William V. Williams$, Thomas Kieber-Emmonsl, David B. WeinerSgll, Donald H. Rubin11 **, .... NaN,, for 1-2 h at 37 "C. The wells were washed; and competitors.
J O U R N TAHL E
OF
BIOLOGICAL CHEMISTRY
Vol. 266, No. 14, Issue of May 15, pp. 9241-9250, 1991 Printed in [J.S.A.
Contact Residuesand Predicted Structureof the Reovirus Type3Receptor Interaction* (Received for publicat.ion, January 29, 1991)
William V. Williams$, Thomas Kieber-Emmonsl, DavidB. WeinerSgll, Donald H. Rubin11 **, and Mark I. Greenell From the Departmentsof $Medicine, TPathology, and J(Microbiology,University of Pennsylvania School of Medicine, the **DeDartment of Medicine, Philadelphia Veterans Affairs Medical Center, and $The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 191044283
Sequence similarity between the reovirus type 3 he- mary step in the infectiousprocess. The reovirus type 3 cellmagglutinin (HA3)and a anti-idiotypic monoclonal an- attachment site is associated with the crl polypeptide, which tibody (87.92.6) has been shown to define the site of is also the reovirus type 3 hemagglutinin (HA3).' This HA3 interaction with a neutralizing (idiotypic) monoclonal epitope for cell attachment is defined by neutralizing monoantibody (9B.G5) and the cellular receptor for the vi- clonal antibody 9B.G5 (1, 2). A murine anti-idiotypic monorus. A synthetic peptide (V, peptide) derived from the clonal antibody developed against 9B.G5, termed 87.92.6, anti-idiotypic sequence inhibits viral binding to the competes withreovirus type 3 for bindingtoits specific receptor. In this study, variants of the VLpeptide were cellular receptors (reovirus type 3 receptors (Reo3R)) (3, 4). utilized to probe specific amino acid residues involvedThe deduced amino acid sequence of HA3 was found to share in binding the neutralizing antibody and the receptor. sequence similarity with a combined determinant comprised These studies indicate that "the O H groups of several of the 87.92.6 heavy and light chain variable region second residues are involved in contacting the reovirus type 3 complementarity-determining regions (CDR 11) ( 5 ) .Synthetic receptor, including Tyr4', Ser50, Ser5', and ThrS3 in peptides have been utilized to determine whether the sequence the anti-idiotypic sequence, corresponding to Tyr326, similarity 87.92.6 and HA3 defines amino acids essential for Ser3", Ser3", and Ser326 inHA3, respectively. In contrast, only Ser" of the anti-idiotypic sequence, corre- epitope recognition. Prior studies (6) indicate that a peptide sponding to Ser3" of HA3, significantly altered neu- corresponding to the87.92.6 light chain CDR I1 (VL peptide) inhibits the interactionsbetween 9B.G5 and HA3,9B.G5 and tralizing antibody binding. Reo3R. A Additional studies implicate sialic acid as a potential 87.92.6, 87.92.6 andthe Reo3R, andHA3and reovirus type 3 receptor on some cells. This includes corresponding peptide derived from the HA3 sequence (reo of eliciting aneutralizing immuneresponse inhibition of binding of reovirus type3 and 87.92.6 to peptide) is capable L cells by heavily sialylated glycoproteins. Sialic acid specific for reovirus type 3 (7). These studies strongly suggest was therefore utilized as a candidate receptor to ana- that this epitope is directly involved in reovirus binding to lyze potential interaction schemes withHA3187.92.6. the Reo3R. Sequence similarity to otherimmunoglobulin strucThe epitope defined by the two peptides appears to have tures with similar sequences allowed modeling of the additional biological functions. Cross-linkingof the Reo3R by three-dimensional structure of these epitopes. These either virus or anti-receptor antibodies results in inhibition structures, in combination with peptide studies, allowof cellular growth and down-modulation of the Reo3R (8). the development of a model of the interaction of these These effects were reproduced by dimeric forms of the VL epitopes with sialic acid, which serves as a reovirus peptide (9). Thus, the interaction between VI. peptide/87.92.6/ type 3 receptor.These models revealthatsimilar HA3 and the Reo3R is alsocapable of transducing transmemamino acid residues and side-chain geometries may be cells. Three-dimensional utilized by the reovirus type 3 and influenza hemag- branesignalsinReo3R-bearing models of the corresponding epitopes on HA3 and in 87.92.6 glutinins in their interactions with cell-surface recephave been developed (6). tors. Studies characterizing theReo3R on murine L cells implicate sialic acid as a potential site for HA3 binding (10, 11). Data consistent withsialic acid participation in reovirus type The attachment of reovirus to both red blood cells (hem- 3 binding include decreased binding of reovirus type 3 followagglutination) and other cells ( i e . neuronal cells) is the pri- ing neuraminidase treatment of L cells as well as inhibition of reovirus type 3 binding by sialylated glycoproteins and * This work was supported by a National Institutes of Health First sialic acid-containing carbohydrates( 1 0 , l l ) . Identification of HA3asthe viralpolypeptideforsialicacid binding was award and grants from the Lupus Foundations of Philadelphia and Pennsylvania and the Center of the Study of Aging (to W. V. W.), accomplished by analysis of reovirus reassortment clones grants from the American Foundation of AIDS Research, theCouncil derived from reovirus type 1 and 3 genetic crosses, suggesting for Tobacco Research,and a NationalInstitutes of HealthFirst a direct interaction between HA3 and sialic acid. award (toD. B. W.), a grant from theAmerican Foundation for AIDS Research (to T. K. E.), and grants from the National Institutes of Health, American Cancer Society, National Cancer Instit.ute, National Eye Institute, and theLucille P. Markey Foundation (to.M. I. G.). The costs of publicat.ion 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.
The abbreviations used are: HA3, type 3 hemagglutinin; Reo3R, reovirus type 3 receptor; CDRs 11, second complementarity-determining regions; BSM, bovine submaxillary mucin; BSA, bovine serum albumin; RIA, radioimmunoassay; PBS, phosphate-buffered saline; IHA, influenza hemagglutinin; anti-Id3, type 3 anti-idiotypic serum; SLC, sialic acid binding conformation.
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PBS; and specific counts/minute hound was determined as noted above. Percent inhibition of binding was calculated by the formula noted above. Flow Cytometry Analysis-The ability of sialylated glycoproteins to inhibit antibody binding cells to was determined by preincubation of the antibody with varying amountsof inhibitor in (in PBS)for 30 min to 1 h a t 23 "C. Cells (either L cells grown as described above or R1.l cells grown in RPMI 1640 medium with 10% fetal calf serum and added antibiotics and L-glutamine from GIBCO) was washed in 1% BSA, PBS, 0.1% NaN:90% homogeneous. Neuraminidase Treatment-L cells were centrifuged, washed twice Monoclonal Antibodies-Neutralizing anti-reovirus type 3 mono- in PBS, and resuspended a t 4-5 X lO5/rnl in PBS. Type VlII neuraclonal antibody 9B.G5 (murine lgGPa,k) or isotype-matched monominidase from Clostridiumperfringens (Sigma)was dissolved in PBS clonal antibody A l l was isolated from the culture supernatant by a t 5 units/ml and added toa final concentration of 20-25 milliunits/ 50%ammoniumsulfateprecipitation; dialyzed against phosphatelo6 cells in tissue culture flasks. Following a 1-h incubation, thecells buffered saline (PBS); bound to a staphylococcal protein A column were centrifuged, washed twice in 1% BSA, PBS, 0.1% NaN:
