JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M504544200 on August 8, 2005

J. Biol. Chem., Vol. 280, Issue 42, 35598-35605, October 21, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/42/35598    most recent
M504544200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Urquiza, M.
Right arrow Articles by Patarroyo, M. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Urquiza, M.
Right arrow Articles by Patarroyo, M. E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Identification of Three gp350/220 Regions Involved in Epstein-Barr Virus Invasion of Host Cells*

Mauricio Urquiza{ddagger}§1, Ramses Lopez{ddagger}§, Helena Patiño{ddagger}§, Jaiver E. Rosas{ddagger}, and Manuel E. Patarroyo{ddagger}§

From the {ddagger}Fundación Instituto de Inmunología de Colombia and §Department of Chemistry and Faculty of Medicine, Universidad Nacional de Colombia, Bogotá 030405, Colombia

Received for publication, April 26, 2005 , and in revised form, July 20, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epstein-Barr virus (EBV) invasion of B-lymphocytes involves EBV gp350/220 binding to B-lymphocyte CR2. The anti-gp350 monoclonal antibody (mAb)-72A1 Fab inhibits this binding and therefore blocks EBV invasion of target cells. However, gp350/220 regions interacting with mAb 72A1 and involved in EBV invasion of target cells have not yet been identified. This work reports three gp350/220 regions, defined by peptide 11382, 11389, and 11416 sequences, that are involved in EBV binding to B-lymphocytes. Peptides 11382, 11389, and 11416 bound to CR2(+) but not to CR2(-) cells, inhibited EBV invasion of cord blood lymphocytes (CBLs), were recognized by mAb 72A1, and inhibited mAb 72A1 binding to EBV. Peptides 11382 and 11416 binding to peripheral blood lymphocytes (PBLs) induced interleukin-6 protein synthesis in these cells, this phenomenon being inhibited by mAb 72A1. The same behavior has been reported for gp350/220 binding to PBLs. Anti-peptide 11382, 11389, and 11416 antibodies inhibited EBV binding and EBV invasion of PBLs and CBLs. Peptide 11382, 11389, and 11416 sequences presented homology with the C3dg regions coming into contact with CR2 (C3dg and gp350 bound to similar CR2 regions). These peptides could be used in designing strategies against EBV infection.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epstein-Barr virus (EBV)2 invades B-lymphocytes through molecular interactions involving the EBV gp350/220 protein. It is known that gp350 binds to B-lymphocyte CR2 (known as C3d receptor) with a 3.2 nM affinity constant (12 nM affinity has been determined for B-lymphocyte surface) (1, 2). This simple 1:1 interaction is involved in adsorption, capping, and EBV endocytosis and is considered to be a primary determinant of EBV tissue tropism (3-5). The CR2 region binding to gp350 overlaps the CR2 region binding to C3dg. The gp350 N-terminal region contains part of the B-lymphocyte binding domain (5).

The gp350 21EDPGFFNVE29 peptide, having significant homology with C3d-amino acid sequences (6), binds to purified CR2 and to CR2(+) but not to CR2(-) B- and T-lymphoblastoid cell lines; this peptide coupled to BSA inhibits CR2 binding to EBV and gp350 (7). This peptide inhibits C3dg binding to B-cells (as well as EBV) and also inhibits C3-induced Raji cell proliferative response (7, 8).

However, the 470-mer N-terminal recombinant protein and gp220, containing this peptide sequence, present single-component binding, whereas gp350/220 presents a higher affinity two-component binding to CR2 (5, 9) and inhibits only 50% of EBV binding and EBV infection of Raji cells (5). Moreover, the monomer gp350 peptide (16IHLTGEDPGFFNVE29) does not inhibit gp350/220 or C3dg binding to CR2(+) cells; BOS-1 monoclonal antibody (recognizing peptide 14SLIHLTGEDPGFFN27) does not inhibit peptide 16IHLTGEDPGFFNVE29 Raji binding or block virus adsorption or infectivity (5). On the other hand, the C3dg region (homologous to this gp350 peptide) is not in contact with CR2 in the reported complex structure (10). It is thus very likely that other gp350/220 regions are involved in EBV binding to B-lymphocytes.

The gp350/220 region containing the epitope recognized by neutralizing monoclonal antibody 72A1 is one of the most important regions involved in gp350/220 binding to B-lymphocyte CR2, because the mAb 72A1 Fab fragment inhibits EBV binding and invasion of host cells (5, 11). Moreover, mAb 72A1 inhibits EBV invasion of monocytes (12), neutrophils (13), and T-cells (14). Anti-gp350/220 mAb 72A1 also inhibits interleukin-6 (IL-6) protein synthesis induced in PBLs by gp350/220 binding to CR2 (15); it also inhibits EBV invasion of B-lymphocytes (5). It is known that the gp350/220 region involved in EBV invasion of host cells is recognized by this monoclonal antibody; however, its precise localization remains unknown. The purpose of this work was thus to identify B-lymphocyte binding sequences that are involved in EBV virus infection of B-lymphocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Peptide Synthesis—Forty-six peptides, covering the total length of EBV gp350-reported sequence (16), were synthesized by the solid phase method (17) with N-terminal t-Boc-protected amino acids. Peptides were cleaved by a low-high hydrogen fluoride protocol (18). These peptides were analyzed by mass spectrometry and reverse-phase high performance liquid chromatography. Synthesized peptide sequences are shown in Fig. 1. Tyrosine (Y) was C-terminally added, allowing those peptides not containing this amino acid to be radiolabeled.

Lymphoblastoid Cell Line Cultures—The cloned Raji (19), Ramos (20), and P3HR-1 (21) lymphoblastoid cell lines and HeLa cells (22) were kept in culture in adjusted RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone), containing 2 mM L-glutamine, 1.5 g/liter sodium bicarbonate, 4.5 g/liter glucose, 10 mM HEPES (Invitrogen), and 1 mM sodium pyruvate. All of the cells were grown at 37 °C in a 5% humidified CO2 atmosphere. HeLa cells, cultured in monolayers, were harvested by adding PBS-EDTA followed by spinning at 1,000 x g for 5 min. Cells were washed three times for 5 min at 1,000 x g with PBS and then counted in a Newbauer chamber, and their viability was assessed by trypan blue staining.

Human Leukocyte Isolation—Cord blood and peripheral blood were collected in EDTA-treated sterile tubes from patients attending the Materno Infantil Hospital in Bogotá. Informed consent was obtained from all participants. Cord blood lymphocytes (CBLs) and peripheral blood lymphocytes (PBLs) were separated by sedimentation on Ficoll-Hypaque gradients. The obtained lymphocytes were washed five times with RPMI 1640, spun at 300 x g for 7 min at room temperature, and counted in a Newbauer chamber (23).

Epstein-Barr Virus—The EBV used for studies was obtained from the American Type Culture Collection (ATCC catalogue no. VR-1492).

125I Peptide Labeling—Peptides were 125I-labeled by the chloramine-T protocol. Briefly, 3.2 µl of Na125I (17.2 mCi/µg) reacted with 28 µg of chloramine-T and 5 µg of peptide. The reaction was stopped by adding 14 µg of sodium bisulfite in isotonic PBS (pH 7.4). The radiolabeled peptide was purified by using size-exclusion chromatography on a Sephadex G-10 column. 125I-Labeled peptide specific activities were between 80 and 160 µCi/nmol.

Peptide Cell Binding Assay—2, 4, 8, and 12 nM concentrations of 125I-labeled peptide were incubated in triplicate with a Raji, Ramos, or P3HR-1 cells or erythrocyte cell suspensions (104 cells µl-1) in the absence (total binding) or presence (nonspecific binding) of unlabeled peptide (1.25 µM) for 1 h at 18°C. Cells were then separated from the medium by spinning at 10,000 x g for 1 min through a dibutylphthalate-dioctylphthalate cushion (d = 1.015 g ml-1). Cell-bound 125I-labeled peptide was measured, and specific binding for each peptide was calculated as the difference between total and nonspecific binding. Cell binding activity for each peptide was defined as the ratio of specific binding over the amount of added radiolabeled peptide. Nemerow's peptide (peptide 11420), which specifically binds to Raji cell (7) (used as the positive binding control peptide), presented 0.034 binding activity. High activity binding peptide saturation curves were performed incubating 104 cells µl-1, with 125I-labeled peptide at a 5-700 nM concentration in the absence or presence of 40 µM unlabeled peptide. Hill analysis was performed as reported previously (24). Briefly, bound (b) and non-bound (free) peptide was measured in saturation assays. Hill analysis were done by determining log(b/(bmax -b) in function of log(free); bmax represents the maximum number of cell receptors to which the peptide could bind (depending on the total number of peptide cell receptors). The slope of this plot gives the Hill coefficient.

