves NeuAcα2-3-containing glycoconjugates.

Reovirus T1L has been shown to adhere to apical surfaces of Caco-2 human colon adenocarcinoma cells in vitro (1). To determine whether glycoconjugates containing NeuAcα2-3 are involved in reovirus T1L binding to these epithelial cells, we used cloned Caco-2_BBe cells that are known to form polarized monolayers with tight junctions and well-differentiated brush borders (54). At 2 dpc, these cells are polarized but relatively undifferentiated; most cells are extended and flat, have not assembled brush borders, and do not have the thick apical glycocalyx typical of enterocytes in vivo (24). We have previously shown that, early after confluence, the apical surfaces of Caco-2_BBe cells resemble those of M cells in that their membranes are relatively accessible to ligand-coated microparticles (21). By 12 dpc most but not all of the cells have differentiated and resemble villus enterocytes in that they are taller and have more narrow, polygonal apical surfaces endowed with well-developed brush borders coated with complex membrane glycoconjugates (21, 24). This in vitro model has proven useful for identifying receptors recognized by bacterial adhesins because the cells do not differentiate synchronously (54). Even at 21 dpc the monolayers display a mosaic pattern of expression of certain membrane enzymes and glycoconjugates, with some cells positive and others negative (7, 24, 54).

Reovirus T1L virions and ISVPs were biotinylated and applied to formalin-fixed and live Caco-2_BBe cell monolayers at 2, 6, and 12 dpc, followed by staining with streptavidin-TRITC. Both virions (data not shown) and ISVPs (Fig. ​(Fig.4A)4A) adhered to a subpopulation of cells in 2- and 6-dpc monolayers, producing a mosaic pattern that ranged from about 10 to over 50% positive cells (Fig. ​(Fig.4A),4A), depending on the experiment. By 12 dpc, binding was dramatically reduced, averaging only 0.3% positive cells per high-power field (data not shown). This suggests that reovirus recognizes a membrane component that is present early and that is either lost or masked as the Caco-2_BBe cells differentiate. Since the viral adherence patterns on fixed and live monolayers were identical, fixed 2-dpc monolayers were used for subsequent experiments.

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FIG. 4.

MAL-II lectin and reovirus T1L colocalized on Caco-2_BBe cell monolayers. Polarized Caco-2_BBe cell monolayers were grown on glass coverslips, fixed at 2 dpc, and incubated with biotinylated T1L ISVPs. After a washing and fixation, cells were labeled with TRITC-conjugated MAL-II lectin, and bound ISVPs were visualized with streptavidin-FITC. The pattern of adherence of ISVPs (A) paralleled that of MAL-II (B). The overlap of virus (green) and lectin (red) binding resulted in yellow fluorescence in merged images (C). Adherence was most evident on relatively undifferentiated cells, which had larger apical surfaces in phase-contrast images (D). Bar, 50 μm.

In an effort to correlate T1L binding with the presence of particular glycoconjugates on polarized Caco-2_BBe cells, we first applied biotinylated T1L virions or ISVPs to 2-dpc monolayers and then applied MAL-II or other selected lectins and anticarbohydrate antibodies. The virus particles and lectins or antibodies were next costained with different fluorophores. The distribution of adherent T1L ISVPs was closely correlated with the binding of the lectins MAL-I and MAL-II (Fig. 4A to C). Both virions (data not shown) and ISVPs (Fig. ​(Fig.4D)4D) bound most avidly to cells that showed a high density of MAL-II binding sites, and these cells had relatively large apical surfaces characteristic of less-differentiated cells (24). The lectin SNA also labeled a subpopulation of the cells (Fig. ​(Fig.5A),5A), but biotinylated virions (data not shown) and ISVPs (Fig. ​(Fig.5B)5B) adhered preferentially to the SNA-negative cells. The binding of Gal- or Fuc-specific lectins (RCA-I and UEA-I), as well as anti-sialyl Lewis^a or anti-sialyl Lewis^x antibodies, showed no correlation with reovirus adherence (data not shown).

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FIG. 5.

SNA did not colocalize with reovirus T1L on Caco-2_BBe cell monolayers, and reovirus T1L did not bind to neuraminidase-treated cells. (A and B) Polarized Caco-2_BBe cell monolayers were incubated with biotinylated T1L ISVPs and labeled with FITC-conjugated SNA. Bound ISVPs labeled with streptavidin-TRITC (B) adhered preferentially to SNA-negative cells (A). (C and D) Polarized Caco-2_BBe cell monolayers were treated with V. cholerae neuraminidase or not treated and incubated with biotinylated T1L ISVPs. ISVPs visualized with streptavidin-TRITC adhered to untreated cells (C) but not neuraminidase-treated cells (D). Bar, 50 μm.

