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Originally published In Press as doi:10.1074/jbc.M702180200 on April 23, 2007

J. Biol. Chem., Vol. 282, Issue 24, 17930-17940, June 15, 2007
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Reovirus Binding Determinants in Junctional Adhesion Molecule-A*Formula

Kristen M. Guglielmi{ddagger}§, Eva Kirchner, Geoffrey H. Holm§||, Thilo Stehle§||, and Terence S. Dermody{ddagger}§||1

From the Departments of {ddagger}Microbiology and Immunology, ||Pediatrics, and the §Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and the Interfakultäres Institut für Biochemie, Universität Tübingen, Hoppe-Seyler-Strasse 4, D-72076 Tübingen, Germany

Received for publication, March 13, 2007 , and in revised form, April 23, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Junctional adhesion molecule-A (JAM-A) serves as a serotype-independent receptor for mammalian orthoreoviruses (reoviruses). The membrane-distal immunoglobulin-like D1 domain of JAM-A is required for homodimerization and binding to reovirus attachment protein {sigma}1. We employed a structure-guided mutational analysis of the JAM-A dimer interface to identify determinants of reovirus binding. We purified mutant JAM-A ectodomains for solution-phase and surface plasmon resonance binding studies and expressed mutant forms of full-length JAM-A in Chinese hamster ovary cells to assess reovirus binding and infectivity. Mutation of residues in the JAM-A dimer interface that participate in salt-bridge or hydrogen-bond interactions with apposing JAM-A monomers abolishes the capacity of JAM-A to form dimers. JAM-A mutants incapable of dimer formation form complexes with the {sigma}1 head that are indistinguishable from wild-type JAM-A-{sigma}1 head complexes, indicating that {sigma}1 binds to JAM-A monomers. Residues Glu61 and Lys63 of beta-strand C and Leu72 of beta-strand C' in the dimer interface are required for efficient JAM-A engagement of strain type 3 Dearing {sigma}1. Mutation of neighboring residues alters the kinetics of the {sigma}1-JAM-A binding interaction. Prototype reovirus strains type 1 Lang and type 2 Jones share similar, although not identical, binding requirements with type 3 Dearing. These results indicate that reovirus engages JAM-A monomers via residues found mainly on beta-strands C and C' of the dimer interface and raise the possibility that the distinct disease phenotypes produced in mice following infection with different strains of reovirus are in part attributable to differences in contacts with JAM-A.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian orthoreoviruses (reoviruses)2 are nonenveloped, icosahedral viruses that contain a genome of 10 double-stranded RNA gene segments encapsulated within two concentric protein shells (1). Reoviruses infect a broad range of mammalian hosts, including humans, but cause disease primarily in the very young (2). The three reovirus serotypes, each represented by a prototype strain, type 1 Lang (T1L), type 2 Jones (T2J), and type 3 Dearing (T3D), differ mainly in the sequence of the viral attachment protein, {sigma}1 (3, 4). Following oral or intramuscular inoculation of newborn mice, strains T1L and T3D invade the central nervous system, yet these viruses use different routes and produce distinct pathologic consequences. T1L spreads to the central nervous system hematogenously and infects ependymal cells (5, 6), resulting in hydrocephalus (7). In contrast, T3D reovirus spreads to the central nervous system by neural routes and infects neurons (5, 6, 8), causing lethal encephalitis (7, 9). Pathways of viral spread (5) and tropism for neural tissues (6, 10) segregate with the viral S1 gene, which encodes the {sigma}1 protein (11, 12). Collectively, these studies suggest that the {sigma}1 protein determines the central nervous system cell types that serve as targets for reovirus infection, presumably through specific receptor binding.

Reovirus attachment protein {sigma}1 is a filamentous, trimeric molecule ~480 Å in length with distinct head-and-tail morphology (13, 14). Reovirus {sigma}1 shares striking structural similarities with the adenovirus attachment protein, fiber (15). Each is a trimer with a tail that partially inserts into the virion at the icosahedral vertices and a head that projects away from the virion surface. Both {sigma}1 and fiber possess an uncommon triple beta-spiral fold in the tail and an 8-stranded beta-barrel structure that composes the head. Discrete regions of reovirus {sigma}1 mediate binding to cell-surface receptors. Sequences in the N-terminal tail bind carbohydrate (16-18), whereas the C-terminal head binds junctional adhesion molecule-A (JAM-A) (19, 20).

JAM-A is a member of the immunoglobulin (Ig) superfamily postulated to regulate formation of intercellular tight junctions (21-23). JAM-A contains two extracellular Ig-like domains, a short transmembrane region, and a cytoplasmic tail possessing a PDZ domain-binding motif (21, 22). Crystal structures of the extracellular region of human (h) JAM-A (24) and murine (m) JAM-A (25) reveal two concatenated Ig-like domains (D1 and D2) (Fig. 1A). Two monomers form a symmetrical dimer with a large interface between apposing D1 domains. The JAM-A dimer interface is concave and primarily composed of four beta-strands (C', C, F, and G). The intermolecular interface is stabilized by four pairs of salt-bridges as well as hydrophobic interactions and two hydrogen bonds (Fig. 1B). Remarkably, JAM-A serves as a receptor for prototype and field-isolate strains of all three reovirus serotypes (19, 26).


Figure 1
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  		FIGURE 1.
Structure and location of residues in the JAM-A D1 domain. A, ribbon drawing of the hJAM-A dimer, with one monomer shown in yellow and the other in green. B, view of the interface between two hJAM-A monomers. The interface is formed by residues on the GFCC' faces of two D1 domains. The view is along a crystallographic dyad. Oxygen atoms are shown in red, nitrogen in blue, and sulfur in purple. Hydrogen bonds and salt-bridges are represented by dashed lines. Amino acids are labeled in single-letter code. C, view of the GFCC' beta-sheet that composes the dimer interface of one JAM-A monomer, labeled as in B. Side chains are shown for residues that were altered in this study. This figure was generated using PyMOL (54).