Immunization Protocol—Each New Zealand rabbit was immunized subcutaneously with 500 µg of a HABP of interest plus 150 µg of the T-helper epitope FISEAIIHVLHSR (25), emulsified with 250 µl Freund's complete adjuvant (500 µl final volume), and boosted on days 20, 40, 60, and 80 with the same antigen dose in Freund's incomplete adjuvant. Blood was drawn 20 days after the second, third, and fourth doses.

ELISA—Plates coated with 1 µg of peptide/well in 100 µl of PBS at 4 °C overnight were subsequently blocked with a 2% of fat-free dry milk in PBS, 0.2% Tween 20 (PBSMT) for 2 h at 37 °C. 100 µl of the appropriate serial rabbit serum dilutions were added and incubated for 2 h at 37 °C. The wells were then incubated with a 1/5,000 dilution of anti-rabbit peroxidase (Vector Laboratories) at 37 °C for 1 h followed by color development with a solution prepared by 3,3',5,5'-tetramethyl-benzidine peroxidase substrate (KPL) for 15 min; absorbency was read at 620 nm. For sandwich ELISA, plates were coated with 100 µl of mAb 72A1 (2.5 µg/ml) in PBS at 4 °C overnight. Plates were then blocked with PBSMT for 1 h at 37°C. Wells were subsequently incubated with increasing peptide solutions (0.3125-10 µg/ml) or with different EBV dilutions (1/10 to 1/320) for 2 h at 18 °C. Wells were then incubated with 100 µl of a 1/4000 anti-EBV or anti-HABP rabbit serum dilution for 2 h at room temperature. Control wells were treated in a similar way but using PBS instead of mAb 72A1. The conditions for the competitive sandwich ELISA (established by using HABP or EBV mAb 72A1 binding curves) were 4 µg/ml 11382, 0.15 µg/ml 11389, or 4 µg/ml 11416 HABPs bound to mAb 72A1 in the presence of 1/20 to 1/1280 EBV dilutions; 1/20 EBV dilution were bound to mAb 72A1 in the presence of 0.0024-10 µg/ml HABPs for 2 h at room temperature. Plates were then incubated with 100 µl of anti-HABP (1/4,000 dilution) or anti-EBV (1/500 dilution) sera for 2 h at 18 °C. Color development was performed as mentioned above (26).

Flow Cytometry Analysis—500,000 cells were washed twice with 0.5% PBS-BSA, spinning at 2,500 rpm for 5 min, for flow cytometry analysis. The cells were then treated with a flow cytometry fixation and permeabilization kit (Dako) according to the manufacturer's instructions. The cellular pellet was suspended in 100 µl of 0.5% PBS-BSA containing a 1/640 serum dilution or with 100 µl of a 1/400 72A-1 monoclonal antibody dilution on ice for 45 min. This mixture was incubated for 50 min at 18 °C. The cells were then washed three times with PBS-BSA, spun at 2,500 rpm for 5 min, incubated with 100 µl of a 1/200 dilution of fluorescein isothiocyanate-labeled goat anti-rabbit IgG F(ab')2 and phycoerythrin-labeled rat anti-human T-cell receptor for 30 min at 4 °C or with 100 µl of a 1/50 dilution fluorescein isothiocyanate-labeled anti-mouse IgG F(ab)2 (Vector Laboratories), and finally washed twice with PBS. Fluorescence cells were read by a FACScan (BD Biosciences).