If reovirus exploits a glycoconjugate recognized by MAL-I and MAL-II lectins, one would predict that binding may be inhibited when the MAL-I and MAL-II binding sites are occupied and may also be inhibited when the oligosaccharide epitopes are destroyed by removal of NeuAc. Caco-2_BBe monolayers at 2 dpc were preincubated with MAL-I or MAL-II at 10, 50, or 200 μg/ml, washed, incubated with biotinylated virions or ISVPs, and stained with streptavidin-TRITC. Pretreatment of monolayers with either lectin at 10 μg/ml completely abolished the binding of ISVPs (data not shown). Virion binding was also inhibited, but only at the higher concentrations (50 and 200 μg/ml) of MAL-I or MAL-II (data not shown). In contrast, the lectin SNA did not inhibit binding of either virions or ISVPs when tested at corresponding concentrations (data not shown). Other 2-dpc Caco-2_BBe monolayers were preincubated with V. cholerae neuraminidase, and removal of NeuAc residues was confirmed by a loss of SNA labeling and an increase in PNA labeling (data not shown), the latter indicating removal of NeuAc and exposure of terminal Gal residues. On the neuraminidase-treated monolayers, the binding of both virions (data not shown) and ISVPs (Fig. ​(Fig.5D)5D) was strongly reduced compared to that for controls (Fig. ​(Fig.5C).5C). These observations indicated that on polarized Caco-2_BBe cells, as on M cells, reovirus T1L binding involves NeuAcα2-3-containing glycoconjugates recognized by the lectins MAL-I and MAL-II.

MAL-II and reovirus T1L share HA profiles.

The distinct carbohydrate binding characteristics of reoviruses T1L and T3D were previously suggested by HA assays in which T1L agglutinated human but not bovine erythrocytes, unlike T3D, which agglutinated bovine erythrocytes preferentially (17, 51, 52). We reasoned that if MAL-II and T1L recognize similar glycoconjugates or oligosaccharide structures on cells, they may show similar HA profiles. We therefore compared the HA activities of T1L virions and ISVPs with those of MAL-II, a known hemagglutinin (32), on human and bovine erythrocytes. T1L virions and ISVPs agglutinated human erythrocytes but failed to agglutinate bovine erythrocytes (data not shown), as was reported previously (51, 58). Similarly, concentrations of MAL-II as low as 0.3 μg/ml agglutinated human erythrocytes, but a concentration of 22.2 μg/ml (~75 times higher) or higher was needed to agglutinate bovine erythrocytes. The fact that the HA activities of MAL-II paralleled those of reovirus T1L is consistent with the hypothesis that the lectin and virus recognize similar glycoconjugates or oligosaccharide structures.

Reovirus T1L uses binding sites on rabbit M cells and polarized Caco-2_BBe cells distinct from those used by reovirus T3D.

Biotinylated particles of reoviruses T1L and T3D were applied in parallel to paraffin sections of rabbit intestinal Peyer's patches in the overlay assay, and bound virus was detected with streptavidin-TRITC. Both virions and ISVPs of T1L, but neither particle type of T3D, bound to apical surfaces of rabbit M cells (Fig. 6A to C; Table ​Table2).2). When the sections were preincubated with the NeuAcα2-3-binding lectins MAL-I or MAL-II before T1L particles were applied, viral binding was eliminated (Table ​(Table2).2). When the NeuAcα2-6-binding lectin SNA was used instead, however, T1L binding was not affected (Table ​(Table2).2). These findings are consistent with the hypothesis that rabbit M cells present NeuAcα2-3-containing glycoconjugates that can be bound by reovirus T1L but not T3D in the overlay assay.

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FIG. 6.

Binding of reoviruses T1L and T3D to rabbit Peyer's patch sections and Caco-2_BBe cell monolayers. Biotinylated ISVPs were applied to sections of a rabbit Peyer's patch (A to C) or to prefixed 2-dpc Caco-2_BBe cell monolayers (D to F) in the presence or absence of competing lectins, and bound ISVPs were visualized with streptavidin-TRITC. In sections of the rabbit Peyer's patch, T1L ISVPs bound specifically to cells in the FAE but not to those in the villus epithelium (VE) (A). A phase-contrast image (B) of the section shown in panel A shows that the virus-positive FAE cells are associated with clusters of intraepithelial lymphocytes, an M cell feature. T3D ISVPs did not bind to the rabbit FAE or villus epithelium (C). ISVPs of both T1L (D) and T3D (F) bound to the Caco-2_BBe cell monolayers. The binding of T1L ISVPs was inhibited by preincubation of the monolayers with the MAL-II lectin (E), but the binding of T3D ISVPs was not inhibited by MAL-II (G). Bar, 50 μm.