 
JAM-A belongs to a structurally related family of cell-adhesion molecules. The most closely related of these proteins to JAM-A are JAM-B and JAM-C (27-29), neither of which serves as a receptor for reovirus (24, 26). Although structural information is not available for JAM-B and JAM-C, sequence analysis suggests that homophilic contacts are conserved between JAM family members (24). The coxsackievirus and adenovirus receptor (CAR) also contains two extracellular Ig-like domains that form similar dimers via the GFCC' interface. Moreover, intermolecular interactions in CAR also include some contacts between charged residues (30, 31).

Chinese hamster ovary (CHO) cells transfected with plasmids encoding chimeric CAR-JAM-A receptor constructs or JAM-A domain-deletion mutants provide evidence that the N-terminal D1 domain of JAM-A is required for reovirus attachment and infection (32). Reovirus binding to JAM-A occurs more rapidly than homotypic JAM-A association and is competed by excess JAM-A in vitro and on cells (32). Chemical cross-linking of JAM-A diminishes the capacity of reovirus to bind JAM-A in vitro and on cells and negates the competitive effects of soluble JAM-A on reovirus attachment (32). These findings suggest that reovirus binds to monomeric JAM-A by engaging the dimer interface to effect stable cell attachment. However, the precise nature of {sigma}1-JAM-A interactions is not understood. Moreover, as all reovirus serotypes bind to JAM-A, it is not apparent how JAM-A binding contributes to the serotype-dependent differences in reovirus tropism in the murine central nervous system.

In this study, we performed a systematic mutagenic analysis of the dimer interface of the D1 domain of JAM-A. We engineered point mutations in the dimer-contributing surface of the extracellular domain of JAM-A, purified the resultant mutants, and characterized effects of the mutations on JAM-A homodimerization and binding to the purified {sigma}1 head domain. In complementary experiments, we tested the capacity of CHO cells expressing full-length JAM-A point mutants to support binding and infection by prototype strains from each of the three reovirus serotypes. Our results indicate that reovirus T3D {sigma}1 engages JAM-A via residues in beta-strands C and C' within the dimer interface and that JAM-A binding requirements differ significantly among the reovirus serotypes. These findings enhance an understanding of reovirus-receptor interactions and suggest that the nature of JAM-A contacts contributes to differences in pathogenesis among reovirus serotypes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells, Viruses, and Antibodies—Spinner-adapted murine L929 cells were cultured as described (33). CHO cells were maintained in Ham's F-12 medium supplemented to contain 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Reovirus strains T1L, T2J, and T3D are laboratory stocks. Strain T3SA- is a reassortant strain containing the S1 gene of T3C44, which does not bind sialic acid (17, 34), and the remaining nine genes of T1L (33). Reoviruses were purified by cesium chloride gradient centrifugation from infected L929 cells as described (13). Particle concentrations were determined by spectrophotometry at 260 nm using a conversion factor of 2.1 x 1012 particles/ml/A260. hJAM-A-specific monoclonal antibody J10.4 was provided by Dr. Charles Parkos (Emory University).

Protein Expression and Purification—The C-terminal head domain of T3D {sigma}1 was purified as described (20). Soluble ectodomains of wild-type and point mutant hJAM-A constructs were fused to an amino-terminal GST affinity tag via a thrombin cleavage site and purified as described (24). Nucleotide sequences corresponding to residues 27-233 of wild-type hJAM-A were cloned by PCR, digested with BamHI and XhoI, and ligated into pGEX-4T-3 (GE Healthcare) for bacterial transformation. Point mutants of hJAM-A were engineered using the QuikChange Site-directed Mutagenesis Kit (Stratagene), as per the manufacturer's instructions. Sequences of primers used to engineer point mutations in hJAM-A are listed in supplemental Table 1. A GST fusion with the D1 domain of hJAM-A was engineered by inserting a XhoI or BamHI restriction site into the D1/D2 linker of hJAM-A in pGEX-4T-3 using site-directed PCR mutagenesis. Digestion with XhoI or BamHI followed by ligation eliminated the D2 domain (residues 131-233) or D1 domain (residues 27-131), respectively, and fused the remaining domain to GST. Bacteria transformed with plasmids encoding GST-JAM-A constructs were cultured in Luria-Bertani broth at 37 °C with shaking, and protein expression was induced with 0.2 mM isopropyl beta-D-thiogalactoside (GE Healthcare) at 25 °C. Bacteria were harvested by centrifugation, solubilized in phosphate-buffered saline (PBS) plus 1% Triton X-100, 2 mM beta-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 100 µg/ml lysozyme, and lysed by sonication. GST-hJAM-A proteins were purified from bacterial lysates by glutathione affinity chromatography. Soluble wild-type and point-mutant hJAM-A ectodomains were liberated from the glutathione resin by incubation with 20 units/ml thrombin (Sigma) at room temperature overnight.

Gel Filtration Chromatography—Gel filtration of purified hJAM-A extracellular domains was performed by loading ~0.5 mg of each protein onto a Superdex 75 column mounted on a BioLogic HR Work station (Bio-Rad). Proteins were resolved in 20 mM Tris (pH 7.5), 100 mM NaCl, 0.01% sodium azide. Gel filtration of the hJAM-A extracellular domain and the {sigma}1 head domain was performed using a Superdex 75 column mounted on a SMART analytical chromatography system (GE Healthcare). The {sigma}1 head (10 µg) in 20 mM HEPES (pH 7.1) and wild-type or mutant forms of the hJAM-A extracellular domain (12.74 µg) in PBS were mixed and incubated at 0 °C for 40 min prior to gel filtration. Mixtures of proteins or individual proteins were resolved in 20 mM Tris (pH 7.5), 100 mM NaCl.