Rabbit Antibody Isolation—Rabbit sera were diluted four times with 60 mM acetate buffer (pH 4.0); the pH was then raised to 4.5 by adding 0.1 N NaOH. 25 µl/ml caprylic acid was added and stirred for 30 min. Samples were then centrifuged at 10,000 x g for 30 min, and the supernatant was separated. A 1/10 volume of 10xPBS was added to the obtained supernatant and the pH adjusted to 7.4 with 0.1 N NaOH. The immunoglobulin fraction was precipitated with 0.35 g/ml ammonium sulfate overnight at 4 °C. The pellet was separated by spinning at 5,000 x g for 15 min at 4 °C and suspended in PBS. The immunoglobulin solution was dialyzed extensively with PBS. The isolated protein concentration was determined by the Bradford test (100-170 µg/ml) and antibody activity by ELISA and immunofluorescence (27).

Epstein-Barr Virus Invasion of CBLs—2x105 cord blood lymphocytes (in 100 µl of RPMI 1640 supplemented with 10% heat-inactivated serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.4 µg/ml cyclosporin A, and 5 mM CaCl2) were incubated with 30 µl of EBV-containing supernatant for 30 min at 37 °C in a 5% CO2 atmosphere. 70 µl of RPMI 1640 medium was then added and the samples incubated for 16 h at 37 °C in 5% CO2 atmosphere. After incubation, cells were washed three times with RPMI 1640 medium (fetal bovine serum-free), and their DNA was obtained for PCR amplification. CBLs were preincubated with 8 µM HABPs for 15 min at 37 °C, or 30 µl of EBV was preincubated with 30 µl of rabbit isolated antibodies (100-170 µg/ml), for 1 h at 37 °C before EBV invasion assays were performed to determine the effect of HABP or anti-HABP antibodies on EBV invasion of CBLs. EBV invasion of CBLs without HABPs or isolated antibodies was used as positive control. CBLs, treated under the same conditions as a positive control but without EBV supernatant, were used as negative control.

EBV DNA Identification by PCR Amplification—DNA was obtained by proteinase K digestion, phenol-chloroform extraction and ethanol precipitation. This DNA was dissolved in 20 µl of TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA), 0.5 mg/ml final concentration (28). PCR was performed in 20 µl of reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and each of the following 200 µM deoxyribonucleotide triphosphate, 0.1-0.5 µg of template DNA, each primer at 0.5 µM, and 1.0 unit of Taq polymerase. Previously reported primers (29, 30) were used to specifically amplify EBV DNA: 5'-TTCATCACCGTCGCTGACT-3' upstream sequence and 5'-ACCGCTTACCACCTCCTCT-3' downstream sequence. These primers specifically amplified a 300-bp DNA fragment from EBV(+) cells (Raji or B95-8), but not EBV(-) cells (CBLs, erythrocyte fraction, or HeLa cells). PCR conditions consisted of 35 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s in a 9600 thermal cycler (PerkinElmer Life Sciences) (29). The amplified fragment was separated and ethidium bromide-stained on 2.5% agarose gels; the PCR product was visualized on a Molecular Imager FX (Bio-Rad).



View larger version (53K):
[in this window]
[in a new window]
  		FIGURE 1.
gp350/220 peptide Raji, Ramos, and P3HR-1 cell binding activities. The column giving peptide numbers uses our laboratory's peptide accession codes. The protein position appears in the second column along with the sequences for gp350/220 peptides used in this study. The black bars represent Raji, Ramos, and P3HR-1 binding activity, calculated as stated under "Materials and Methods."

 
Producing mAb 72A1—Female 4-week-old Balb/c mice were immunized with 200 µl of Pristane (ICN) and 10 days later with 1 x 106 hybridoma cells (ATCC HB-168) in 200 µl of sterile PBS. Ascitic fluid was drained between the 15th and 30th day following hybridoma cell immunization. Antibody presence was determined by ELISA, sandwich ELISA, and cytometry analysis.

