Isolating the Epstein-Barr Virus gp350/220 Binding Site on

Isolating the Epstein-Barr Virus gp350/220 Binding Site onComplement Receptor Type 2 (CR2/CD21)*

Received for publication, July 31, 2007, and in revised form, October 9, 2007 Published, JBC Papers in Press, October 9, 2007, DOI 10.1074/jbc.M706324200

Kendra A. Young‡, Xiaojiang S. Chen§, V. Michael Holers‡, and Jonathan P. Hannan‡1

From the ‡Department of Medicine and Immunology, University of Colorado at Denver and Health Sciences Center,Aurora, Colorado 80045 and the §Department of Molecular and Computational Biology, University ofSouthern California, Los Angeles, California 90089

Complement receptor type 2 (CR2/CD21) is essential for theattachment of Epstein-Barr virus (EBV) to the surface of B-lym-phocytes in an interactionmediated by the viral envelope glyco-protein gp350. The heavily glycosylated structure of EBV gp350has recently been elucidated by x-ray crystallography, and theCR2 binding site on this protein has been characterized. Toidentify the corresponding gp350 binding site on CR2, we haveundertaken a site-directed mutagenesis study targeting regionsof CR2 that have previously been implicated in the binding ofCR2 to the C3d/C3dg fragments of complement component C3.Wild-type or mutant forms of CR2 were expressed on K562cells, and the ability of these CR2-expressing cells to bind gp350wasmeasured using flow cytometry. Mutations directed towardthe two N-terminal extracellular domains of CR2 (SCR1-2)reveal that a large contiguous surface of CR2 SCR1-2 is involvedin gp350 binding, including a number of positively charged res-idues (Arg-13, (Arg-28, (Arg-36, Lys-41, Lys-57, Lys-67, andArg-83). These data appear to complement the CR2 binding siteon gp350,which is characterized by a preponderance of negativecharge. In addition to identifying the importance of charge inthe formation of a CR2-gp350 complex, we also provide evi-dence that both SCR1 and SCR2make contact with gp350. Spe-cifically, two anti-CR2 monoclonal antibodies, designated asmonoclonal antibodies 171 and 1048 whose primary epitopesare located within SCR2, inhibit binding of wild-type CR2 toEBV gp350; with regard to SCR1, both K562 cells expressing anS15P mutation and recombinant S15P CR2 proteins exhibitdiminished gp350 binding.

Epstein-Barr virus (EBV)2 is a human �-herpesvirus that isubiquitously found inmost of the adult population of theworld.

Primary infection normally occurs within the first few years oflife and is usually asymptomatic, but if infection is delayed untiladolescence or later ages, then it may present as infectiousmononucleosis. Long term carriage of EBVhas been implicatedin the development of a number of other more serious diseasestates including B cell lymphomas, nasopharyngeal carcinoma,and gastric carcinoma (for review, see Refs. 1–4). EBV has alsobeen linked as a potential etiologic agent in the development ofa number of autoimmune conditions including systemic lupuserythematosus, rheumatoid arthritis, and multiple sclerosis(5–7). EBV predominantly infects two major cell types: B lym-phocytes and epithelial cells. Generally, more is known aboutthe mechanism of entry of EBV into B lymphocytes than intoepithelial cells. Preferential binding to B-lymphocytes is pri-marily mediated by the binding of the EBV viral envelope pro-tein gp350 to complement receptor type 2 (CR2/CD21) on thesurface of B cells (8–14) and through the binding of a secondglycoprotein gp42 to human leukocyte antigen class II mole-cules (15, 16) which triggers fusion with the host cell in a proc-ess mediated by three additional viral glycoproteins, gB, gH,and gL (17–19). Additional viral ligands have also been impli-cated in the attachment and invasion of cells by EBV independ-ent of the gp350/CR2 and gp42/human leukocyte antigen IIpathways. EBVhas been demonstrated in vitro to infectCR2(�)cells, and gp350 knock-outmutant forms of EBVhave also beenshown to infect primary B-lymphocytes, lymphoid cell lines,and epithelial cell lines that are susceptible to wild-type EBVinfection, albeit with a significantly reduced efficiency of infec-tion (20).EBV gp350/220 is an extensively glycosylated polypeptide

(907 residues) that is expressed in two alternatively splicedforms of �350 and 220 kDa (21) and has been identified as thedominant protein in the extracellular virus envelope (22). TheN-terminal 470 residues of gp350 have been shown to be essen-tial for the binding interaction with CR2 (14). In addition,monoclonal antibodies generated against gp350 have beenshown to effectively inhibit infectivity of peripheral B lympho-cytes with EBV (23), and some forms of this protein have beenshown to act successfully as a vaccine against EBV-associateddiseases in animal models (24, 25). The three-dimensionalstructure of the CR2 receptor binding domain of gp350 hasrecently been determined by x-ray crystallography (26). Threedistinct domains were identified, each comprising an anti-par-allel �-barrel structure and adjoined by two long linker regions,each of 11 amino acids. The domains are tightly packed againsteach other, forming a distinctive L-shaped arrangement that is

* This work was supported by National Institutes of Health Grant R0-1R01CA053615 (to V. M. H.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article must there-fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact.

1 To whom correspondence should be addressed: Division of Rheumatology,P. O. Box 6511, Mail Stop B-115, Dept. of Medicine and Immunology, Bar-bara Davis Center for Childhood Diabetes, University of Colorado at Den-ver and Health Sciences Center, 1775 N. Ursula St., Aurora, CO 80045. Tel.:303-724-7605; Fax: 303-724-7581; E-mail: [email protected].

2 The abbreviations used are: EBV, Epstein-Barr virus; CR2, complementreceptor type 2; SCR, short consensus repeat; PBS, phosphate-bufferedsaline; FITC, fluorescein isothiocyanate; PE, phycoerythrin; MFI, meanfluorescence intensity; mAb, monoclonal antibody; MBP, maltose-bind-ing protein; ELISA, enzyme-linked immunosorbent assay; Bis-Tris,2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 50, pp. 36614 –36625, December 14, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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almost uniformly glycosylated. Site-directed mutagenesis tar-geting one of the few glycan-free areas of gp350 has identifiedthe CR2 binding site to be located within a negatively chargedregion of this molecule, with mutant forms of gp350 exhibitingdiminished or negligible ability to bind CR2 (26). Convincingevidence that this is indeed the correct receptor binding site isprovided by the fact that themutations targeting the CR2 bind-ing site also disrupt the ability of gp350 to recognize its majorneutralizing monoclonal antibody, 72A1 mAb (26, 27). Theepitope for 72A1 mAb within gp350 has been identified as oneof the key areas involved in EBV gp350 binding to host cellsbecause 72A1 mAb can successfully inhibit EBV binding andinvasion of B-lymphocytes, Raji cells, monocytes, neutrophils,and T-cells (14, 28–30). Also, a recent peptide mapping studybyUrquiza et al. (31) has identified three gp350 regions that areinvolved in EBV binding to B-lymphocytes, two of whichdirectly overlap with the CR2 binding site identified in the crys-tal structure of EBV gp350.CR2 is a �145-kDa transmembrane glycoprotein expressed

