INTRODUCTION

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Mammalian reoviruses display broad cell and tissue tropism in vivo (reviewed in reference 43) and infect numerous types of cultured cells (reviewed in reference 43). Viral attachment is mediated by outer-capsid protein 1 (23, 26, 46), which is encoded by the S1 gene segment (27, 28, 49). Some strain-dependent patterns of reovirus spread and tropism in newborn mice segregate with the S1 gene (20, 44, 47, 48), indicating that 1 plays a key role in the pathogenesis of reovirus-induced disease. Within the murine central nervous system, type 1 reovirus infects ependymal cells, whereas type 3 reovirus infects neurons (47). This difference in cell tropism is determined by receptor specificities of type 1 and type 3 1 proteins (13, 41). The 1 protein also is responsible for strain-dependent differences in the attachment of virions to mammalian erythrocytes (9, 12, 49) and murine erythroleukemia (MEL) cells (9, 38). Sialic acid serves as a receptor for type 3 reovirus on MEL cells (9, 38) and also can function as a receptor on murine L929 (L) cells (11, 18, 30, 32, 33, 37). Type 3 1 protein also binds another receptor in addition to sialic acid (6, 8, 29, 30), but the identity of this receptor has not been determined.

The 1 protein is a homo-oligomer located at the vertices of the virion icosahedron (3, 16, 17, 24, 40). Electron microscopic analyses of virion-associated 1 (17), 1 isolated from virions (16, 17), and expressed 1 (1) reveal that 1 protein is a fibrous molecule consisting of an elongated tail domain and a virion-distal globular head domain. Five distinct, tandemly arranged morphologic regions of 1, designated T(i), T(ii), T(iii), T(iv), and H, have been discerned using digitized image enhancements of 1 electron micrographs (16). These morphologic regions correlate well with predictions of 1 secondary structure (31). Sequences represented by morphologic regions in the tail are proposed to form an amino-terminal short (~25-residue) -helical coiled-coil and turn/loop [T(i)], a long (~150-residue) -helical coiled-coil [T(ii)], an eight-stranded cross -sheet [T(iii), ~65 residues], and two short regions of -helical coiled-coil (two to three heptad repeats each) that flank a four-stranded cross -sheet [T(iv), ~75 residues]. Amino acid sequences carboxy terminal to morphologic region T(iv) (~145 residues) are predicted to assume a more complex arrangement of secondary structures corresponding to the globular head domain (H) of 1.

Previous studies of type 3 1 protein indicate that sequences in the tail bind sialic acid. Treatment of virions of reovirus strain type 3 Dearing (T3D) with protease to generate infectious subvirion particles (ISVPs), which are intermediates in reovirus disassembly, results in cleavage of 1 and loss of the head and part of the T(iv) domain (8, 30). ISVPs retain the capacity for hemagglutination, demonstrating that sequences amino terminal to the cleavage site, which has been identified as residue 245 (8), are sufficient to bind sialic acid. Concordantly, sequence polymorphism within the fourth predicted -strand of morphologic region T(iii), amino acid residues 198 to 204, determine the capacity of type 3 reoviruses to mediate hemagglutination and bind and infect MEL cells (9, 12). However, it is not known whether sequences within T(iii) constitute part of a sialic acid-binding domain or control this function at another site. Hemagglutination mediated by type 1 reovirus virions also is dependent on carbohydrate binding (25), although the specific carbohydrate has not been identified. Available evidence indicates that type 1 reovirus does not bind sialic acid (9, 12, 32, 38). Moreover, nothing is known about sequences in type 1 1 that mediate receptor binding.

To identify sequences in 1 protein that bind carbohydrate, we generated chimeric and truncated 1 proteins using the 1 sequences of reovirus strains type 1 Lang (T1L) and T3D. Expressed 1 proteins were tested in assays of carbohydrate binding, and morphologic region T(iii) was identified as the minimum structural domain in type 3 1 required to bind sialic acid. In contrast, sequences in the T(iv) region of type 1 1 protein were found to be required for hemagglutination, indicating that the minimal carbohydrate-binding domain in type 1 1 resides in the carboxy-terminal one-half of the molecule. Results from this study confirm the presence of discrete receptor-binding domains in the head and tail domains of type 3 1 and indicate that the topology of receptor-binding domains differs between type 1 and type 3 1 proteins.