IL-6 Protein Quantification—300 µl 1 x 106 PBLs were incubated with 150 µl of EBV or each HABP at 13.3 µM for 96 h at 37 °C. The supernatant thus obtained was stored at -20 °C until IL-6 protein was determined by ELISA according to the manufacturer's instructions. The effect of 10 µg/ml mAb 72A1 on IL-6 protein synthesis induced by HABP-11382 or -11389 (11 µM) or EBV was determined. In other experiments, EBV was treated for 15 min at 4 °C with 100-160 µg/ml anti-HABP or anti-EBV isolated antibodies or with 10 µg/ml mAb 72A1 to determine the effects of these antibodies on EBV-induced IL-6 protein synthesis. Also, the effect of 10 µg/ml mAb 72A on EBV-induced IL-6 protein synthesis was determined in the presence of 8 µM HABP-11382, -11389, or -11416.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identifying B-lymphocyte-binding Regions in gp350—gp350/220 specifically binds to the CR2 molecule, which has been found on Raji and Ramos but not P3HR-1 cells (31, 32). Raji and Ramos cells were thus chosen as positive cell binding control and P3HR-1 as negative cell binding control for selecting B-lymphocyte-binding gp350 peptides. The CR2 molecule was determined on Raji and Ramos cells, but not on P3HR-1, by using flow cytometry analysis with the previously reported CR2-recognizing monoclonal antibody (33) (data not shown).

Peptides 11381, 11382, 11388, 11389, and 11394 from the gp350 N-terminal region and 11415 and 11416 from the gp350 C-terminal region presented CR2(+) Raji and Ramos cell binding activity higher than or equal to 0.034 (the known value for the binding activity of Nemerow's peptide), but these peptides did not bind to CR2(-) P3HR-1 cells (Fig. 1). These peptides were named CR2(+) cell HABPs, of which 11381, 11382, 11388, 11389, and 11416 presented conserved amino acid sequences in the gp350/220 protein reported to date, whereas the HABPs 11394 and 11415 presented Lys401 and Pro812 amino acid variations in their respective sequences. Raji cell affinity constants determined for these CR2(+) cell HABPs (except for HABP-11381 for which the affinity constant could not be determined from the obtained data) were in the 40-340 nM range, with HABP-11415 exhibiting the highest binding affinity (Kd = 40 nM), slightly higher than control peptide 11420 (Kd = 68 nM). HABPs 11388, 11389, and 11394 showed affinity constants of around 300 nM and HABPs 11382 and 11416 around 150 nM. HABPs 11382, 11388, 11389, 11394, and 11416 presented Hill coefficients of around 1, suggesting a single type of receptor-ligand interaction, but HABP-11415 presented a Hill coefficient of 2.0, suggesting positive cooperativity (TABLE ONE).


View this table:
[in this window]
[in a new window]
  		TABLE ONE
Peptide binding constants and Hill coefficients to Raji cells

 
CR2(+) Cell HABPs Inhibited EBV Attachment to B-lymphocytes—gp350/220 binding to CR2 on PBLs induces IL-6 synthesis (15, 34, 35). PBLs were thus exposed to CR2(+) cell HABPs, and IL-6 protein synthesis level was determined in the supernatant of these cell cultures. PBLs exposed to HABPs 11381, 11388, 11389, 11394, or 11415 showed IL-6 protein levels similar to that of non-treated PBLs, suggesting that these peptides did not have any effect on IL-6 synthesis. On the contrary, PBLs exposed to HABPs 11382 and 11416 increased four times the IL-6 protein levels, similar to IL-6 protein levels induced in PBLs by exposure to EBV (Fig. 2A).

It is probable that mAb 72A1 binds to some of these HABP sequences. ELISA revealed that HABPs 11382, 11389, and 11416 specifically bound to mAb 72A1 in a dose-dependent way, showing 72, 100, or 55% EBV binding to mAb 72A1, respectively, with antibody titers higher than or equal to 25,600 at 10 µg/ml peptide. On the other hand, HABPs 11381, 11388, 11394, and 11415 presented no significant binding to mAb 72A1 at the same peptide concentrations (less than 20% of the binding obtained with EBV) (Fig. 2B). Moreover, HABPs 11382, 11389, and 11416, but not the nonrelevant peptide, inhibited mAb 72A1 binding to EBV in a dose-dependent manner, just as EBV did, as determined by ELISA (Fig. 2C). PBLs were also exposed to HABP-11382, -11389, or -11416 or EBV in the presence or absence of mAb 72A1, and IL-6 protein levels were then determined; mAb 72A1 inhibited more than 50% of IL-6 protein synthesis induced by HABP-11382 or -11416 or EBV (Fig. 2D). Furthermore, the effect of mAb 72A1 on EBV-induced IL-6 synthesis was eliminated when mAb 72A1 had been incubated previously with HABP-11382, -11389, or -11416 (Fig. 2E).