mainly on B-lymphocytes, follicular dendritic cells, and someT-cell subtypes. On B-cells CR2 either forms part of a CR2/CD81/CD19 complex or is otherwise associated with CR1 andplays an essential role in cell activation and the initiation ofnormal immune responses to pathogens (32–35). Interaction offoreign antigen that is coated by covalently attached C3d withCR2 results in a cell-signaling event occurring through CD19.This significantly lowers the signaling threshold for B-cell acti-vation via a signal transduction cascade. CR2 also plays a criticalrole in the development of humoral immune responses to T-dependent antigens. Thus, CR2 acts as a bridge between theadaptive and innate immune systems (36). CR2 is a member ofthe structural family of C3/C4 receptor and regulatory proteinsknown as the regulators of complement activation (RCA fam-ily).Members of this family are characterized by the presence ofshort repeating domains of �70 amino acids, known as SCR orCCP modules. Each of these compact units contains a numberof conserved residues including four cysteines and an invarianttryptophan residue. The conserved cysteines form a pattern ofdisulfide bridges that connect Cys-1—Cys-3 andCys-2—Cys-4.Themodular composition of CR2 is well known and consists ofa 15- or 16-SCR extracellular domain, a 24-amino acid trans-membrane domain, and a short 34-amino acid intracellularC-terminal tail. In addition to EBV gp350, high affinity ligandsfor CR2 include the proteolytic fragments of complement com-ponent C3, C3d, C3dg, and iC3b (37, 38), the low-affinity IgEreceptor, CD23 (39, 40), and interferon � (41, 42). Peptide andepitope mapping experiments have indicated that all knownCR2 ligands bind within the first two N-terminal SCR domains(SCR1-2) at overlapping binding sites (43–45), although anadditional glycosylation-dependent interaction with CD23 alsoinvolves SCR5–8 (40).Recently, we have utilized a site-directed mutagenesis strat-

egy to examine the binding interaction between C3dg and full-length CR2 (46). This work involved expressing wild-type orsingle site mutant forms of CR2 on the surface of K562 eryth-roleukemia cells and then measuring the relative binding affin-ities of these cells for recombinant C3dg-biotin tetramers usingmulticolor flow cytometry. Mutagenesis targets for this study

were directed by previously reported solution phase studies ofthe CR2-C3d interaction and by the available co-crystal struc-ture of the CR2 SCR1-2-C3d complex (43–45, 47–50). Ourdata revealed that in addition to the CR2-C3d interfaceobserved in the co-crystal structure, which involved only SCR2of CR2, a number of positively charged residues on the SCR1domain were also essential for a significant CR2-C3dg interac-tion to occur. Several of these residues had not been implicatedin CR2-ligand binding by previous experimental studies,although a recent study utilizing theoretical electrostaticpotential and apparent pKa calculations observed that the asso-ciation between CR2 and C3d was dependent not only on theapparent CR2 SCR2-C3d interface observed in the structuralelucidation but also on the overall respective net charges of CR2SCR1-2 and C3d (51). The binding interactions between CR2and C3d and between CR2 and gp350 have previously beenindicated to occur at overlapping binding sites (43–45). How-ever, a number of intriguing differences between these bindingsites or the nature of the binding interaction between CR2 andthese ligands must occur. Murine CR2 SCR1-2 shares a �61%sequence identity with human CR2 SCR1-2, and althoughmurine CR2 can bind human C3d and iC3b with an equal affin-ity to the human form, it cannot bind EBV (43, 47). Datareported by Martin et al. 52 and Prota et al. 53 have proposedthat two areas on CR2 are critical in the binding interactionwith EBV gp350. Substitutions among residues 8–15within thefirst inter-cysteine region of SCR1 have suggested this area is astructural determinant in gp350 binding; murine CR2 containsa N-linked glycosylation site at position 66 within the linkerregion connecting SCR1 and SCR2, and this has been proposedto sterically inhibit the interaction between CR2 and EBVgp350. To gain a better understanding of the binding interac-tion between CR2 and its ligand EBV gp350 we have utilized acombination of site-directedmutagenesis and epitopemappingto determine those residues on CR2 that are likely to play a rolein the adsorption of EBV to B-lymphocytes.

EXPERIMENTAL PROCEDURES

Cloning and Expression of EBV gp350—EBV genomic DNAwas extracted from previously obtained cell supernatants of themarmoset HB95-8 leukocyte cell-line (American type CultureCollection) using a QIAampUltraSens virus kit (Qiagen). DNAcorresponding to residues 1–470 of EBV gp350/220 was PCR-amplified using the primers 5�-GCG GCC CAG CCG GCCGAGGCAGCCTTGCTTG-3� (incorporating an Sfi site) and5�-G ATA GTT TAG CGG CCG CAT TCT TAT GGT GGATAC AGT GGG-3� (incorporating a NotI site). In addition, afragment of the Escherichia coli biotin carboxyl carrier proteincorresponding to residues 70–156 was also PCR-amplifiedusing the primers 5�-CGG GGG GCC CCC CAT GGA AGCGCC AGC AGC-3� and 5�-CTG GGG CCC CTA CTC GATCGAGACCAGC-3� both of which incorporate a DraII site forcloning. PCR fragments were ligated into the pSecTag2/HygroB eukaryotic expression vector (Invitrogen), which encodes anMyc epitope and a hexahistidine tag at the 3�-end of theinserted DNA. cDNA for the gp350-biotin construct wassequenced by the University of Colorado Cancer CenterSequencing Core (Denver, CO) to confirm the insert was in-