GST Capture Assays—Swelled, washed, glutathione-coated beads (Sigma) were incubated with 20 µg of purified GST or GST-hJAM-A fusion proteins and diluted in 200 µl of PBS plus 1% Tween 20 at 4 °C for 1 h. Glutathione beads with captured GST fusion proteins were washed twice with PBS plus 1% Tween 20 and incubated with 10 µg of purified T3D {sigma}1 head domain in 200 µl of PBS plus 1% Tween 20 at 4 °C for 1 h, followed by two additional washes. After the final wash, buffer was aspirated carefully, and beads with bound proteins were resuspended in 30 µl of sample buffer (313 mM Tris (pH 6.8), 4% SDS, 10% beta-mercaptoethanol, 20% glycerol, 0.01% bromphenol blue). Samples (15 µl/lane) were loaded into wells of a SDS 4-20% continuous gradient polyacrylamide gel and electrophoresed at 120 V until the dye front reached the bottom of the gel. Proteins were visualized by staining with Coomassie Brilliant Blue.

Assessment of {sigma}1-JAM-A Interactions Using Surface Plasmon Resonance (SPR)—A BIAcore CM5 chip (Pharmacia Biosensor AB) was coated with mouse ascites containing monoclonal GST-specific antibody (Sigma) to ~1800 resonance units of IgG by amine coupling. Purified GST or wild-type or mutant GST-hJAM-A ectodomain fusion proteins at a concentration of 2 µM in HEPES-buffered saline (pH 7.0) were captured by injection across individual flow cells of an antibody-coated chip for 3 min at 30 µl/min using a BIAcore 2000 (GE Healthcare). Purified T3D {sigma}1 head domain was injected across the conjugated chip surface at 30 µl/min. Following {sigma}1 binding, chip surfaces were regenerated with a 20-µl pulse of 10 mM glycine (pH 2.5). Affinity constants for {sigma}1 binding to hJAM-A were determined using separate kon and koff nonlinear regression with BIAevaluation 3.0 software (GE Healthcare), assuming a 1:1 Langmuir binding model (35). Molar concentrations of {sigma}1 constructs were calculated based on {sigma}1 forming a homotrimer (20, 36).

Flow Cytometric Analysis of Receptor Expression and Virus Binding—CHO cells (106) were transiently transfected with receptor-encoding plasmids using Lipofectamine and PLUS reagent (Invitrogen) according to the manufacturer's instructions and incubated for 24 h to allow receptor expression. Cells were detached from plates by incubation with 20 mM EDTA in PBS and incubated with hJAM-A-specific monoclonal antibody J10.4 at 10 µg/ml or incubated with reovirus T1L, T2J, or T3SA- (105 particles/cell) on ice for 1 h. Virus-adsorbed cells were washed with PBS containing 0.1% bovine serum albumin and incubated with clarified, combined T1L/T3D antiserum (37) at 1:1000 dilution on ice for 1 h. Samples were washed with PBS containing 0.1% bovine serum albumin and incubated with Alexa 488-conjugated goat anti-rabbit or goat anti-mouse IgG (Invitrogen) at a 1:1000 dilution on ice for 1 h. Cells were washed twice with PBS containing 0.1% bovine serum albumin and fixed with 4% paraformaldehyde in PBS. Cells were analyzed for antibody or virus binding using a FACSCalibur flow cytometer (BD Biosciences).

Transient Transfection and Infection of CHO Cells—Point mutations were engineered into full-length hJAM-A in pcDNA3.1 (24) using the QuikChange Site-directed Mutagenesis Kit (Stratagene) and the same primers used to engineer mutations in the hJAM-A extracellular domain in pGEX-4T-3 (supplemental Table 1). CHO cells (2 x 105) were transiently transfected with receptor-encoding plasmids using Lipofectamine and PLUS reagent (Invitrogen) and incubated for 24 h to allow receptor expression. Cells were infected with reovirus at multiplicities of infection of 1 (T1L), 5 (T2J), and 10 (T3SA-) plaque forming units/cell and incubated at 37 °C for 18-20 h. Infected cells were processed for indirect immunofluorescence as described (33). Images were captured at x200 magnification using a Zeiss Axiovert 200 microscope. For each experiment, three fields of view were scored from three independently transfected wells. Mean values from three independent experiments were compared using the unpaired Student's t test as applied using Microsoft Excel. p values of less than 0.05 were considered statistically significant.