	MATERIALS AND METHODS

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Cells and viruses. Spinner-adapted L cells were grown in either suspension or monolayer cultures in Joklik's modified Eagle's minimal essential medium (Irvine Scientific, Santa Ana, Calif.) that was supplemented to contain 5% fetal bovine serum (Intergen, Purchase, N.Y.), 2 mM L-glutamine, 100 U of penicillin per ml, 100 µg of streptomycin per ml, and 0.25 µg of amphotericin (Irvine) per ml. Spodoptera frugiperda Sf21 and Trichoplusia ni Tn High Five insect cells were grown in either suspension or monolayer cultures using Grace's medium (Gibco, Grand Island, N.Y.) supplemented to contain 10% fetal bovine serum and 100 U of penicillin, 100 µg of streptomycin, and 0.25 µg of amphotericin per ml. Reovirus strains T1L and T3D are laboratory stocks. Purified virion preparations of reovirus were made using second- and third-passage L-cell lysate stocks of twice-plaque-purified reovirus as previously described (17). To obtain purified virions containing ³⁵S-labeled proteins, Easy Tag Express-[³⁵S] protein labeling mix (NEN, Boston, Mass.) was added to cell suspensions (~12.5 µCi per ml) at the initiation of infection. Baculovirus vector strains were derived from Autographa californica nuclear polyhedrosis virus (Clontech Laboratories, Palo Alto, Calif.). Recombinant baculoviruses containing wild-type (wt) and mutant S1 gene cDNAs were generated and propagated as previously described (8).

Construction of recombinant S1 gene cDNAs. Using primers specific for noncoding regions of the S1 gene, 1-encoding S1 gene cDNAs were generated by reverse transcription-PCR (22) and cloned into the pCR2.1 vector (Invitrogen, San Diego, Calif.). Cloned T1L and T3D S1 gene cDNAs were used as template to generate chimeric and truncated S1 genes. Cloned chimeric S1 gene cDNAs were used as template to generate two additional chimeric constructs, 1-1-3-3-1 and 1-1-3-1-1.

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Chimeric and truncated 1 proteins used for studies of carbohydrate binding. (A) Model of 1 structure depicting predicted secondary structures and correlating primary amino acid sequence with morphologic regions of 1 (T(i), T(ii), T(iii), T(iv), and H) seen in computer-processed electron micrographic images of 1 protein isolated from virions (16, 31). In the simplified version of this model shown below, -helical regions of the tail domain are indicated by horizontal bars and regions of -strand/-turn are symbolized by ovoid shapes. The globular head domain (H) is depicted as a circle. (B) Sequence features of chimeric and truncated 1 constructs. White symbols represent sequences derived from T1L 1, and black symbols represent sequences derived from T3D 1. Constructs are named according to the parental origin of 1 morphologic regions as previously described (16, 31): 1, sequences derived from T1L 1; and 3, sequences derived from T3D 1. Sequences corresponding to morphologic regions T(i), T(ii), T(iii), T(iv), and H are represented by the first, second, third, fourth, and fifth characters, respectively, of construct names. , deleted sequences. The sequences of T1L and T3D 1 comprising each construct are denoted by numbers corresponding to 1 amino acid residues reported by Nibert et al. (31) (T1L) and Bassel-Duby et al. (2) (T3D).

Expression of and purification of recombinant 1 proteins. Second- or third-passage stocks of recombinant baculovirus were used to infect insect cell monolayers (2.4 × 10⁷ cells) at a multiplicity of infection of 5 PFU per cell. After 72 h of incubation, cells were harvested and resuspended in 2 ml of phosphate-buffered saline (PBS; 140 mM NaCl, 2.7 mM KCl, 8.3 mM Na₂HPO₄, 1.5 mM KH₂PO₄) containing 5 mM phenylmethylsulfonyl fluoride and Complete, Mini, EDTA-free protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, Ind.). Cells were lysed by sonication, and supernatants were cleared of debris by centrifugation. To produce ³⁵S-labeled 1 proteins, culture medium was replaced after 24 h incubation with methionine-free Grace's medium (Gibco) supplemented to contain 10% fetal bovine serum, 100 U of penicillin, 100 µg of streptomycin, and 0.25 µg of amphotericin per ml, and 10 µCi of Easy Tag Express-[³⁵S] protein labeling mix per ml. Cells were harvested after 48 h of incubation in ³⁵S-containing medium. Expressed 1 proteins containing an intact head domain were recovered from cell lysates using T1L 1-specific monoclonal antibody (MAb) 5C6 (45) or T3D 1-specific MAb 9BG5 (6) conjugated to cyanogen bromide-activated Sepharose (Pharmacia, Uppsala, Sweden). Beads containing adsorbed 1 protein were washed five times with buffer consisting of 50 mM Tris (pH 8), 1.2 M NaCl, 0.4% sodium dodecyl sulfate (SDS), 0.2% Triton X-100, and 5 mM EGTA, followed by three washes with a solution of 50 mM triethanolamine (pH 11.6), 0.5 M NaCl, and 0.1% Triton X-100. Beads then were washed three times with virion storage buffer (150 mM NaCl, 10 mM MgCl₂, 10 mM Tris [pH 7.5]). Truncation mutants of 1 lacking the head domain were purified from cell lysates by precipitation with rabbit antiserum raised to virions of strain T1L or T3D. Precipitates were recovered using protein A-Sepharose (Pharmacia) and washed as described above.