Mutational Analysis of the EBV-CR2 Interaction

DECEMBER 14, 2007 • VOLUME 282 • NUMBER 50 JOURNAL OF BIOLOGICAL CHEMISTRY 36615


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frame with the plasmid DNA and also that it contained thedesignated sequences for the gp350 and biotin carboxyl carrierprotein proteins outlined above. Plasmid DNA was subse-quently transfected into human embryonic kidney 293f free-style cells (Invitrogen) for soluble expression of recombinantEBV gp350 into the media. The recombinant EBV gp350 wasconcentrated and concurrently exchanged into a 20mMsodiumphosphate buffer, pH 7.4, containing 10 mM imidazole and 0.5M NaCl and applied to a Ni2� charged HisTrap column (GEHealthcare). Bound protein was eluted with a 0.5 M imidazolegradient, concentrated, and subsequently purified by gel filtra-tion on a Hiload 26/60 Superdex 200 prep grade column usingan AKTA fast protein liquid chromatography system (GEHealthcare). After purification the EBV gp350 was biotinylated(EBV gp350-biotin) using biotin ligase (Avidity) according themanufacturer’s instructions and then conjugated to Phyco-erythrin-NeutrAvidin (Molecular Probes), generating fluoro-chrome tagged-gp350 monomers for flow cytometric bindinganalysis.Expression of Wild-type and Mutant Forms of Full-length

CR2 on K562 Cells—Wild-type or mutant full-length rCR2 wasexpressed on human K562 erythroleukemia cells with theeukaryotic expression vector pSFFV-neo, as previouslydescribed (44, 46). Briefly, CR2-expressing transfectants wereincubated in the presence of biotinylated anti-CR2 HB5 mAband then sorted using streptavidin-coated magnetic beads(Dynabeads, Dynal) to establish stable populations of cells withCR2 protein expression. Point mutations within the first twoSCR domains of CR2 were carried out utilizing a QuikChangesite-directed mutagenesis kit (Stratagene). Mutated full-lengthCR2 cDNA was sequenced by the University of Colorado Can-cer Center Sequencing Core (Denver, CO) and transfected intoK562 human erythroleukemia cells for binding analysis by flowcytometry. Mutations were generated targeting: 1) Arg-83, aresidue that plays a prominent role in the ligation of C3d toSCR2 of CR2 (R83A and R83E), 2) the OKB7 mAb epitope onSCR1 of CR2 (Pro-8—Ser-15) contained within the first inter-cysteine region of SCR1, specifically Asn-11, Arg-13, andSer-15 (N11A, R13A, R13E, and S15P); Tyr-16 and Ser-32,which are in close spatial proximity to this Pro-8—Ser-15region of SCR1, were also selected as candidates for substitu-tion (Y16A and S32A); 3) the eight-residue linker region con-necting SCR1 to SCR2 (residues 63–70); Tyr-64, Lys-67, andTyr-68 were chosen from this region for site-directedmutagenesis (Y64A, K67A, K67E and Y68A); 4) a patch ofconserved positively charged residues within SCR1 locatedoutside the OKB7 epitope. In this case residues Arg-28, Arg-36, Lys-41, Lys-50, and Lys-57 were chosen for substitutionanalysis (R28A, R28E, R36A, R36E, K41A, K41E, K50A,K50E, K57A, and K57E).Expression of Wild-type, N11A, R13A, S15P, R28A, R36A,

K41A, K57A, K67A, and R83A CR2 SCR1-2 Recombinant Pro-teins in E. coli—DNA corresponding to residues 1–133 of wild-type CR2 (SCR1-2) was PCR-amplified using the primers5�-CCG GAA TTC CGG ATT TCT TGT GGC TCT CCT-3�incorporating an EcoRI site and 5�-CCC AAGCCTGGGTCATCA CTC GAG AGG GAA AAC ACT-3� incorporating twostop sequences and a HindIII site after the codon correspond-

ing to residue 133. Resulting PCR fragments were ligated intothe prokaryotic expression vector pMAL-p2x (New EnglandBiolabs) as previously described (26), which encodes amaltose-binding protein (MBP) tag at the 5�-end of the inserted DNA.This plasmid includes the malE gene with its signal sequenceallowing the export of fusion proteins to the bacterialperiplasm, facilitating folding and disulfide bond formation totake place. Plasmid DNA was subsequently transformed intoE. coli BL21 cells, and wild-type recombinant MBP-CR2SCR1-2 was produced as follows. Ampicillin-resistant colonieswere used to start overnight cultures thatwere expanded to 1–3liters and grown at 37 °C until an A600 of 0.3 was obtained.Cultures were induced with 0.3 mM isopropyl-�-D-thiogalacto-side at 30 °C for 4 h before harvesting by centrifugation. Har-vested pellets were resuspended in a column buffer comprising20 mM Tris-HCl, pH 7.4, 0.2 M NaCl, 1 mM EDTA and lysed bysonication. The lysate was clarified by centrifugation and thenapplied to an amylose resin column. Bound MBP-CR2 SCR1-2was eluted from the column using column buffer containing 10mMmaltose. Finally, theMBP-CR2 SCR1-2 was purified by sizeexclusion chromatography using the protocol described abovefor EBV gp350.Recombinant N11A, R13A, S15P, R28A, R36A, K41A, K57A,

K67A, and R83A CR2 SCR1-2 DNA was produced from wild-type MBP-CR2 SCR1-2 DNA utilizing a QuikChange site-di-rected mutagenesis kit (Stratagene) according to the manufac-turer’s instructions. Plasmid DNA containing the mutant CR2SCR1-2 insert was then transformed into E. coli BL21, andrecombinant mutant CR2 SCR1-2 proteins were expressed andpurified as per the protocol laid out for wild-type MBP-CR2SCR1-2 above.Flow Cytometry—Flow cytometric experiments were carried

out using K562 erythroleukemia cells transfected with full-length wild-type or mutant human CR2. Binding analyses werecarried out using gp350-biotin. For each condition, 5 � 105human CR2-transfected K562 cells were first incubated withfluorescein isothiocyanate (FITC)-conjugated anti-CR2 HB5mAb at 1�g/ml on ice for 1 h. The primary epitope for HB5 hasbeen identified within the N-terminal SCR3-4 extracellulardomains of CR2 and, accordingly, does not interferewith ligandbinding. During this incubation 100 �l of gp350-biotin mono-mers in PBS, 0.1% bovine serum albumin , 0.01% sodium azidewere prepared for each condition by adding the appropriateamount of recombinant gp350-biotin and 0.4 �g of PE-conju-gated NeutrAvidin (Molecular Probes) and incubating at roomtemperature for 30 min. gp350-biotin concentrations usedwere 0.5g, 0.25, 0.125, 0.0625, and 0.03125 �g. After washingof the FITC-stained K562 cells, 100 �l of monomeric PE-conjugated gp350-biotin were added to each sample of cellsand incubated for 30 min on ice. After washing, the cellswere fixed and analyzed by multicolor flow cytometry in theUniversity of Colorado Cancer Center Flow Cytometry CoreFacility (Denver, CO).Cells were divided into high, medium, and low CR2 expres-

sion using 101 as the lower limit and gating on the intermediate18% of CR2 expressing cells (FITC-positive). Monomericgp350-biotin binding was determined by PE mean channel flu-orescence. A minimum of four separate experiments was car-