Figure 2
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  		FIGURE 2.
Characterization of the {sigma}1 head domain binding to GST-JAM-A. A, binding of the {sigma}1 head domain to GST-JAM-A. Purified {sigma}1 head domain, at concentrations of 167 (red), 16.7 (yellow), or 1.67 nM (green), was injected for 5 min across a biosensor surface on which GST fused to the JAM-A ectodomain previously had been captured. Buffer alone was injected for 6.7 min. The baseline is 167 nM {sigma}1 head domain binding to GST (gray). The KD of {sigma}1 head binding to GST-JAM-A is ~2.4 x 10-9 M. B, binding of the {sigma}1 head domain to GST-JAM-A Ig-like domains. Purified {sigma}1 head domain, at a concentration of 167 nM, was injected for 5 min across a biosensor surface on which GST-JAM-A fusion proteins previously had been captured. Buffer alone was injected for 6.7 min. The resultant traces show binding to GST-JAM-A (yellow), GST-D1 (red), and GST-D2 (green). The baseline is 167 nM {sigma}1 head domain binding to GST (gray). Binding is expressed in resonance units (RU).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of {sigma}1 Head Binding to GST-JAM-A—For studies of {sigma}1-JAM-A interactions, we purified an N-terminal GST fusion with the extracellular region of JAM-A (GST-JAM-A) (24). Bacteria were transfected with plasmids encoding GST-JAM-A, harvested by centrifugation, and lysed by sonication. GST-JAM-A constructs were purified from bacterial lysates using glutathione affinity chromatography (32). To determine whether GST-JAM-A is capable of binding {sigma}1, we captured GST-JAM-A on the surface of a biosensor chip via a covalently linked GST-specific antibody. We then injected a purified C-terminal fragment of T3D {sigma}1 at three different concentrations and detected association with GST-JAM-A by SPR (Fig. 2A). The injected fragment of {sigma}1 incorporates residues 293 to 455, which comprise the globular beta-barrel head domain and one beta-spiral repeat of the tail and is referred to as the {sigma}1 head (20). The {sigma}1 head did not show any specific binding to the GST-specific antibody or to GST captured on the biosensor surface (data not shown). However, the {sigma}1 head bound GST-JAM-A with an affinity of ~2.4 x 10-9 M, which approximates previous calculations of {sigma}1 affinity for JAM-A (19, 20). To determine whether the isolated D1 Ig-like domain of JAM-A is capable of {sigma}1 engagement, we expressed and purified only the D1 or D2 Ig-like domains of JAM-A with an N-terminal GST tag. We assessed the capacity of the fusion proteins to bind the {sigma}1 head using the SPR strategy employed for GST-JAM-A. Whereas the {sigma}1 head bound GST-D1 with an affinity similar to GST-JAM-A, GST-D2 was incapable of capturing the {sigma}1 head (Fig. 2B and data not shown). In accordance with data obtained using chimeric receptor molecules (32), these data indicate that the membrane distal D1 domain of JAM-A is sufficient for high affinity interactions with {sigma}1.


Figure 3
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  		FIGURE 3.
Precipitation of the {sigma}1 head domain with purified GST-JAM-A point mutants. Glutathione-agarose was incubated with buffer alone (NP), GST, or GST fused to the JAM-A D1, D2, entire extracellular domain (JAM-A), or point mutants shown at 4 °C for 1 h. The glutathione-agarose was pelleted, washed, and incubated with purified T3D {sigma}1 head at 4 °C for an additional 1 h. The glutathione-agarose was washed, pelleted, resuspended in SDS sample buffer, and boiled. Samples were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue to visualize captured proteins. Input T3D {sigma}1 head also was resolved following boiling in SDS sample buffer. Molecular masses of protein standards in kDa are indicated on the left. Proteins are labeled on the right.

 
Generation and Characterization of JAM-A Dimer Interface Point Mutants—To identify specific residues in the D1 domain required for {sigma}1 binding, we generated individual alanine substitutions of residues with surface-exposed side chains in the JAM-A dimer interface (Fig. 1C). Two groups of residues were targeted for mutagenesis: (i) those that form direct protein-protein intersubunit contacts: Arg59, Glu61, Lys63, Leu72, Tyr75, Met110, and Glu121, and (ii) those that make indirect contacts: Asp65, Thr70, Asn76, Thr106, Thr108, Ser112, and Lys123. GST-JAM-A point mutants were purified by glutathione affinity chromatography and used in GST precipitation assays to identify mutations that ablate{sigma}1 binding. In these experiments, glutathione-coated beads bound GST fusion proteins, which in turn were used to capture the {sigma}1 head domain. As anticipated, the{sigma}1 head was bound efficiently by GST-JAM-A and GST-D1 but not detectably by beads alone, GST, or GST-D2 (Fig. 3). The majority of JAM-A dimer interface point mutants were capable of binding the {sigma}1 head. However, mutants E61A, K63A, L72A, and N76A were incapable of binding the {sigma}1 head as detected by Coomassie Blue staining or immunoblotting, despite similar capture of the GST-JAM-A mutants on the beads (Fig. 3 and data not shown). Residues Leu72 and Asn76 are located on beta-strand C' and the C'C'' loop, respectively (Fig. 1C). Leu72 participates in a hydrophobic interaction with Tyr119 on an apposing JAM-A monomer. Residues Glu61 and Lys63 are located on beta-strand C and participate in salt-bridge interactions that stabilize JAM-A dimers. These data suggest that the {sigma}1 head engages JAM-A via residues near the top of (as oriented in Fig. 1C) and within the dimer interface.


Figure 4
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  		FIGURE 4.
Binding of the {sigma}1 head to GST-JAM-A point mutants. Purified {sigma}1 head domain, at a concentration of 167 nM, was injected for 5 min across a biosensor surface on which GST-JAM-A fusion proteins, as indicated, had been captured. Buffer alone was injected for 6.7 min. The resultant traces show binding to GST-JAM-A and GST-JAM-A point mutants, as indicated. The resultant sensograms are grouped based on binding kinetics: A, kon and koff similar to wild-type JAM-A; B, faster kon than wild-type JAM-A; C, faster kon and koff than wild-type; and D, no specific binding. The baseline is 167 nM {sigma}1 head domain binding to GST (gray). Binding is expressed in resonance units (RU).