Protease treatment of expressed 1 proteins. Aliquots of 1-containing Sepharose beads in virion storage buffer were incubated at 15°C with 0, 0.1, 1.0, or 10 µg of N-p-tosyl-L-sulfonyl phenylalanyl chloromethyl ketone-treated bovine trypsin (Sigma Chemical Co., St. Louis, Mo.) per ml for 75 min. Reaction mixtures were mixed 1:1 with 2× protein sample buffer (3) and incubated at 100°C for 10 min. Reaction products were resolved by polyacrylamide gel electrophoresis (PAGE) in an SDS-10% polyacrylamide gel and visualized by autoradiography.

Assessment of 1 multimerization status during SDS-PAGE. ³⁵S-labeled purified reovirus virions (5 × 10¹⁰ particles) or expressed 1 proteins adsorbed to MAb-conjugated Sepharose were suspended in protein sample buffer (62.5 mM Tris, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue [3]) adjusted to pH 6.8 or 8.3 and incubated at 100°C (pH 6.8) or 60°C (pH 8.3) for 10 min. Proteins were resolved in SDS-10% polyacrylamide gels and visualized by autoradiography.

Hemagglutination assay. Clarified insect cell lysates containing expressed 1 proteins were prepared as described above. Lysates were aliquoted into 96-well U-bottom microtiter plates (Costar, Cambridge, Mass.) and serially diluted twofold in 0.05 ml of PBS. Human type O⁺ erythrocytes or calf erythrocytes (Colorado Serum Co., Denver, Colo.) were washed twice in PBS and resuspended at a concentration of 1% (vol/vol). Erythrocytes (0.05 ml) were added to wells containing expressed protein and incubated at 4°C for at least 2 h. A partial or complete shield of erythrocytes on the well bottom was interpreted as a positive hemagglutination result; a smooth, round button of erythrocytes was interpreted as negative. The highest dilution of lysate sufficient to produce hemagglutination was designated to equal 1 hemagglutination unit (HA unit).

Glycophorin-binding assay. Human type MN glycophorin or asialoglycophorin (Sigma) was biotinylated using ENZOTIN reagent (Enzo Diagnostics, Farmingdale, N.Y.) according to the supplier's instructions. Expressed 1 proteins containing intact head domains were purified from insect cell lysates using MAb-conjugated Sepharose as described above, and 0.004 ml of the 1 preparation was diluted in a total volume of 0.1 ml of PBS supplemented to contain 0.05% Tween 20 (J. T. Baker, Phillipsburg, N.J.) (PBS-T) and 0.034 µg of biotinylated glycophorin or biotinylated asialoglycophorin per ml. Beads were incubated at room temperature (RT) for 15 min, rinsed with three washes in 0.5 ml of PBS-T, then incubated at RT in 0.1 ml of PBS-T with 0.2 U of streptavidin-peroxidase conjugate (Boehringer) per ml for 15 min, and rinsed again with three washes in 0.5 ml of PBS-T. Finally, beads were incubated at RT in 0.1 ml of solution of chromogenic substrate [0.04% (wt/vol) 2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS; Sigma) in 0.05 M phosphate-citrate buffer (pH 5.0) containing 0.0075% H₂O₂] for 15 min. Absorbance at 405 nm was determined using a Thermomax microplate reader (Molecular Devices, Crawley, United Kingdom). Nonspecific binding of biotinylated glycophorin and biotinylated asialoglycophorin was determined exactly as above, using MAb-conjugated Sepharose without 1 protein.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Expression and folding of chimeric and truncated 1 proteins. To identify structural domains in 1 that bind carbohydrate, the 1 proteins of reovirus strains T1L and T3D, seven T1L-T3D chimeric 1 proteins, and three 1 truncation mutants shown in Fig. 1 were expressed in insect cells using baculovirus vectors. Sequences exchanged among the 1 chimeras correspond to morphologic regions of 1 identified in electron micrographs (16). The specific exchange loci were at conserved positions between sequence regions distinguished by unique patterns of repeating apolar amino acids (31). MAbs specific for T1L and T3D 1 proteins, 5C6 (45) and 9BG5 (6), respectively, were used to purify expressed 1 proteins from cell lysates. The reactivity of MAbs 5C6 and 9BG5 with chimeric 1 proteins demonstrates that these antibodies recognize epitopes in the 1 head (Fig. 2). MAb 5C6 bound wt T1L 1 and chimera 3-3-3-3-1, which has head-forming sequences derived from only T1L 1. In contrast, MAb 9BG5 bound wt T3D 1 and chimera 1-1-1-1-3, which has head-forming sequences from only T3D 1. Consistent with this pattern, chimera 3-3-3-3-1 was not bound by MAb 9BG5, and chimera 1-1-1-1-3 was not bound by MAb 5C6. Either MAb 5C6 or MAb 9BG5 was capable of recovering 1 proteins containing a head domain (Fig. 3 to 5), thus confirming expression of our panel of recombinant S1 gene constructs. Additionally, these antibodies bind conformationally sensitive epitopes in 1 (4, 35, 45), which suggests that expressed 1 proteins are properly folded.