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ried out for each mutation. The whole cell populations and/orthe normalized gp350-biotin binding data for each of the inter-mediate wild-type and mutant populations are given.Anti-CR2 mAb Inhibition of EBV gp350 Binding to Full-

length CR2—Wild-type CR2-expressing cells were incubatedwith FITC-conjugated anti-CR2 HB5 mAb at a concentrationof 1 �g/ml on ice for 1 h. After this initial incubation, cells werewashed, and 100 �l of PBS solution containing one of the anti-CR2monoclonal antibodies, 171, 1048, or 629mAb, was addedto the cell mix at a concentration of 0.5 �g/ml and allowed tostand on ice for 1 h. After this period cells were again washed,and 100 �l of EBV gp350-biotin monomers conjugated to Phy-coerythrin-NeutrAvidin was added to the CR2-expressingK562 cells as described above. Cells were then fixed, and EBVgp350-biotin binding was measured by multicolor flowcytometry.Wild-type andMutant CR2 SCR1-2-gp350 and CR2 SCR1-2-

C3d ELISA Assays—Plates were coated overnight at 4 °C with 5�g/ml gp350-biotin or 5 �g/ml recombinant C3d expressedand purified as previously described (50) in 20 mM sodiumbicarbonate buffer, pH 8.0. After coating the plates wereblocked using 0.1% bovine serum albumin in a diluted 1/3 PBSsolution, pH 7.4 (containing 45.6 mM NaCl, 2.7 mM Na2HPO4,0.9mMKCl, 0.5mMKH2PO4) for 1 h at room temperature. Theplates were then washed and incubated with either wild-typeMBP-CR2 SCR1-2, R13A MBP-CR2 SCR1-2, or S15P MBP-CR2 at concentrations ranging from2 to 0.016�g/ml in 1/3 PBSsolution for 1 h at room temperature. After further washing,wild-type or mutant MBP-CR2 binding was detected usingcommercially available horseradish peroxidase-conjugatedanti-MBP mAb (New England Biolabs) according to the man-ufacturer’s instructions.Production of the Anti-gp350 Monoclonal Antibody 72A1—

The HB-168 hybridoma cell line was obtained from the ATCC.The anti-gp350 monoclonal antibody 72A1 was subsequentlyobtained from the spent culture medium of hybridoma cellsgrown in RPMI 1640 supplemented with 2mM L-glutamine andadjusted to contain 1.5 g/liter sodium bicarbonate, 4.5 g/literglucose, 10 mMHEPES, 1.0 mM sodium pyruvate, and 10% fetalbovine serum. The antibody was then purified by affinity chro-matography using protein G-Sepharose 4 Fast Flow resin (GEHealthcare) according to the manufacturer’s instructions. Theresulting purified 72A1 mAb was exchanged into PBS, pH 7.4,and finally concentrated to give a stock solution containing 1mg/ml antibody as determined by UV-visible spectrophotom-etry and stored at �20 °C until required.EBV Pull-down Binding Assay—The B95-8 strain of EBVwas

obtained from the clarified culture medium of marmosetB-lymphocytes (GM07404) obtained from Coriell Cell Reposi-tories. EBV-containing supernatant was subsequently passedthrough a 0.88-�m (pore size) filter and then ultracentrifugedat 25,000 � g at 4 °C for 4 h. Virus-rich pellets were then resus-pended in 1/20 original volume PBS, pH 7.4. 1 ml of the result-ing concentrated EBV solution was incubated with 10 �g ofrecombinant wild-type or mutant (N11A, R13A, S15P, R28A,R36A, K41A, K57A, K67A, and R83A) MBP-CR2 SCR1-2 atroom temperature for 30min. Subsequent to this stage, 40�l ofa pre-equilibrated 50% slurry of amylose beads were added to

the MBP-CR2 SCR1-2/EBV solution, and the mixture allowedto incubate for another 20 min. The amylose beads were thenpelleted by centrifugation at 10,000 � g for 30 s, and the super-natant containing unbound virus was aspirated off. The amy-lose beads were subsequently washed a total of three times inPBS. After washing, 30�l of an elution buffer (20mMTris-HCl,pH 7.4, 0.2 M NaCl, 1 mM EDTA, 10 mM maltose) was added tothe beads, and after 10 min, the beads were pelleted a final timeand the elution buffer containing recombinant wild-type ormutant MBP-CR2 SCR1-2 and EBV virus that had been boundwas removed for SDS-PAGE and Western blotting.Samples obtained from the pulldown experiment were

diluted with loading buffer (Invitrogen), and any virus presentwas inactivated by heating at 80 °C for 10 min. For detection ofwild-type and mutant forms of MBP-CR2, samples wereapplied to a 10% Bis-Tris gel (Invitrogen), and levels of CR2 inthe eluentwere detected byCoomassie staining. To detect EBV,samples were first separated on 3–8%Tris acetate gels (Invitro-gen) by electrophoresis before being transferred to nitrocellu-lose membranes. After blocking overnight using a 10% milksolution, the membranes were washed in PBS-Tween (0.5%)and then incubated with the anti-gp350 mAb, 72A1, at a con-centration of 1 �g/ml. Upon further washing, the membranewas incubated with goat anti-mouse heavy and light chain per-oxidase-conjugated IgG at a concentration of 0.5 �g/ml (Jack-son ImmunoResearch Laboratories), washed again, and finallydeveloped using ECL reagents (GE Healthcare).