 
Kinetics of {sigma}1 Head Binding to GST-JAM-A Dimer Interface Point Mutants—To quantitatively assess the effects of mutations in the JAM-A dimer interface on interactions with {sigma}1, GST-JAM-A point mutants were captured on a biosensor surface and tested for {sigma}1 binding by SPR. Based on the kinetics and affinity of the interaction with {sigma}1, the majority of JAM-A mutants clustered into one of four groups. The first group (I) includes point mutants that display binding kinetics similar to wild-type (Fig. 4A and Table 1). Group I includes mutants of Thr70 in beta-strand C' and nearby residue Thr106 in beta-strand F, which do not contribute to intersubunit interactions at the JAM-A dimer interface. The second group of GST-JAM-A mutants (II) exhibited faster association kinetics than wild-type JAM-A by ~5-10-fold, resulting in higher calculated affinities for {sigma}1 than wild-type JAM-A (Fig. 4B and Table 1). Initially upon injection of wash buffer, much of the {sigma}1 was removed from these mutants. However, specifically bound {sigma}1 protein exhibited a similar off-rate to wild-type GST-JAM-A. Group II includes mutants T108A, M110A, and E121A. Thr108, Met110, and Glu121 all are located within the dimer interface in proximity to one another on beta-strands F and G (Fig. 1C). Glu121 participates in salt-bridge interactions that stabilize the dimer interface (24). Met110 participates in a hydrophobic interaction with Met110 on an apposing JAM-A monomer. The third group (III) includes GST-JAM-A mutants R59A, Y75A, and N76A (Fig. 4C), which exhibited a faster {sigma}1 on-rate than wild-type JAM-A but also a faster off-rate, resulting in affinities similar to wild-type. The N76A mutant exhibited a slower on-rate and a lower level of {sigma}1 binding, relative to the other mutants in this group, and binding returned to baseline levels over several minutes of wash buffer injection. This result agrees with results obtained in the GST precipitation assay, in which the N76A mutant of JAM-A was incapable of capturing the {sigma}1 head domain (Fig. 3). Arg59, Tyr75, and Asn76 are located on beta-strands C and C' in the dimer interface (Fig. 1C). The final group of JAM-A point mutants includes E61A, K63A, and L72A (Fig. 4D), which showed very low levels of {sigma}1 binding above background. Upon injection of wash buffer, the binding signal returned to baseline levels immediately, indicating that the observed binding was not specific. We were unable to find suitable binding models for the interaction of these mutants with the {sigma}1 head using BIAevaluation software (data not shown). These results also are in accord with results obtained in the GST precipitation assay (Fig. 3), in which no {sigma}1 head was bound detectably by the E61A, K63A, or L72A GST-JAM-A point mutants. These data reinforce the importance of Glu61, Lys63, and Leu72 as mediators of {sigma}1-JAM-A interactions and suggest that these residues serve as contact points for the {sigma}1 head.


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  		TABLE 1
Kinetics of purified {sigma}1 head binding to GST-JAM-A point mutants Association and dissociation rates and dissociation constants were calculated using BIAevaluation 3.0 software. Mutants are grouped as in Fig. 4. Group IV mutants did not display specific binding.

 


Figure 5
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  		FIGURE 5.
Characterization of {sigma}1 head/JAM-A extracellular domain mixtures using gel filtration chromatography. Purified T3D {sigma}1 head and wild-type JAM-A (A) or JAM-A point mutants S112A (B), E121A (C), and E61A (D) were mixed in a 3:1 molar ratio and incubated on ice for 40 min. The mixtures (yellow) or individual purified proteins (red and blue) were applied to a Superdex 75 gel-filtration column, and proteins in the column fractions were detected by A280. Dimeric forms of wild-type JAM-A and S112A elute earlier than monomeric forms E121A and E61A. Wild-type JAM-A, S112A, and E121A each form a complex with {sigma}1 that elutes from the column at approximately the same time, thus with similar apparent molecular weight, for each protein mixture.

 
Oligomeric State of JAM-A Point Mutants—Because two residues that participate in salt-bridge interactions at the JAM-A dimer interface are required for efficient {sigma}1 binding, we asked whether mutation of these residues or other residues in the dimer interface alters the capacity of JAM-A to form dimers. To answer this question, the extracellular domains of wild-type and selected JAM-A point mutants were released from the immobilized GST tag by thrombin cleavage and further purified using gel filtration. The extracellular domain of wild-type JAM-A eluted as a single peak, with an apparent molecular mass of ~48 kDa, indicating that it forms a dimer (Table 2 and data not shown) (24). Likewise, alanine substitutions of Ser112 and Lys123, which participate in indirect contacts at the intersubunit interface, do not alter the oligomeric state of JAM-A (Table 2). In sharp contrast, JAM-A extracellular domains with alanine substitutions of Arg59, Glu61, Lys63, or Glu121, which form salt-bridges across the dimer interface, or position Tyr75, which forms a hydrogen bond with Glu114 from an apposing monomer, eluted from the column as a single species with an apparent molecular mass of ~30 kDa. This elution peak likely represents solely monomeric JAM-A. These data indicate that each salt-bridge pair and Tyr75 individually is required for stabilization of JAM-A dimers.


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  		TABLE 2
Characterization of selected point mutants of the JAM-A dimer interface by gel filtration Apparent molecular masses were calculated by graphing Ve - Vo against the log10 Mr of protein standards, performing linear regression, and back-calculating, based on elution time.

 
To determine whether {sigma}1 engages monomeric or dimeric forms of the JAM-A extracellular domain, we assessed binding of the {sigma}1 head to the JAM-A extracellular domain in solution using gel-filtration chromatography. Incubation of purified wild-type JAM-A and the {sigma}1 head in a 3:1 molar ratio (three JAM-A monomers to one {sigma}1 trimer), results in formation of a higher molecular weight complex that contains both JAM-A and the {sigma}1 head (Fig. 5A and data not shown). Similar findings were made following incubation of the extracellular domain of dimeric JAM-A mutant S112A or the monomeric mutant E121A with the {sigma}1 head (Fig. 5, A-C). In both cases, a complex was formed that eluted from the size-exclusion column at the same volume as the complex observed following incubation of wild-type JAM-A with the {sigma}1 head. As expected, monomeric JAM-A point mutant E61A does not form a detectable complex when incubated with the {sigma}1 head (Fig. 5D). Because mutant forms of JAM-A that are incapable of forming dimers retain the capacity to interact with {sigma}1, we conclude that the {sigma}1 head can engage monomeric forms of JAM-A.


Figure 6
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  		FIGURE 6.
Reovirus T3SA-binding to CHO cells expressing JAM-A mutants. CHO cells (106) were transiently transfected with plasmids encoding the indicated receptor constructs. Following incubation for 24 h to permit receptor expression, cells were lifted from plates using EDTA and stained with hJAM-A-specific monoclonal antibody J10.4 or adsorbed with reovirus T3SA- (105 particles/cell). Cell-surface expression of receptor constructs and virus binding was assessed by flow cytometry.

 
Reovirus Binding and Infectivity of CHO Cells Expressing Mutant Forms of JAM-A—To determine the capacity of reovirus to bind JAM-A mutants in cell culture, we transiently transfected CHO cells, which are poorly permissive for reovirus infection (26, 32), with plasmids encoding wild-type (24) or mutant forms of full-length JAM-A. Transfected cells were incubated for 24 h to allow receptor expression and adsorbed with reovirus virions. Surface expression of JAM-A and reovirus binding were assessed by flow cytometry (Fig. 6). Wild-type JAM-A and each of the JAM-A mutants were expressed at the surface of transfected cells at approximately equivalent levels, providing confidence that the mutant constructs are properly folded. Virions of T3SA-, a non-sialic acid-binding reovirus strain with a serotype 3 {sigma}1 identical in sequence to T3D in the {sigma}1 head domain (33), bound poorly to vector-transfected CHO cells, whereas specific binding to CHO cells expressing wild-type JAM-A was observed. We used T3SA-rather than T3D in these studies to diminish background binding to cells due to interactions with carbohydrate. T3SA-binding approximated JAM-A expression at the surface of CHO cells expressing the majority of the point mutants. However, T3SA-binding to cells expressing the K63A or L72A mutants was diminished, and no binding above background was observed with cells expressing the E61A mutant form of JAM-A. These results closely mimic the binding of the T3D {sigma}1 head to JAM-A mutants in the GST precipitation and SPR assays (Figs. 2 and 3). Interestingly, mutation of Glu61 to aspartate or Lys63 to arginine was not associated with the binding defects observed for the E61A and K63A mutants (Fig. 6), indicating that the acidic or basic properties of these residues are important for reovirus engagement.


Figure 7
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  		FIGURE 7.
Reovirus T1L, T2J, and T3SA-binding to CHO cells expressing JAM-A mutants. CHO cells (106) were transiently transfected with plasmids encoding the indicated receptor constructs. Following incubation for 24 h to permit receptor expression, cells were lifted from plates using EDTA and stained with hJAM-A-specific monoclonal antibody J10.4 or adsorbed with reovirus T1L, T2J, or T3SA- (105 particles/cell). Cell-surface expression of receptor constructs and virus binding was assessed by flow cytometry.

 
To compare JAM-A binding determinants for reoviruses of the three serotypes, we assessed binding of virions of strains T1L and T2J to CHO cells expressing wild-type and mutant forms of JAM-A by flow cytometry. Because no serotype 1 or 2 reoviruses incapable of binding carbohydrate have been isolated, we used prototype strains T1L and T2J to represent serotypes 1 and 2. Similar to T3SA-, each virus bound poorly to vector-transfected cells, whereas T1L and T2J binding to cells expressing wild-type JAM-A approximated JAM-A expression (Fig. 7). In contrast to T3SA-, T1L and T2J showed approximately wild-type binding to CHO cells expressing each of the JAM-A point mutants (data not shown). To determine whether T1L and T2J might share similar binding determinants with T3D and T3SA- but with less stringency, we constructed double point mutants R59A/E61A and E61A/K63A. As anticipated, T3SA- showed no specific binding to CHO cells expressing either of the JAM-A double point mutants (Fig. 7). Although not with the same magnitude as T3SA-, both T1L and T2J showed diminished binding to CHO cells expressing the double point mutants, with T1L exhibiting highly impaired binding to CHO cells expressing the R59A/E61A JAM-A mutant.


Figure 8
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  		FIGURE 8.
Reovirus infection of CHO cells expressing JAM-A mutants. CHO cells (2 x 105) were transiently transfected with empty vector (pcDNA) or plasmids encoding mutant forms of JAM-A. Following incubation for 24 h to permit receptor expression, cells were adsorbed with reovirus at a multiplicity of infection of 1 (T1L), 5(T2J), or 10 (T3SA-) plaque-forming units/cell at room temperature for 1 h. Cells were washed with PBS, incubated in complete medium at 37 °C for 20 h, and stained for reovirus antigen. Infected cells were identified by indirect immunofluorescence and quantified by counting cells exhibiting cytoplasmic staining in three confluent fields of view per experiment. The results are expressed as the mean fluorescent focus units (FFU) per field of view for triplicate experiments. Error bars indicate S.D. *, p < 0.05 in comparison to cells expressing wild-type JAM-A.

 
To assess the functional significance of mutations in JAM-A that alter reovirus binding, we quantified infectivity of T1L, T2J, and T3SA-following adsorption to CHO cells expressing full-length wild-type or mutant forms of JAM-A by indirect immunofluorescence (33). Infectivity of T3SA- was significantly decreased (~50%) following infection of CHO cells expressing the E61A and K63A mutants in comparison to cells expressing wild-type JAM-A (Fig. 8). Consistent with the flow cytometry experiments (Fig. 7), conservation of charge at residues Glu61 and Lys63 by substituting aspartate and arginine, respectively, rescued the infectivity defect. T3SA-infectivity was decreased almost to the level of background following adsorption to CHO cells expressing the double point mutants R59A/E61A and E61A/K63A. As predicted by the binding results, T1L also showed a striking loss of infectivity in cells expressing the R59A/E61A JAM-A mutant and a slight decrease in infectivity in cells expressing the K63A or E61A/K63A mutants. T2J showed only a very modest decrease in infectivity, less than 25%, in CHO cells expressing the R59A mutant or either of the two double point mutants, in accordance with the flow cytometry results. Taken together, these data reinforce the validity of results obtained from binding assays using the T3D {sigma}1 head domain and suggest that T1L, T2J, and T3SA-engage JAM-A using non-identical residues.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The goal of this study was to identify reovirus binding determinants in JAM-A. We generated alanine substitutions of residues with solvent-exposed side chains in the JAM-A dimer interface in the context of a GST-JAM-A fusion. Analysis of the point mutants using GST precipitation assays and SPR experiments led to the identification of Glu61, Lys63, and Leu72 that individually are required for efficient binding of the T3D {sigma}1 head. Glu61 and Lys63 have charged side chains and participate in salt-bridge contacts that stabilize the JAM-A dimer interface (24). Leu72 is located in proximity to Glu61 and Lys63 and participates in hydrophobic interactions with Tyr119 from the apposing JAM-A monomer (24). SPR findings were concordant with these results and indicate that mutation of specific residues in the JAM-A dimer interface can enhance or diminish the rate of association and dissociation of the T3D {sigma}1 head. Specifically, T108A, M110A, and E121A mutations enhanced association of {sigma}1, leading to higher calculated binding affinities in comparison to wild-type JAM-A. On the other hand, R59A and Y75A displayed faster association with the {sigma}1 head but also faster dissociation. The same charged residues required for JAM-A-{sigma}1 interactions in biochemical assays, Glu61 and Lys63, also are required for serotype 3 {sigma}1 interactions with JAM-A in cultured cells.