View larger version (63K): [in this window] [in a new window]   FIG. 2.   Binding of anti-1 MAbs to expressed 1 proteins. 35S-labeled wt and chimeric 1 proteins were expressed in insect cells, and cell lysates were incubated with T1L 1-specific MAb 5C6 and T3D 1-specific MAb 9BG5 conjugated to Sepharose. Sepharose was washed under stringent conditions of salt, detergent, and pH to remove nonspecifically associated proteins, followed by incubation in protein sample buffer at 100°C to release antibody-bound 1 protein. Samples were resolved in an SDS-10% polyacrylamide gel and visualized by autoradiography. Positions of molecular weight standards (in kilodaltons) are shown.

View larger version (69K): [in this window] [in a new window]   FIG. 3.   Effect of trypsin treatment on wt and chimeric 1 proteins. 35S-labeled 1 proteins were purified from insect cell lysates using anti-1 MAb 5C6 or 9BG5 conjugated to Sepharose followed by treatment at 15°C with 0, 0.1, 1.0, or 10 µg of bovine trypsin per ml for 75 min. Reaction mixtures were heated at 100°C in protein sample buffer; digestion products were resolved in an SDS-10% polyacrylamide gel and visualized by autoradiography. Sequences corresponding to the T(iv) region of proteins shown in panel A are derived from T1L 1, whereas sequences corresponding to the T(iv) region of proteins shown in panel B are derived from T3D 1. , 0 to 10 µg of trypsin [TRY] per ml. Positions of molecular weight standards (in kilodaltons) are shown.

Identification of a protease-sensitive domain in morphologic region T(iv) of T3D 1. To identify protease-sensitive domains in T3D 1, and to confirm that sequences in chimeric 1 proteins fold into their native conformations, each protein was purified from cell lysates and treated with trypsin. Trypsin cleaves expressed (15) or virion-associated (8) T3D 1 at Arg²⁴⁵ within morphologic region T(iv). T1L 1 is resistant to cleavage by trypsin (5, 8, 14, 15, 30). Likewise, wt T1L 1 and chimeric proteins containing the T(iv) region of T1L 1 were resistant to cleavage (Fig. 3A). T1L 1 truncation mutant 1-1-1-1-, which contains tail-forming sequences only, was resistant to trypsin cleavage at lower enzyme concentrations but exhibited partial cleavage susceptibility at higher concentrations (~10 µg of trypsin per ml) (Fig. 4); the predominant cleavage product migrated slightly faster than untreated protein. The wt T3D 1 protein was cleaved by trypsin, as was each 1 construct containing the T(iv) region of T3D 1 (Fig. 3B and 4). Trypsin treatment of this group of expressed 1 proteins resulted in the generation of stable cleavage products of approximately 25 kDa, which is characteristic of trypsin-treated wt T3D 1 (8, 14, 15, 24, 30, 52). Thus, the pattern of susceptibility of chimeric 1 proteins to cleavage by trypsin confirms the location of a protease-sensitive region in T3D 1, T(iv), and is consistent with native folding of these molecules.

View larger version (58K): [in this window] [in a new window]   FIG. 4.   Effect of trypsin treatment on truncated 1 proteins. 35S-labeled 1 proteins were purified from insect cell lysates using antireovirus serum plus protein A-Sepharose, followed by treatment at 15°C with 0, 0.1, 1.0, or 10 µg of bovine trypsin per ml for 75 min. Reaction mixtures were heated at 100°C in protein sample buffer; digestion products were resolved in an SDS-10% polyacrylamide gel and visualized by autoradiography. , 0 to 10 µg of trypsin [TRY] per ml. Positions of untreated 1 deletion mutants are indicated by arrows. Positions of molecular weight standards (in kilodaltons) are shown.

Identification of a domain in 1 important for multimer stability. As an additional test of protein folding, expressed 1 proteins were examined for the capacity to maintain oligomeric structure during SDS-PAGE. In previous studies, it was shown that virion-associated (3, 51) and expressed (3, 24, 40) T3D 1 protein migrates as an oligomer in SDS-polyacrylamide gels after solubilization in protein sample buffer under specific conditions of temperature and pH. When virions of T1L and T3D were disrupted at 60°C in pH 8.3 sample buffer, the 1 protein of T1L migrated as a monomer, whereas T3D 1 migrated as an oligomer (Fig. 5A). Incubation of virions at 100°C in pH 6.8 sample buffer resulted in the appearance of 1 monomers only. This pattern was replicated by wt T1L and T3D 1 proteins expressed in insect cells (Fig. 5B and C). Chimeric and truncated 1 proteins containing T1L T(i) and T(ii) sequences migrated as monomers under these conditions, and 1 proteins with T(i) and T(ii) sequences derived from T3D migrated as oligomers. Thus, results obtained using chimeric and truncated 1 proteins show that sequences constituting morphologic regions T(i) and T(ii), which are predicted to form almost exclusively -helical coiled coil, determine the difference in stability of T1L and T3D 1 oligomers in SDS-polyacrylamide gels. Additionally, these results indicate that native tertiary and quaternary structures are maintained in the amino-terminal aspect of the tail domain of expressed 1 proteins used for our studies.

View larger version (75K): [in this window] [in a new window]   FIG. 5.   Oligomer stability of chimeric and truncated 1 proteins. (A) 35S-labeled purified T1L and T3D virions were incubated at 100°C in pH 6.8 protein sample buffer or 60°C in pH 8.3 protein sample buffer for 10 min. Reaction products were resolved in an SDS-10% polyacrylamide gel and visualized by autoradiography. Viral structural proteins are labeled. (B and C) 35S-labeled full-length wt and chimeric 1 proteins were purified from insect cell lysates using anti-1 MAb 5C6 or 9BG5 conjugated to Sepharose and treated as for panel A. Positions of molecular weight standards (in kilodaltons) are shown. (D) 35S-labeled truncated 1 proteins were purified from insect cell lysates using MAb 9BG5 conjugated to Sepharose or antireovirus serum plus protein A-Sepharose and treated as for panel A. Positions of molecular weight standards (in kilodaltons) are shown. 1*, bands corresponding to 1 oligomers.

Hemagglutination activity of chimeric 1 proteins. To identify sequences in 1 that bind carbohydrate, chimeric and truncated proteins derived from T1L and T3D 1 were expressed in insect cells, and 1 proteins contained in cell lysates were tested for the capacity to mediate hemagglutination. In previous studies, it was shown that virions of T1L agglutinate human but not bovine erythrocytes, whereas virions of T3D agglutinate bovine erythrocytes more efficiently than human erythrocytes (9, 12). In accordance with this profile, wt T1L 1 agglutinated human but not bovine erythrocytes, whereas wt T3D 1 exhibited the reverse pattern (Fig. 6).

View larger version (26K): [in this window] [in a new window]   FIG. 6.   Hemagglutination activity of chimeric and truncated 1 proteins. Insect cells (1.2 × 107) were inoculated with 1-expressing recombinant baculoviruses, and cultures were harvested after 4 days of incubation. Cells were resuspended in 1 ml of PBS supplemented with protease inhibitors and disrupted by sonication, followed by centrifugation to clarify lysates. Supernatants were diluted twofold serially, and either human O+ or calf erythrocytes were added to each well. Hemagglutination reactions were scored after incubation at 4°C for at least 2 h. The hemagglutination pattern of each expressed 1 protein was determined in 8 to 20 independent experiments, and a positive result was defined as 8 HA units of activity per 100 µl of lysate. For inclusion in the analysis, any given construct was required to display hemagglutination in two or more experiments; otherwise, the construct was deemed hemagglutination negative and assigned an HA titer of zero. Results are expressed as the mean log2 HA titer. Error bars indicate standard deviations of the mean.

Binding of expressed 1 proteins to sialic acid. To confirm that sequences in morphologic region T(iii) of type 3 1 protein bind sialic acid, we performed a quantitative assay to assess sialic acid-dependent binding of expressed 1 proteins to glycophorin. Glycophorin is a highly sialylated glycoprotein on the erythrocyte surface (10), and sialic acid residues of human erythrocyte glycophorin are bound by type 3 reovirus (34). Expressed 1 proteins were captured on Sepharose beads conjugated to MAb 5C6 or 9BG5 and treated with biotinylated human glycophorin or asialoglycophorin. Expressed 1 proteins segregated into two groups according to their capacity to bind glycophorin relative to asialoglycophorin (Fig. 7). The first group, which includes wt T1L 1 and chimeric 1 proteins 1-1-1-1-3 and 3-3-1-1-1, did not exhibit appreciable binding to glycophorin over background. The second group, which includes wt T3D 1 and recombinant 1 proteins 3--3-3-3, 1-1-3-3-3, 3-3-3-1-1, 1-1-3-3-1, 3-3-3-3-1, and 1-1-3-1-1, bound glycophorin 2- to 40-fold more efficiently than asialoglycophorin. Thus, the latter group of recombinant 1 proteins exhibited sialic acid-dependent glycophorin binding. The only type 3 1 sequences common to all members of this group correspond to morphologic region T(iii), which indicates that T(iii) sequences are sufficient to mediate binding of sialic acid by type 3 1 protein. Accordingly, expressed proteins that exhibited only background binding to glycophorin (wt T1L, 1-1-1-1-3, and 3-3-1-1-1) contain T(iii) sequences derived from type 1 1. It was not possible to test truncation mutants 1-1-1-1- and 3-3-3-3- in the glycophorin-binding assay since these experiments would require domain-specific antibodies that bind the 1 tail, and such antibodies currently are unavailable. Results of the glycophorin-binding and hemagglutination assays demonstrate that sialic acid is bound by sequences within morphologic region T(iii) of the type 3 1 tail domain, amino acid residues 175 to 234. 