RESULTS

Anti-CR2 SCR1-2 Monoclonal Antibody Inhibition—Wild-type CR2 expressing K562 erythroleukemia cells that wereinitially incubated with the FITC-conjugated anti-CR2monoclonal antibody HB5 and subsequently incubated withgp350-biotin PE-NeutAvidin at a range of gp350-biotin con-centrations ranging from 0.5 to 0.0313 �g exhibited a typicaldose-dependent binding curve as measured by dual-color flowcytometry (Fig. 1, A and E). However, when cells were preincu-bated with the anti-CR2 SCR1-2 monoclonal antibodies 629,171, and 1048 before adding gp350-biotin, the latter two anti-bodies were found to exhibit significantly compromise gp350-biotin binding (Fig. 1, B–E). Preincubation with 171 mAb inparticular inhibited binding of gp350-biotin to wild-type CR2with an mean fluorescence intensity (MFI) of only �3%,whereas 1048 mAb demonstrated an MFI of 19% relative towild-type CR2 (at a concentration of 0.5 �g of gp350-biotin/0.4�g of PE-NeutrAvidin) (Table 1). No decrease in gp350-biotinbinding was observed as a result of preincubating K562 cellswith 629 mAb. The primary epitopes for 171 and 1048 mAbshave previously been identified on the SCR2 extracellulardomain of CR2, specifically 86TPYRH90 for 171 mAb and111WCQANNMW118 for mAb 1048, as determined by peptidemapping (54). It should be noted that the epitope for 171 mAbon SCR2 partly overlaps with that of the C3d binding site onSCR2, which involves residues 80YKIRGSTP88, as revealed bythe co-crystal structure of the CR2-C3d complex (50). Thesedata coupled with previous studies are suggestive that thegp350 binding site is located immediately adjacent to, ordirectly overlapping with both the 171 mAb and C3d binding




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sites. In contrast, the 1048 mAb primary epitope is geographi-cally located on the opposite side of the CR2 molecule relativeto the 171mAb and theC3d binding sites. Only a single residue,Trp-118, which forms part of the interface between SCR1 andSCR2, is located on the same visage as both the C3d and 171mAb sites. It is likely, therefore, that 1048 mAb inhibits theinteraction between CR2 and gp350 via steric or allostericmeans. A monoclonal antibody, which was not part of the cur-rent study, designated as 944mAb has previously been found toinhibit the interaction between CR2 and its ligands, and thismonoclonal antibody shares most of its primary epitope(110VWCQANNM117) with that of 1048 mAb (54).SCR2-C3d Interaction Site and the Linker Region Connecting

SCR1 and SCR2—The SCR2 interface with C3d is dominatedbymain-chain interactions, oftenmediated bywatermolecules.From the CR2 perspective, only Arg-83 appears to contribute asignificant side-chain interaction to C3d binding. In the CR2-

C3d co-crystal structure the side chain of Arg-83 is insertedinto an anion hole formed by the carbonyl atoms of residuesIle-115, Leu-116, Glu-117, and Gln-119. To investigate thedegree of overlap between the C3d and gp350 binding sites onCR2, we generated R83A and R83E CR2 mutants expressed onthe surface of K562 cells and assessed their capacity to interactwith gp350-biotin. Both of these mutants exhibited decreasedability to bind gp350-biotin relative to wild-type CR2 with anMFI of �33 and 41%, respectively (Table 1, Fig. 2). These datataken in context with the monoclonal antibody inhibition data

FIGURE 1. Relative ability of anti-CR2 SCR1-2 monoclonal antibodies toblock gp350 binding by CR2 expressed on the surface of K562 erythro-leukemia cells. Shown are representative whole cell populations expressingwild-type CR2 preincubated with no anti-SCR1-2 antibody (control) (A), 0.5�g/ml 629 mAb (B), 0.5 �g/ml 171 mAb (C), and 0.5 �g/ml 1048 mAb (D)before being incubated with PBS containing 0.5 �g of gp350-biotin/0.4 �g ofPE-NeutAvidin. E, binding affinities of varying concentrations of gp350-biotin(0.5, 0.25, 0.125, 0.0625, and 0.0313 �g, each with 0.4 �g of PE-NeutAvidin) forwild-type CR2 in the absence and in the presence of 629, 171, and 1048 mAb.Average and S.E. of the normalized values for the MFI of the intermediate CR2expressing population (18%) are demonstrated.

FIGURE 2. Binding affinities of varying concentrations of gp350-biotin(0.5, 0.25, 0.125, 0.0625, and 0.0313 �g each with 0.4 �g of PE-NeutAvidin)with R83A and R83E CR2 mutants. The average and S.E. of the normalizedvalues for the mean fluorescence intensity of the intermediate CR2 mutantexpressing population (18%) relative to wild-type are demonstrated. In theCR2-C3d co-crystal structure Arg-83 was identified as providing the only sub-stantial side-chain contribution from the CR2 side of the CR2-C3d interaction.The numbering system used within this article for CR2 is based on the previ-ously reported amino acid sequence for the mature protein (55).

TABLE 1Intermediate CR2 expressionShown are the percentage EBV gp350-biotin binding affinities to mutant CR2-expressing K562 cells relative to K562 cells expressing wild-type CR2. Also shownare the results of anti-CR2 monoclonal antibody inhibition experiments. �� ,90–120%; ��, 89.9–70%; ��, 69.9–40%;�, 39.9–20%; �, 19.9–0%.

Mutation MFI (gp350-biotinbinding)

S.E. (gp350-biotinbinding) Weighting

N11A 112.6 11.6 ��R13A 7 0.4 �R13E 7.2 0.6 �S15P 5.4 0.4 �Y16A 66.7 4 ��R28A 21.9 1.8 �R28E 7.8 0.9 �S32A 108 12.9 ��R36A 29 4.8 �R36E 12 1 �K41A 20.8 1.8 �K41E 7.2 1.3 �K50A 59.2 1.9 ��K50E 51.2 8.8 ��K57A 36.5 3.7 �K57E 64.3 8.8 ��Y64A 84.5 3.4 ��K67A 42.1 3.9 ��K67E 22.6 1.8 �Y68A 111.4 7.4 ��R83A 32.9 4.9 �R83E 41.2 2.5 ��171 mAb 3.4 0.1 �629 mAb 101.9 12.2 ��1048 mAb 19 6.1 �




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described above using 171 and 1048 mAbs and also withprevious epitope mapping and peptide binding studies indi-cate that SCR2 is essential for the attachment and subse-quent fusion of EBV with B-lymphocytes and also that thereis likely to be some overlap with the known C3d binding siteon this domain (45, 47, 54).The linker region between SCR1 and SCR2 is one of the long-

est of all known SCR connectors comprising eight residues(63EYFNKYSS70). Previous work in separate studies by Martinet al. 52 and Prota et al. 53 has implicated this linker region inplaying a significant role in EBV binding. Specifically, thesequence 66NKY68, as found in human CR2, whenmutated to asequence identical to the corresponding murine sequence of66NKT68, causes glycosylation of Asn-66. Significantly, murineCR2 is unable to bind EBV or gp350. Subsequent introduction