We also tested the binding of T1L and T3D particles to polarized Caco-2_BBe cell monolayers. In contrast to the findings with M cells, virions and ISVPs of both strains bound to apical surfaces of the Caco-2_BBe cells (Fig. 6D and F; Table ​Table2).2). When the monolayers were preincubated with MAL-I or MAL-II before virus was applied, the binding of T1L was eliminated (Fig. ​(Fig.6E;6E; Table ​Table2)2) but binding of T3D was not affected (Fig. ​(Fig.6G;6G; Table ​Table2).2). When the monolayers were preincubated with SNA, the binding of T1L and T3D was not affected (Table ​(Table2).2). We conclude that T3D utilizes distinct apical binding sites on polarized Caco-2_BBe cells and that the binding of neither strain strictly requires the NeuAcα2-6-containing epitopes recognized by SNA.

Patterns of binding to rabbit M cells and polarized Caco-2_BBe cells are specific to the reovirus serotype.

We tested two additional type 1 isolates, three type 2 isolates, and two additional type 3 isolates for binding in the overlay assay on rabbit intestinal sections and on polarized Caco-2_BBe cell monolayers. Both additional type 1 isolates, but neither additional type 3 isolate, bound specifically to rabbit M cells, and two of the three type 2 isolates bound to all epithelial cells in the sections including M cells (Table ​(Table2).2). The M cell binding of both additional type 1 isolates was eliminated by MAL-I and MAL-II, but the binding of the two type 2 isolates was not affected (Table ​(Table2).2). Although all type 1 and type 3 isolates adhered to the Caco-2_BBe monolayers, only the type 1 isolates bound in a MAL-I- and MAL-II-sensitive manner (Table ​(Table2).2). SNA had no detectable effect on the binding of any of the isolates (Table ​(Table2).2). These findings indicate that the patterns of reovirus binding to rabbit M cells and polarized Caco-2_BBe cells are specific to the virus serotype. Since serotype specificity is determined by the σ1 protein encoded by the S1 genome segment (reviewed in references 38 and 44), the results suggest that the pattern of virus binding to these cells is likely determined by σ1, the reovirus protein responsible for attachment to other cell types.

The presence of T1L σ1 protein in reassortant virus particles and recoated cores is associated with binding to rabbit M cells and polarized Caco-2_BBe cells.

The differences in binding behavior of reoviruses T1L and T3D described above provided the opportunity to test the role of σ1 by using T1L-T3D reassortant strains. ISVPs of reassortants containing the T1L S1 genome segment on a T3D background (3HA1) or the T3D S1 on a T1L background (1HA3) were applied to rabbit Peyer's patch sections and polarized Caco-2_BBe cell monolayers. The 3HA1 ISVPs bound to rabbit M cells (Fig. ​(Fig.7A;7A; Table ​Table3)3) and the Caco-2_BBe cells (Table ​(Table3)3) in a MAL-I/II-sensitive manner. In contrast, the 1HA3 ISVPs did not bind to rabbit M cells (Fig. ​(Fig.7B;7B; Table ​Table3).3). 1HA3 ISVPs also bound to the Caco-2_BBe cells, but binding was not reduced by MAL-I or -II (Table ​(Table3).3). SNA had no effect on the binding of either reassortant (Table ​(Table3).3). With regard to all examined binding activities, therefore, 3HA1 behaved like T1L and 1HA3 behaved like T3D. This suggested that the S1 genome segment is the genetic determinant of the observed differences. Moreover, since the indicated properties were mediated by purified virus particles and since only the σ1 translation product of S1 is a particle-associated protein, the differences in binding among these strains are likely to be based in the attachment activities of σ1. Because only a limited set of reassortants was tested, however, more-definitive evidence was required to support this conclusion.

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FIG. 7.

Binding of T1L-T3D reassortant strains and recoated cores containing the T1L or T3D σ1 protein to rabbit Peyer's patch sections. Biotinylated particles were applied to sections of a rabbit Peyer's patch, and bound particles were visualized with streptavidin-TRITC. ISVPs of 3HA1, a reassortant containing T1L σ1, bound specifically to M cells in the FAE (A), whereas ISVPs of 1HA3, a reassortant containing T3D σ1, did not bind (B). ISVP-like particles derived from recoated cores containing T1L σ1 also bound preferentially to M cells in the FAE (C), but ISVP-like particles derived from recoated cores containing T3D σ1 did not bind (D). Bar, 50 μm.

TABLE 3.

ISVP binding profiles of T1L-T3D reassortants and recoated cores^a

Viral isolate or particleb	Binding to:
	M cells in rabbit Peyer's patch sections				Polarized Caco-2BBe monolayers
	Control	Treated with lectin:			Control	Treated with lectin:
		MAL-I	MAL-II	SNA		MAL-I	MAL-II	SNA
Reassortants
3HA1 with T1L σ1	+	−	−	+	+	−	−	+
1HA3 with T3D σ1	−	−	−	−	+	+	+	+
Recoated coresl
T1L with T1L σ1	+	−	−	+	+	−	−	+
T1L with T3D σ1	−	−	−	−	+	+	+	+

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^aISVPs derived from the different isolates or particles were assayed for binding as indicated for Table ​Table22.

^bThe natures of the different isolates and particles are described in the text.

To obtain direct evidence for the specific role of σ1 in binding to rabbit M cells and polarized Caco-2_BBe cell monolayers, we made use of recoated cores (11, 12). In each case for these experiments, the cores were derived from T1L virions and recombinant forms of the T1L μ1 and σ3 proteins were used to recoat them in vitro. However, the recombinant σ1 protein used for recoating was from T1L for one preparation and from T3D for another, and in a third preparation no σ1 was added. When ISVP-like particles derived from these different types of particles were applied to rabbit Peyer's patch sections, the particles containing T1L σ1 bound to rabbit M cells, but the particles containing T3D σ1 did not (Fig. 7C, D; Table ​Table3).3). Particles containing either T1L σ1 or T3D σ1 bound to the Caco-2_BBe cells (Table ​(Table3),3), but only the binding of the T1L σ1-containing particles was sensitive to MAL-I and MAL-II (Table ​(Table3).3). Particles lacking σ1 showed no binding to either rabbit M cells or the Caco-2_BBe cells (data not shown). These data strongly support the conclusion that σ1 played an essential role in binding to epithelial cells in these experiments, as well as in determining the serotype-specific patterns of binding to these cells.

JAM1 is not involved in the MAL-I/II-sensitive binding of reovirus T1L to apical membranes of polarized Caco-2_BBe cells.

JAM1 has been identified as a receptor for reoviruses T1L and T3D (3, 56). Because JAM1 is located on the basolateral membranes of polarized epithelial cells (3), we considered it unlikely that it would be involved in the MAL-I/II-sensitive binding of T1L on Caco-2_BBe monolayers. We nevertheless tested this possibility by applying anti-JAM1 MAb J10.4 to our polarized Caco-2_BBe cell monolayers in staining and inhibition experiments. Staining of fixed, impermeable monolayers with this MAb showed that JAM1 was not detectable on apical membranes. A few fixed monolayers showed a “chicken wire” pattern of staining (data not shown), consistent with exposure of JAM1 due to shrinkage of cells and opening of tight junctions, but these monolayers were not used in the experiments with virus. When intact monolayers were pretreated with the anti-JAM1 MAb, binding by T1L virions and ISVPs to apical Caco-2_BBe cell surfaces was not visibly affected. Pretreatment of monolayers with a mixture of an anti-JAM MAb and MAL-II resulted in complete loss of viral binding. We conclude that the MAL-I/II-sensitive binding of reovirus T1L to apical membranes of intact, polarized Caco-2_BBe cells is not dependent on JAM1.

T1L binding to L929 cells is not inhibited by NeuAc-specific lectins.

Previous studies have shown that infection of L929 cells by T1L virions or ISVPs is not NeuAc dependent (46). To verify that NeuAc epitopes are not required for T1L adherence to this fibroblast line, L929 cells were grown on glass coverslips, briefly fixed to stabilize plasma membranes and prevent endocytosis, exposed to biotinylated T1L virions and ISVPs under the same conditions used for polarized Caco-2_BBe cells and rabbit Peyer's patch sections, and stained with streptavidin-FITC. Preincubation of the fixed L929 cells with NeuAc-specific lectin MAL-I, MAL-II, SNA, or LFA at 10 μg/ml prior to viral overlay had no effect on binding (data not shown). These results are consistent with the capacity of reovirus T1L to use an alternative, NeuAc-independent binding mechanism to attach to L929 cells.

Anna Helander and Katherine Silvey contributed equally to this study.

This work was supported by NIH Research Grants HD17557 (to M.R.N.) and AI46440 (to M.L.N.) and NIH Center Grant DK34854 to the Harvard Digestive Diseases Center. K.J.S. was partially supported by an NIH Training Grant to the Committee on Immunology, Harvard Medical School.

We are indebted to David M. Knipe, Department of Microbiology and Molecular Genetics, Harvard Medical School, and to Jessica Wagner for technical assistance.