View larger version (16K): [in this window] [in a new window]   FIG. 7.   Binding of expressed 1 proteins to glycophorin and asialoglycophorin. Expressed wt and chimeric 1 proteins were purified from insect cell lysates using MAb-conjugated Sepharose, followed by treatment of equivalent amounts of 1 with biotinylated glycophorin or biotinylated asialoglycophorin. Sepharose beads then were incubated with streptavidin-peroxidase conjugate, followed by the addition of chromogenic substrate. The reaction was allowed to develop for 15 min, and supernatant absorbance at 405 nm was determined using a microplate reader. Nonspecific binding of glycophorin and asialoglycophorin was determined using MAb-conjugated Sepharose without 1 protein, and raw absorbance data were corrected accordingly to derive values of specific absorbance. The results are presented as the mean log10 ratio of specific glycophorin binding to specific asialoglycophorin binding by 1. Error bars represent the standard deviation of three independent experiments.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

This study was designed to identify the minimal domain in reovirus attachment protein 1 capable of binding carbohydrate. Using a panel of expressed chimeric and truncated 1 proteins derived from strains T1L and T3D, we found that sequences predicted to form an eight-stranded -sheet in the T3D 1 tail are sufficient to mediate binding of sialic acid by T3D 1; the head domain, which contains sequences that bind an unidentified receptor on L cells (8, 14, 29, 30, 42, 50, 52), is dispensable for sialic acid binding. Carbohydrate binding by type 1 1 protein also is mediated by sequences in the tail predicted to form a -sheet structure; however, these sequences are contained in a morphologic region of the tail, T(iv), that is distinct from that involved in binding sialic acid by type 3 1. The organization of receptor-binding domains in 1 protein has important implications concerning mechanisms used by reovirus to achieve a stable virus-receptor complex that facilitates viral entry into cells.

T3D 1 protein expressed in prokaryotic or eukaryotic cells retains the capacity to bind sialic acid (29, 34) and mediate hemagglutination (1, 26). Our approach was to exploit differences in carbohydrate specificity of type 1 and type 3 1 and unique hemagglutination patterns mediated by these proteins to identify sequences in 1 that bind carbohydrate. We took advantage of the unique domain organization of 1 protein (8, 16, 30, 31) to design chimeric molecules and truncation mutants that were likely to isolate independent functional units of sequence. Exchange loci in chimeric proteins were created at positions of conserved residues located between sequence units corresponding to morphologic regions seen in electron micrographic images of 1 protein (16) (Fig. 1). Assays to test proper folding of 1 sequences, including oligomer stability, susceptibility to protease cleavage, and binding to conformationally sensitive MAbs, indicate that natural 1 conformation was preserved in the amino-terminal portion of the tail domain, carboxy-terminal portion of the tail domain, and head domain, respectively, of baculovirus-expressed 1 proteins (Fig. 2 to 5). These results support a model of modular 1 structure (31) and validate the use of chimeric and truncated 1 proteins to define structure-function relationships.

MAbs 5C6 and 9BG5, specific for type 1 and type 3 1 proteins, respectively (6, 45), were used to test fidelity of 1 folding and for purification of 1 from insect cell lysates. MAb 9BG5 binds sequences in the T3D 1 head domain (4, 14, 29, 42, 52). Consistent with the epitope specificity of 9BG5, wt T3D 1 and chimeric and truncated 1 proteins containing the T3D head were bound efficiently and specifically by this MAb (Fig. 2 to 5). Prior to this study, the domain in T1L 1 bound by MAb 5C6 was not known. MAb 5C6 specifically bound expressed 1 proteins containing the T1L 1 head domain, and sequences in the head but not the tail were sufficient for 5C6 binding (Fig. 2). Therefore, these results indicate that MAb 5C6 binds the head domain of T1L 1.