CR2(+) Cell HABPs Induced Antibodies Recognizing EBV-infected Cells and Inhibited IL-6 Protein Synthesis Induced by EBV Binding to PBLs—Each rabbit was immunized with one of the CR2(+) cell HABPs; these peptides induced antibodies with anti-HABP antibody titers between 6,400 and 51,200 as determined by ELISA. These anti-HABP antibodies also recognized EBV by ELISA but with lower antibody titers (between 3,200 and 25,600) (Fig. 3A). On the other hand, anti-EBV antibodies were able to recognize these HABPs with antibody titers between 1,600 and 6,400.

Anti-HABP antibodies isolated from rabbit sera through precipitation with caprylic acid and ammonium sulfate specifically recognized a 11.3 ± 3.2% mean of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated B95-8 cells (EBV-infected cells) but only 1.6 ± 0.5% of CBLs (non-EBV-infected cells) by flow cytometry. Anti-EBV antibodies recognized around 31.64% of B95-8 cells and only 3.86% of CBLs. On the contrary, antibodies isolated from sera obtained before the first immunization or anti-nonrelevant peptide antibodies recognized only a 2.6 ± 1.8 mean of the B95-8 cells or CBLs (Fig. 3B). EBV binding to PBLs induced IL-6 protein synthesis in these cells; but when EBV was treated previously with anti-HABP-11382, -11389, or -11416 antibodies, the IL-6 protein synthesis induction diminished between 50 and 70%, in the same way that anti-EBV antibodies did. This effect was not seen with anti-nonrelevant peptide antibodies (Fig. 4).

CR2(+) cell HABPs 11382, 11389, and 11416 or Their Anti-HABP Antibodies Inhibited EBV Invasion of CBLs—EBV was able to invade CBLs not only because the virus presence in these cells could be determined by PCR amplification of the highly specific 300-bp DNA fragment but also because anti-gp250/350 mAb 8174 (Chemicon) recognized 31.6% of EBV-treated CBLs while recognizing only 2% of EBV-nontreated CBLs by flow cytometry analysis (data not shown).

EBV invasion of CBLs was performed in the presence of each CR2(+) cell-HABP or anti-HABP antibody. HABPs 11381, 11388, 11394, and 11415, or their anti-HABP antibodies, did not have a significant effect on EBV invasion of CBLs, because EBV DNA was detected by PCR in these cells (Fig. 5). On the contrary, EBV invasion of CBLs in the presence of HABPs 11382, 11389, and 11416, or their anti-HABP antibodies, was significantly inhibited, because EBV DNA was not detected in these cells (Fig. 5).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The aim of this work was to find gp350/220 regions involved in EBV binding to CR2(+) cells by using synthetic peptides. This approach has been used previously for defining protein regions involved in virus and host cell interactions (36-43). The initial screening binding assay was performed using Raji cells because they express CR2, bind EBV, and support virus-cell fusion (44). Nemerow's peptide was used as positive control because it specifically binds to these cells (7). Binding assays were performed at low peptide concentrations (2-12 nM) for identifying high affinity and high capacity binding peptides.

Raji binding peptides 11378, 11410, 11411, and 11412, which also bound to CR2(-) P3HR-1 cells, and 11401-11405 and 11423, which did not bind to CR2(+) Ramos cells, were discarded from further study, as gp350 specifically binds to CR2(+) cells but not to CR2(-) cells. However, peptides 11401-11405 from the repeat region (residues 502-621) could be involved in EBV invasion of B-lymphocytes, because the 576-mer N-terminal fragment binds to Raji cells, inhibits EBV binding to lymphocytes, and blocks EBV infection more efficiently than the 470-mer N-terminal fragment (lacking the 471-576 region) (5). Furthermore, the VTTPTPNATSPTLGKT sequence is the target for anti-EBV neutralizing monoclonal antibodies (45). HABPs 11402, 11403, and 11404 contain this reported epitope (underlined in the following: SAVTTPTPNATSPTLGKTSPT); HABPs 11401 and 11405 contain only part of this epitope (AVTTPTPNATSPT). The differences in cell binding tropism between B95-8 EBV (containing the repeat region) and P3HR-1 EBV (which does not contain this region) could be due to this binding region (46, 47).