of this glycan moiety to human CR2either in conjunction with muta-tions targeting residues 8PILN-GRIS15 of the first inter-cysteineregion of SCR1 or in their absencehas been demonstrated to cause asubstantial decrease in gp350 bind-ing to CR2 (52, 53). To test theimportance of the SCR1-SCR2linker region in binding to gp350-biotin, we generated four pointmutations targeting a total of threeresidues within the linker region.These mutations were Y64A, K67A,K67E, and Y68A. Cells transformedwith plasmid DNA containing anadditional Y68T mutation gener-ated with the goal of causing Asn-66to be glycosylated failed to sort withstreptavidin-coated magnetic beadsafter being incubated with biotiny-lated anti-CR2 HB5 mAb and were,accordingly, rejected from thestudy. Whole cell populations ofK562 cells expressing Y64A, K67A,K67E, and Y68Amutants are shown(Table 1, Fig. 3). Neither Y64A norY68A mutants demonstrated anypronounced decrease in gp350-bio-tin binding affinity relative to wild-type CR2. Binding curves for gp350-biotin binding to Y64A and Y68Amutants over the full range ofgp350-biotin concentrations areshown in Fig. 3E. However, alanineand opposite charge substitutionstargeting Lys-67, also locatedwithinthe linker region (K67A and K67E),showed a decreased capacity forbinding gp350-biotin relative towild-typeCR2withMFI values of 42and �23% for K67A and K67Emutants, respectively (Fig. 3F).

Interestingly, in the available structures of CR2, Lys-67 is ori-ented toward the same face on CR2 as Arg-83 and the 171mAbepitopes, whereas the side chains of Tyr-64 and Tyr-68 are ori-ented behind and away from this region (50, 53).

8PILNGRIS15 Region of SCR1—The region within the firstinter-cysteine area of SCR1, previously defined as one of themajor epitopes of the inhibitory monoclonal antibody OKB7and located within residues Pro-8—Ser-15, has been impli-cated in a number of studies as being essential for the ligation ofCR2 to C3d/C3dg and also to EBV/gp350 (44–47, 52). In par-ticular, work byMartin et al. 52 demonstrated it was possible toget murine forms of CR2 to bind EBV when residues 8–15(8EVKNARKP15) contained a single point mutation of P15S.Constructs of this P15S mutant form of murine CR2 were ableto bind EBV irrespective of whether or not theN-glycanmoiety

FIGURE 3. Flow cytometric analysis of the interaction between EBV gp350-biotin and full-length mutantforms of CR2 targeting the 8-residue linker (residues 63–70) connecting SCR1 and SCR2. Whole cellpopulations of K562 erythroleukemia cells expressing Y64A (A), K67A (B), K67E (C), and Y68A (D) are shown afterbeing incubated with 0.5 �g of EBV gp350-biotin, 0.4 �g of PE-NeutrAvidin. Normalized binding affinities ofY64A and Y68A (E) and K67A and K67E (F) relative to wild-type CR2 are shown. The average and S.E. of thenormalized values for the MFI of the intermediate CR2 expressing population (18%) are given.




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at Asn-66 was removed by site-directed mutagenesis (52). Toinvestigate the role that residues 8–15 play in binding gp350,we generated a total of 6 mutations targeting 5 residues withinor in close spatial proximity to the 8PILNGRIS15 region of

SCR1. Mutations generated wereN11A, R13A, R13E, S15P, Y16A,and S32A. Whole cell populationsfor the four mutations within thePro-8—Ser-15 region are shown inFig. 4, A–D. In addition, dose-dependent binding curves for all ofthe mutants over the range of 0.5–0.0313 �g of gp350-biotin areshown in Fig. 4, E–H. These datareveal that a number of residueswithin the OKB7 primary epitopeare essential for binding gp350.R13A and R13E mutants both dem-onstrate greatly decreased bindingaffinities relative to wild-type CR2,with binding levels of �7%. In addi-tion, when Ser-15wasmutated to itscorresponding murine amino acid,Pro-15, a minimal value of�5%wasobserved for gp350-biotin binding.Of the othermutants studied, N11Aand S32A both demonstrated simi-lar binding curves of gp350-biotinto that of wild-type CR2, whereasY16A showed slightly reduced bind-ing with anMFI of �67% relative towild-type CR2.Because Arg-13 has not previ-

ously been implicated in the associ-ation of gp350 with CR2 and otherstudies have postulated that thelinker region between SCR1 andSCR2 is themajor site of attachmentfor gp350, we decided to generate alimited number of additional muta-tions targeting residues Arg-13 andSer-15 and expressed using anE. coli system. Equal quantities ofpurified wild-type, R13A, and S15Pforms of MBP-CR2 SCR1-2 wereassessed by SDS-PAGE, and theircapacity to bind plate-bound gp350-biotin or plate-bound C3d was sub-sequently measured by ELISA anal-ysis (Fig. 5). Both R13A and S15Pmutants demonstrated levels ofgp350 ligand binding consistentwith that seen using the K562mutant analysis. R13A exhibitedinsignificant gp350-biotin bindingat all of the MBP-CR2 SCR1-2 con-centrations used in the ELISA(2–0.016�g/ml), whereas S15P also

exhibited a considerably reduced capacity to bind gp350-biotin,especially at concentrations below 0.5 �g/ml. It is not knownwhether the decrease in binding affinity associated with theS15P mutation is the result of a localized structural rearrange-

FIGURE 4. gp350-biotin binding analysis for mutations selected around and within the 8PILNGRIS15

region of SCR1. Whole cell populations of K562 erythroleukemia cells expressing N11A (A), R13A (B), R13E (C),and S15P (D) are shown after being incubated with 0.5 �g of EBV gp350-biotin, 0.4 �g of PE-NeutrAvidin.Normalized binding affinities of R13A (E), R13E (F), N11A and S15P (G), and Y16A and S32A (H) relative towild-type CR2. Average and S.E. of the normalized values for the MFI of the intermediate CR2 expressingpopulation (18%) are given.




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ment disrupting a binding interaction with gp350 or whetherSer-15 itself is directly involved in gp350 ligation. However,these data are highly suggestive that SCR1 undergoes a directinteraction with the surface of gp350 especially around Arg-13.In contrast to these data the R13A binding curve for plate-bound C3d demonstrated appreciable, although reduced bind-ing at levels similar to those seen in our previous CR2-C3dgtetramer binding studies (46), whereas the S15P mutant exhib-ited C3d binding similar to that seen for wild-type CR2. Com-parison of theCR2-gp350 and theCR2-C3dELISAbinding datasuggest that there are likely to be intrinsic differences betweenthe binding sites on CR2 for gp350 and for C3d.Positively Charged Surface of SCR1—Human CR2 SCR1-2