In a previous study, we found that a sequence polymorphism in the T(iv) region of type 3 1 protein, isoleucine or threonine at amino acid position 249, is a determinant of T3D 1 cleavage susceptibility at Arg²⁴⁵ during treatment of virions with trypsin to generate ISVPs (8). In T3D 1, position 249 is occupied by a threonine residue, which interrupts a heptad repeat sequence predicted to form -helical coiled coil (31). Although the specific amino acid at position 249 regulates cleavage susceptibility of T3D 1 protein, it is possible that sequences outside the T(iv) region are also required. The cleavage profiles of chimeric and truncated 1 proteins demonstrate that all sequences necessary for cleavage of expressed T3D at Arg²⁴⁵ by trypsin are contained within morphologic region T(iv) (Fig. 3 and 4). These results support the hypothesis that the isoleucine-threonine polymorphism at position 249 is the minimal determinant of T3D 1 susceptibility to protease.

Previous studies of T3D 1 oligomerization demonstrated that oligomer stability under conditions of SDS-PAGE is mediated by sequences corresponding to the amino-terminal half of T3D 1 (24, 40). In agreement with these findings, baculovirus-expressed T3D 1 tail-forming sequences representing morphologic regions T(i) through T(iv) migrated as oligomers in SDS-polyacrylamide gels (Fig. 5C and D). Interestingly, the T1L 1 oligomer is labile under the same conditions. This difference in oligomer stability was mapped to sequences corresponding to the T(i) and T(ii) regions of T1L and T3D 1 proteins, which is consistent with the finding that the amino-terminal 161 amino acids of T3D 1 protein form a stable oligomer (24) and that destabilizing mutations in T3D 1 selected during persistent reovirus infection of cultured L cells occur in the T(ii) region (51). Thus, differences in T1L and T3D 1 oligomer stability are determined by strain-specific properties of the extended region of predicted -helical coiled coil in the tail domain. Accordingly, T3D 1-derived deletion mutant 3--3-3-3, in which about 75% of region T(ii) sequences are missing, migrated as a monomer under these conditions of SDS-PAGE. Since the entirety of region T(i) sequences are intact in this deletion mutant, these results suggest that region T(ii) sequences alone determine oligomer stability.

The hemagglutination characteristics of chimeric and truncated 1 proteins used for these studies is the strongest evidence to date that morphologic region T(iii) contains the complete hemagglutination domain of type 3 1 protein (Fig. 6). These results agree with our previous findings indicating that the hemagglutination domain of type 3 1 protein is located amino terminal to Arg²⁴⁵ in the tail (8, 30) and that sequence polymorphism within T(iii) is a determinant of hemagglutination capacity (9, 12) and sialic acid-dependent viral infectivity (9). An intriguing finding from the domain mapping experiments was that the T1L hemagglutination pattern segregated with sequences in morphologic region T(iv). Prior to this study, no functions had been ascribed to any specific region of type 1 1 protein, but these results indicate that a carbohydrate-binding domain resides in the carboxy-terminal aspect of the tail.

Because a construct corresponding to sequences found only in the T1L 1 tail was unable to mediate hemagglutination in our experimental system, it is possible that the head domain is required for proper folding of T(iv) sequences into a conformation functional for carbohydrate binding. Irregular folding of the 1-1-1-1- T(iv) region is suggested by results of trypsin treatment of this construct; at higher concentrations of trypsin, a cleavage product that migrated slightly faster than untreated 1-1-1-1- was produced, which is indicative of subterminal cleavage, possibly within morphologic region T(iv) (Fig. 4). The loss of T1L 1 hemagglutination activity by truncation of head-forming sequences contrasts with the behavior of construct 3-3-3-3-, in which hemagglutination activity remains fully intact (and expanded to include human erythrocytes) in the absence of a head domain. Perhaps this differential effect of the head domain on hemagglutination by type 1 and type 3 1 proteins reflects the relative proximity of the hemagglutination domain to the head and the potential differences in steric interactions that would follow.

Using wt and chimeric 1 proteins in a quantitative assay of sialic acid binding, we were able to show that specific binding to sialic acid by type 3 1 also segregates with morphologic region T(iii) (Fig. 7). This finding confirms and extends the results of hemagglutination assays as a biologically relevant assessment of 1 function. Sialic acid is the minimal determinant of type 3 reovirus attachment (33), and results obtained using expressed 1, glycophorin, and asialoglycophorin indicate that virus binding to sialic acid occurs through direct interactions between this carbohydrate and sequences in morphologic region T(iii). In previous studies (9, 12), we identified three amino acid residues within a single predicted -strand of T(iii)Asn¹⁹⁸, Arg²⁰², and Pro²⁰⁴that determine the capacity of reovirus to bind sialic acid and carry out sialic acid-dependent infection of cultured cells. Findings made in the present study indicate that the effect of these residues on receptor binding is a local one, confined to region T(iii), and provide support for a model in which Asn¹⁹⁸, Arg²⁰², and Pro²⁰⁴ comprise part of the 1 sialic acid-binding domain.

Among 1 constructs containing the T3D T(iii) region, only truncation mutant 3-3-3-3- and chimeric 1 protein 1-1-3-3-3 agglutinated human erythrocytes in addition to bovine erythrocytes (Fig. 6). Despite this observation, all constructs containing morphologic region T(iii) from T3D 1 were able to bind sialic acid presented on human glycophorin (Fig. 7), which is the sialylated glycoprotein bound by type 3 reovirus on human erythrocytes (34). The glycophorin-binding assay was designed to test the capacity of expressed 1 proteins to specifically bind sialic acid, and the inability of most 1 proteins to agglutinate human erythrocytes may reflect the more complex 1-receptor interactions that occur during hemagglutination when virion-associated 1 molecules cross-link cell surface glycophorin, and perhaps other sialoglycoconjugates, on adjacent cells.

The capacity of constructs 3-3-3-3- and 1-1-3-3-3 to agglutinate both bovine and human erythrocytes suggests that sequences in the head domain of T3D 1 influence hemagglutination capacity of sequences in the tail and that certain combinations of sequences from T1L and T3D 1 proteins enhance agglutination of human erythrocytes by T3D 1-derived sequences. Alternatively, because T3D 1 protein has a lower avidity for human than for bovine erythrocytes (9, 12), relative amounts of chimeric and truncated 1 proteins in insect cell lysates may influence the capacity of T3D 1-derived sequences to mediate agglutination of human erythrocytes.

We observed that sialic acid-dependent binding of glycophorin by expressed 1 proteins spanned a range of 20-fold. This variability may be due to the heterogeneous sequence contexts of 1 proteins containing morphologic region T(iii) of type 3 1, leading to differences in individual affinities for sialic acid. Although the amount of expressed 1 protein used for the glycophorin-binding assays was not quantitated, the results of sialoglycophorin binding were normalized to asialoglycophorin binding for each construct, which should have minimized the effects of 1 concentration differences on the binding results.

Our findings permit discrete topographical assignment of receptor-binding activities of type 3 1 protein to morphologic region T(iii) of the tail (this study) and the head domain (8, 14, 29, 30, 42, 50, 52) (Fig. 8). These results explain our previous findings that ISVPs of T3D containing a cleaved 1 protein bind sialylated cellular receptors (8, 30) and that sequences in morphologic region T(iii) determine viral capacity for sialic acid binding (9). Although the two receptor-binding domains are clearly distinct in images of isolated 1 protein (16), the steric relationship of the head and T(iii) on the virion surface is not known. The 1 protein exhibits considerable flexibility (1, 7, 16, 17), and it is possible that sequences in these two regions of the molecule approximate one another when 1 rests in its virion-associated conformation. If so, the relative proximity of receptor-binding domains in the head and tail may influence viral receptor specificity, stability of viral attachment, or subsequent events, such as endocytic uptake of virus and intravesicular viral disassembly. The mechanism of receptor engagement employed by type 3 1 protein may not be shared by type 1 1 since the carbohydrate-binding domain of the latter was mapped to more head-proximal sequences, morphologic region T(iv). Thus, diversity in viral attachment strategies may account for some serotype-dependent patterns of reovirus biology that segregate with 1 protein (9, 13, 36, 38, 41, 44, 48). As higher-resolution models of 1 structure are developed, the individual and corporate activities of 1 receptor-binding domains will become clearer. This information will have particular import in the area of reovirus pathogenesis, where viral cell tropism at the level of receptor recognition determines the pathologic outcome of infection (13, 41, 48).

View larger version (33K): [in this window] [in a new window]   FIG. 8.   Functional domains of the reovirus attachment protein. Morphologic regions of 1 [T(i), T(ii), T(iii), T(iv), and H] seen in computer-processed electron micrographic images of 1 protein isolated from virions (16) are correlated with 1 predicted secondary structure (31). (A) Type 1 1 protein. Tail-forming sequences adjacent to the head, morphologic region T(iv), are required for binding to carbohydrate, the nature of which has not been identified for type 1 1. (B) Type 3 1 protein. All sequences necessary to bind sialic acid are contained in a predicted region of -sheet in the tail constituting morphologic region T(iii). The sialic acid-binding domain is discrete from other sequences located in the head that bind an unidentified receptor on L cells (8, 14, 29, 30, 42, 50, 52) and determine viral tropism in the murine central nervous system (CNS) (4, 21, 39). These two receptor-binding domains are bridged by a region of sequence, T(iv), that confers susceptibility of strain T3D 1 to cleavage by trypsin. Filled circles denote amino acid residues Asn198, Arg202, and Pro204, which determine viral capacity for sialic acid-dependent binding and infectivity (9). An arrow denotes the predicted location of amino acid residue 249, which is the minimal determinant of 1 susceptibility to cleavage by intestinal proteases (8). Stability of T3D 1 oligomers in SDS-polyacrylamide gels was mapped to sequences corresponding to morphologic regions T(i) plus T(ii).