Gel-filtration chromatography of purified, mutant JAM-A extracellular domains revealed that mutation to alanine of any residue in the JAM-A dimer interface that contributes to salt-bridge interactions as well as one residue, Tyr75, which makes a hydrogen-bond contact with Glu114 (24), abolishes the capacity of JAM-A to form dimers in solution at neutral pH. These data indicate that each salt-bridge interaction and the Tyr75-Glu114 hydrogen-bond interaction are essential mediators of JAM-A dimer stabilization. Both monomeric and dimeric point mutants of JAM-A are capable of binding the {sigma}1 head. Because residues required for {sigma}1 head binding are found within the dimer interface, we hypothesize that a monomeric form of JAM-A is the relevant {sigma}1 binding partner. Based on elution profiles, it is formally possible that the {sigma}1-JAM-A complex contains dimeric JAM-A and monomeric {sigma}1. However, we think it unlikely that dimeric JAM-A is in the complexes, because the monomeric E121A mutant of JAM-A forms complexes with {sigma}1 that are indistinguishable by gel filtration from wild-type JAM-A-{sigma}1 complexes. It also is unlikely that monomeric {sigma}1 interacts with JAM-A, as a {sigma}1 point mutant that is incapable of trimer formation is incapable of binding JAM-A (20).


Figure 9
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  		FIGURE 9.
Predicted serotype 3 reovirus binding region in JAM-A. View of the JAM-A D1 domain dimer. One monomer is oriented as in Fig. 1C and is shown as a space-filling representation. The apposing monomer is shown as a red ribbon drawing. Residues Glu61, Lys63, and Leu72 (dark green) are required for efficient serotype 3 reovirus engagement of JAM-A and are proposed to serve as critical contacts for T3D {sigma}1. Residues Arg59, Tyr75, and Asn76 (light green) are proposed to serve as additional contacts. This figure was generated using PyMOL (54).

 
Our data support a model of reovirus-JAM-A interactions in which the T3D {sigma}1 head domain directly engages the dimer interface of JAM-A via critical contacts with Glu61 and Lys63 in beta-strand C and Leu72 in beta-strand C' and additional, less-critical contacts with nearby residues Arg59, Tyr75, and Asn76 (Figs. 1C and 9). In this model, {sigma}1 is anticipated to bind with high affinity only to JAM-A monomers. Comparisons of the hJAM-A and mJAM-A crystal structures suggest that small movements of one JAM-A monomer with respect to the apposing monomer can occur in a physiologic context (24). We envision two possible scenarios to explain how {sigma}1 accesses monomeric forms of JAM-A. In the first, small movements of JAM-A monomers with respect to one another might provide {sigma}1 the opportunity to interact weakly with residues closer to the solvent-exposed regions of the JAM-A dimer interface, such as Arg59, Tyr75, and Asn76, as the dimer shifts slightly to a more open conformation. This interaction could potentially orient {sigma}1 to access the dimer interface, where interactions that lead to high-affinity binding occur. In support of this model, mutation of Arg59, Tyr75, or Asn76 to alanine leads to reduced levels of binding and faster association and dissociation kinetics between JAM-A and the {sigma}1 head, whereas mutation of Glu61, Lys63, or Leu72 to alanine ablates {sigma}1 binding altogether. In the second, reovirus might exploit an equilibrium between monomeric and dimeric forms of JAM-A at the cell surface to engage the dimer interface of JAM-A monomers.

Our binding and infectivity data from CHO cells transfected with JAM-A mutants highlight differences in JAM-A engagement among the reovirus serotypes. Residues Arg59 and Glu61 are required for efficient T1L binding and infectivity, but only when both residues are altered simultaneously, whereas a small infectivity diminishment is observed when Lys63 is altered. In contrast, binding and infectivity of JAM-A-expressing CHO cells by reovirus strain T3SA-requires residues Glu61 or Lys63, with some additional contribution from Arg59. In a previous study, T1L virions showed diminished binding to JAM-A mutants S57K and Y75A in a plate-based binding assay (32). These two residues are located in proximity to Glu61, with Ser57 residing at the NH2 terminus of beta-strand C (24). Thus, it appears that T1L utilizes a binding site that overlaps with, but is not identical to, the serotype 3 binding site. Based on our binding and infectivity studies with CHO cells, it does not appear that T2J shares contact points with serotype 1 or 3 reoviruses. T2J showed a slight decrease in binding to CHO cells transfected with JAM-A double point mutants R59A/E61A and E61A/K63A, but infectivity was not significantly diminished in these cells. These findings constitute the first evidence that different serotypes of reovirus engage JAM-A at distinct sites or via different contact residues and, therefore, potentially with different specificities and affinities. Of note, differences in affinity for the primary cellular receptor {alpha}-dystroglycan have been shown to mediate strain-specific differences in tropism for lymphocytic choriomeningitis virus (38). It is possible that a similar mechanism contributes to serotype-dependent differences in reovirus pathogenesis. For example, a reovirus serotype that has a higher affinity for JAM-A might be capable of infecting cells that express JAM-A at a low level, whereas a reovirus serotype with a lower affinity for the receptor may not. Instead, the tropism of a reovirus with a lower affinity for JAM-A might be more dependent on the carbohydrate binding specificity of the virus or engagement of unidentified receptors on cells expressing low levels of JAM-A.