contains a number of conserved positively charged residues,primarily concentrated on a single external face of SCR1. Wehave previously used site-directed mutagenesis to highlight theimportance that positive charge plays in the ligation of CR2 toC3d (46). To evaluate the role that this same region may alsoplay in the binding of EBV to CR2, we decided to utilize ourpreviously generated alanine and opposite charge substitutionstargeting residues Arg-28, Arg-36, Lys-41, Lys-50, and Lys-57on SCR1 for our gp350-biotin binding analysis. Data for R28A,R28E, R36A, R36E, K41A, K41E, K50A, K50E, K57A, and K57Eare given (Table 1, Fig. 6). Three of these pairs of mutations inparticular appeared to demonstrate decreased gp350-biotinbinding; that is, R28A andR28E, R36A andR36E, andK41A andK41E all demonstrated less than 30% gp350-biotin binding rel-ative to wild-type CR2 for the alanine substitutions and lessthan or equal to 12% for the glutamic acid point mutations.Each of these three sets of mutations demonstrated a substan-tial decrease in gp350-biotin binding with the transition fromthe wild-type arginine or lysine positively charged side-chaingroup to a neutral alanine side-chain followed by a further

decrease in binding as a negativelycharged glutamic acid side chainwas introduced. This stepwisedecrease in gp350-biotin bindingsuggests that electrostatic interac-tions involving residues Arg-28,Arg-36, and Lys-41 in conjunctionwith residue Arg-13 from the previ-ously defined OKB7 mAb epitopedescribed above, as shown in Fig.7A, are likely to play an importantrole in the association of gp350 toCR2. The remaining two pairs ofmutants used in this analysis, pairK50A and K50E and pair K57Aand K57E, also show decreasedgp350-biotin binding relative towild-type CR2 SCR1-2, but to alesser degree. K50A and K50Emutants demonstrated an MFI of�59 and �51%, respectively,whereas K57A and K57E exhibitedan MFI of �37 and �64%, respec-tively. The data for the Lys-57mutations are unusual in that the

opposite charge point mutant has a demonstrably higherbinding affinity than the alanine substitution mutant and assuch should be treated with caution. However, in general itwould appear that Lys-50 and Lys-57 are less critical to theCR2-gp350 interaction either from the perspective of beingproximal rather than central to a gp350 binding site or asweaker contributors to a long-range electrostatic interactionleading up to the formation of an encounter complex.Intact EBV Binding—Our cell binding data, summarized in

Table 1 and Fig. 7, was used to direct the generation of furthermutant forms of recombinantMBP-CR2SCR1-2 (N11A, R13A,S15P, R28A, R36A,K41A,K57A,K67A, andR83A) expressed inE. coli. With the exception of N11A, which was made as a con-trol mutation, all of the other mutants generated were pre-dicted to exhibit compromised ability to bind intact EBV. TheWestern blot assay used to measure the capacity of mutantforms of MBP-CR2 SCR1-2 to bind EBV demonstrated thatindeed the majority of the mutations generated displayed com-promised binding capacity when incubated with concentratedvirus (Fig. 8). Only N11A demonstrated binding levels similarto that of wild type. These data are indicative that our cell bind-ing data mapping a potential gp350 binding interface on CR2 isalso likely to be relevant to the interaction of CR2 with intactEBV.

DISCUSSION

We have used a large number of instructive full-length CR2mutants expressed on the surface ofK562 erythroleukemia cellsto map out those residues within the two N-terminal extracel-lular SCR domains that are essential for the attachment of theEBV envelope gp350 protein to B-lymphocytes. The objectiveof this study was to complement the recent three-dimensionalstructural determination of gp350, which identified the major

FIGURE 5. A, SDS-PAGE of wild-type MBP-CR2 SCR1-2, R13A MBP-CR2 SCR1-2, or S15P CR2 SCR1-2 recombinantproteins in which 5 �g of sample has been loaded onto the gel matrix. B, ELISA demonstrating wild-type (WT),R13A MBP-CR2 SCR1-2, and S15P MBP-CR2 SCR1-2 binding to plate-bound gp350. C, ELISA demonstratingwild-type, R13A MBP-CR2 SCR1-2, and S15P MBP-CR2 SCR1-2 binding to plate-bound C3d. The average and S.E.of the normalized values relative to wild-type gp350 binding are given.




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CR2 binding site within this molecule to be a glycan-free,negatively charged region within the N-terminal 470 resi-dues (26). A summary of the site-directed mutagenesis bind-ing data and the primary inhibitory monoclonal antibodyepitopes used in this study is shown (Fig. 7, B and C). Both ofthe N-terminal extracellular SCR domains of CR2 appear tobe essential in the binding interaction with gp350. Ourepitope mapping data confirmed previous studies whichdemonstrated that the anti-CR2 monoclonal antibodies1048 mAb, and particularly, 171 mAb, significantly inhibitbinding of gp350 to CR2 (54). The primary epitope for 171mAb overlaps directly with the previously determined C3d/C3dg ligand binding site on SCR2, and these data along withthe available R83A and R83E mutant-gp350-biotin bindingcurves allow us to propose that SCR2 plays an important rolein the ligation of CR2 to gp350.

A number of studies have high-lighted the ability of human CR2 tobind EBV or gp350, whereas themurine formof the protein is unableto do so. This decreased capacity tobind virus or viral protein on thepart of murine CR2 has beenlinked to differences in two majorareas of CR2; that is, the 8-aminoacid linker region connectingSCR1 and SCR2 comprising resi-dues 63EYFNKYSS70 for the humanprotein and 63EESVNKTIS70 for themurine protein and within residues8–15 of SCR1, which in humanCR2delineates one of themajor epitopesfor the no longer available anti-CR2monoclonal antibody OKB7 mAb.The linker region adjoining the twoSCR domains in murine CR2 differsfrom the human form of the proteinwith the presence of an additionalN-glycanmoiety at position 66. Thisglycan has been shown to interferewith EBV binding by studies inwhich receptor bearing cells ex-pressing human CR2 containing aY68T point mutation exhibitedcompromised EBV binding (53).Although we were unable to gener-ate our own stable population ofK562 cells expressing an identicalY68T mutant, we did successfullyproduce valuable gp350-biotinbinding data of Y64A, K67A, K67E,and Y68A point mutants within thehuman CR2 linker region. Our dataindicate that substitution of resi-dues on the outside of the linkerregion (Y64A and Y68A) which areoriented away from the tightV-shaped structure resolved in the

available x-ray structures of CR2 do not appear to have anydeleterious effect on gp350-biotin binding. However, alanineand opposite charge substitutions targeting Lys-67, which isoriented toward the tightly packed structure, resulted in greaterdecreases in gp350-biotin binding (Fig. 7, B and C). Within theOKB7 mAb epitope of CR2, we were able to analyze gp350-biotin binding for a large number of mutations directed at andimmediately proximal to this area. R13A, R13E, and S15Pmutant forms of CR2 expressed on K562 cells all exhibitedinsignificant levels of gp350-biotin binding. Other mutationstargeting this Pro-8—Ser-15 area all exhibited gp350-biotincurves binding identical to, or only slightly reduced from wild-type CR2. Using recombinant wild-type and mutant forms ofMBP-CR2 SCR1-2 produced using a prokaryotic expressionsystem, we were able to replicate our cell binding data for theR13A and S15P mutants by ELISA. Although we are unable to

FIGURE 6. Mutagenesis screening of residues located within the positively charged face of CR2. Shownare the normalized binding affinities relative to wild-type CR2 of R28A and R28E (A), R36A and R36E (B), K41Aand K41E (C), K50A and K50E (D), and K57A and K57E (E). The average and S.E. of the normalized values for theMFI of the intermediate CR2 expressing population (18%) are given.




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state if the negligible gp350 binding observed in this study forthe S15Pmutant is the result of a localized structural perturba-tion, resulting in an altered gp350 binding site or if Ser-15 is

actually involved in a binding interaction with gp350 itself, thecombined mutagenesis data available for Ser-15 and Arg-13make it likely that SCR1 undergoes a contact interaction withgp350. Our MBP-CR2 SCR1-2 ELISA methodology whenextended to probe C3d ligand binding also allowed us to isolateresidues on CR2 that are essential for gp350 binding but not forthat of C3d.One of the most striking features of our data is the impor-

tance that charge plays in the attachment of gp350 to CR2. Inaddition to residues Arg-13, Lys-67, and Arg-83 we also foundextremely low gp350-biotin apparent binding affinities for anumber of point mutations targeting positively charged aminoacids on SCR1, namely Arg-28, Arg-36, and Lys-41 and, to alesser extent, Lys-50 and Lys-57. In this study we cannot ascer-tain whether the observed charge dependence associated withthe formation of a CR2-gp350 complex is a result of a long-range electrostatic attraction between the two molecules,which has previously been proposed as one of the mechanismsbehind theCR2-C3d interaction (51) or is rather a consequenceof localized ion-pair formation at a CR2-ligand binding inter-

FIGURE 7. Surface representations of the CR2 SCR1-2 molecule in its C3d ligand-bound state (C3d not shown) as determined by x-ray crystallography(PDB accession code 1GHQ (49)). A and D, electrostatic representation of the structure of CR2 SCR1-2 demonstrating the preponderance of positive chargeassociated with this molecule. D, as for A except the molecule has been rotated about the y axis by 90°. B and E, alanine and proline substitutions mapped ontothe surface of CR2 SCR1-2. The scheme used to color mutants shown in B, C, E, and F represents the percentage of gp350-biotin monomer binding of mutantsrelative to wild-type CR2 (at a concentration of 0.5 �g of gp350-biotin, 0.4 �g of PE-NeutrAvidin). E, as for B, except the molecule has been rotated about they axis by 90°. C and F, negative charge substitutions and inhibitory monoclonal antibody epitopes mapped onto the surface of CR2 SCR1-2. The primaryepitopes for 171 and 1048 mAbs on SCR2 of CR2 have been identified as 86TPYRH90 for 171 mAb and 111WCQANNMW118 for 1048 mAb and are shown in greenand cyan, respectively (54). Also indicated is Trp-118, which is the only residue in the 1048 mAb epitope that is located on the same face as the C3d ligandbinding site. F, as for C except the molecule has been rotated about the y axis by 90°. This figure was generated using the PYMOL Molecular Graphics System(Delano Scientific, San Carlos, CA).

FIGURE 8. SDS-PAGE and Western blot demonstrating capacity of recom-binant wild-type and mutant forms of MBP-CR2 SCR1-2 to bind B95-8EBV virus, as detected by the anti-gp350 monoclonal antibody 72A1.Upper panels in A and B represent levels of wild-type (WT) or mutant MBP-CR2eluted from the amylose beads, whereas lower panels indicate levels of EBVidentified using 72A1. A, shown are mutations targeting residues identifiedwithin SCR1 (R13A, S15P, R28A, R36A, K41A, K57A) and the linker region con-necting SCR1 and SCR2 (K67A) as being important in the interaction betweenCR2 and gp350. Also shown is N11A, which demonstrates binding identical towild-type as a positive control. B, shown is a mutant targeting Arg-83 (R83A)within SCR2 that has also been demonstrated to be important in the gp350binding interaction and exhibits reduced binding relative to wild type.




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face. One likely scenario would involve a combination withlong-range and short-range electrostatic interactions leading tothe formation of a stable CR2-gp350 complex.The overall binding site on CR2 delineated by our site-di-

rected mutagenesis targeting the binding of the N-terminalthree domains of gp350 also appears to reflect the binding sitefor the full-length form of gp350 and for intact EBV. Our EBVpulldown assay in which MBP-CR2 point mutants were incu-bated with concentrated virus demonstrated that all of the res-idues on CR2 implicated by our cell binding data to play a sig-nificant role in gp350 binding also exhibited a reduced capacityto bind intact virus.Our data also suggest that it is likely that themajor CR2 binding site on gp350 is primarily located within theN-terminal 470 residues, in agreement with previous studies(14), although we cannot rule out the possibility that an addi-tional binding site located on theC terminus of gp350 and com-prising residues 822–841, as identified by Urquiza et al. (31),plays a peripheral role in binding.In summary, our site-directed mutagenesis analysis of the

CR2-gp350-biotin interaction has revealed a number of resi-dues on both SCR1 and SCR2 that are critical for binding tooccur. Many of these have not previously been associated withgp350 binding, particularly a number of positively charged res-idues on SCR1. It is interesting to note that themajority of theseresidues lie on a single face of CR2, although whether this faceaccurately delineates the gp350 binding site on this moleculedepends on the mechanism of the electrostatic interactionbetween the two. It is hoped that ourmutagenesis data definingthe essential roles that residues Arg-13, Arg-28, Lys-41,andArg-83 play in the binding of CR2 and EBVwill assist in under-standing of viral-receptor interactions.

Acknowledgment—We thank the University of Colorado at Denverand Health Sciences Center Cancer Center Flow Cytometry Core forassistance.

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Kendra A. Young, Xiaojiang S. Chen, V. Michael Holers and Jonathan P. HannanType 2 (CR2/CD21)

Isolating the Epstein-Barr Virus gp350/220 Binding Site on Complement Receptor

doi: 10.1074/jbc.M706324200 originally published online October 9, 20072007, 282:36614-36625.J. Biol. Chem.

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