Although our data suggest non-identical binding sites in JAM-A for different serotypes or strains of reovirus, we find it noteworthy that both T1L and T2J are capable of agglutinating human erythrocytes (39), presumably via carbohydrate engagement, whereas T3SA- is unable to bind sialic acid or agglutinate mammalian erythrocytes (33, 34). Reovirus utilizes an adhesion-strengthening mechanism of attachment to host cells, in which initial low affinity interactions with carbohydrate are thought to tether the virus to the cell surface where it can diffuse laterally until it encounters and engages JAM-A in a high affinity interaction (19, 33). Therefore, it is possible that engagement of carbohydrates on the surface of CHO cells by T1L and T2J reovirus provides an advantage in the capacity of these viruses to encounter JAM-A mutants. The proximity to JAM-A afforded by carbohydrate binding, combined with avidity effects, might lead to detectable binding by T1L and T2J but undetectable binding by T3SA- to some JAM-A mutants expressed at the cell surface.

Whereas the precise nature of {sigma}1-JAM-A interactions will not be known until the structure of the attachment protein bound to the receptor is available, data generated in this study allow some speculation about the nature of these interactions. Because JAM-A serves as a receptor for all serotypes of reovirus (19, 26), we previously hypothesized that conserved residues in {sigma}1 might contribute to {sigma}1-JAM-A interactions (26, 36). While possible, our data suggest that different serotypes of reovirus engage JAM-A via distinct but perhaps overlapping residues, a result that has led us to extend our search for the JAM-A binding site in {sigma}1. Residues at positions 61 and 63 of JAM-A are critical mediators of efficient interactions with {sigma}1. However, efficient binding and infectivity of CHO cells expressing mutant forms of JAM-A is achieved whether there is an aspartate or glutamate at position 61 or an arginine or lysine at position 63. These results demonstrate that the side chain charge of residues at positions 61 and 63 is important for {sigma}1 binding, whereas the length of the side chain is not. Because the charged residues required for efficient {sigma}1 binding participate in salt-bridge interactions that stabilize the dimer interface, we think that charged residues in {sigma}1 form similar salt-bridge interactions with JAM-A, mimicking the dimer interface. There are several clusters of solvent-exposed charged residues in {sigma}1. Furthermore, beta-sheet BADG in {sigma}1 can be superimposed onto JAM-A beta-sheet GFCC' with low root mean square deviations (31). Using a newly developed plasmid-based reverse genetics system for reovirus (40), we can make directed mutations in the JAM-A binding region of {sigma}1 to directly test this hypothesis. Non-JAM-A-binding reoviruses might prove invaluable as tools to define the independent contributions of JAM-A and carbohydrate co-receptors to tropism in vivo.

In this study, we identified reovirus binding determinants in the most membrane-distal Ig-like domain of JAM-A. We also provided the first evidence that reoviruses of different serotypes engage JAM-A via distinct contact residues. It is interesting that so many viruses have adapted to utilize Ig superfamily members as attachment moieties (19, 41-50). Even more intriguing is the observation that viruses as evolutionarily diverse as adenovirus, HIV, and reovirus bind the same structural regions of Ig superfamily molecules (51, 52). Perhaps this family of molecules, which mediate diverse protein-protein recognition functions such as cell-cell adhesion and high affinity antigen binding, are well suited to serve as virus receptors due to the adhesive nature of their D1 domains (53). Future studies with reovirus and other viruses may serve to enhance an understanding of the co-evolution of cell-adhesion molecules, viral attachment proteins, and immune recognition.


   FOOTNOTES
 
* This work was supported by United States Public Health Service Awards T32 GM08554 (to K. M. G.), R37 AI38296 (to T. S. D.), R01 GM67853 (to T. S. D. and T. S.), and the Elizabeth B. Lamb Center for Pediatric Research. Additional support was provided by the Vanderbilt-Ingram Cancer Center and the Vanderbilt Diabetes Research and Training Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1. Back

1 To whom correspondence should be addressed: Lamb Center for Pediatric Research, D7235 MCN, 1161 21st Ave. S., Nashville, TN 37232-2581. Tel.: 615-343-8911; Fax: 615-343-9723; E-mail: terry.dermody{at}vanderbilt.edu.

2 The abbreviations used are: reoviruses, mammalian orthoreoviruses; T1L, type 1 Lang; T2J, type 2 Jones; T3D, type 3 Dearing; JAM-A, junctional adhesion molecule-A; Ig, immunoglobulin; hJAM-A, human JAM-A; mJAM-A, murine JAM-A; CAR, coxsackievirus and adenovirus receptor; CHO, Chinese hamster ovary; GST, glutathione S-transferase; PBS, phosphate-buffered saline; SPR, surface plasmon resonance; GST-JAM-A, NH2-terminal glutathione S-transferase fusion with the extracellular region of JAM-A; HIV, human immunodeficiency virus. Back


   ACKNOWLEDGMENTS
 
We acknowledge Annukkah Antar, Jim Chappell, Takeshi Kobayashi, Sameep Naik, and Bernhard Paetzhold for helpful discussions and ideas. We thank Hannah Fish and the technicians at the Veterans Affairs Flow Cytometry Resource Center for expert technical assistance.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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