HABPs 11381, 11382, 11388, 11389, 11394, 11415, and 11416 specifically bound to CR2(+) cells (Raji and Ramos) but not to CR2(-) cells (P3HR-1), and taking into account that gp350/220 specifically binds to CR2 molecules, these HABPs were therefore considered putative CR2-binding sequences. CR2(+) HABPs 11381, 11382, 11388, 11389, and 11394 were located in the gp350/220 576-mer N-terminal fragment, containing part of the Raji-binding region, because this fragment bound to Raji cells in a similar way as to the entire gp350/gp220 (5, 48). HABPs 11415 and 11416 were located in the C-terminal region, specifically in the immunodominant region recognized by anti-EBV human neutralizing antibodies (49, 50).

The CR2(+) HABPs showed saturable binding, having a finite number of HABP-binding sites per Raji cell, thus supporting the idea of specific binding receptors. The affinity constants were in the 40 to 340 nM range (TABLE ONE), showing strong interaction between these HABPs and Raji cells, which could be very important for efficient EBV binding to B-lymphocytes (except for HABP-11381 for which the affinity constant could not be obtained). HABPs 11382, 11388, 11389, 11394, and 11416 presented a single type of interaction. On the contrary, HABP 11415 presented a 2.0 Hill coefficient, suggesting positive cooperativity, and could be seen as a parallel increase in affinity accompanied by increased HABP binding to Raji cells. This peptide perhaps bound with different affinity to at least two different receptors that could also interact with each other, i.e. CD21 and MHC class II, which are known to bind peptides having this length (51, 52).



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 2.
HABP binding to PBLs and its effect on IL-6 protein synthesis. HABP-11382, -11389, and -11416 sequences probably contain the gp350/220-binding region that interacts with CR2 on PBLs. A, PBLs were exposed to EBV (C(+)) or to CR2(+)-cell HABPs. IL-6 protein levels were determined by ELISA. It is shown that HABPs 11382 and 11416 induced IL-6 protein synthesis in the same way as did EBV (C(+)). B, CR2(+) cell HABP binding to mAb 72A1 at different concentrations was determined by ELISA. It is shown that HABP-11382, -11389 or -11416, but not HABP-11381, -11388, -11394, or -11415, were specifically recognized by mAb 72A1. C, mAb 72A1 binding to EBV was performed in the presence of nonrelevant peptide 25684, HABPs 11382, 11389, and 11416, or EBV. HABPs 11382, 11389, and 11416, nonrelevant peptide 25684, or EBV. It is clearly shown that HABP-11382, -11389, or -11416 inhibited mAb 72A1 binding to EBV in a dose-dependent manner; on the contrary, HABP-25684 did not. D, PBLs were exposed to HABP-11382, -11389, or -11416 in the presence (white bars) or absence (gray bars) of mAb 72A1; this inhibited IL-6 protein synthesis induced by HABPs 11382 and 11416. A nonrelevant mAb (mAb NR) did not inhibit IL-6 induced by EBV. E, PBLs were exposed to EBV in the presence or absence of a nonrelevant mAb (which did not inhibit IL-6 induced by EBV) or mAb 72A1 (which inhibited IL-6 induced by EBV) plus HABP-11382, -11389, or -11416. It is shown that HABPs 11382, 11389, and 11416 blocked the effect of mAb 72A1 on EBV-induced IL-6 protein synthesis in PBLs.

 



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 3.
Anti-HABP antibody reactivity against peptides and EBV-infected cells by ELISA and flow cytometry analysis. A, anti-HABP or anti-EBV antibody serum titers from rabbits immunized with CR2(+) cell HABPs were determined by ELISA. B, the percentage of EBV-infected or noninfected cells that became fluorescent when anti-HABP antibody recognition was determined by flow cytometry analysis.

 



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 4.
The effect of anti-HABP antibodies on EBV binding to PBLs (CR2(+) cells). PBLs were exposed to EBV in the presence (white bars) of anti-11382, -11389, or -11416 antibodies or antibodies isolated from preimmune sera (gray bars); then, IL-6 protein levels were determined by ELISA. Anti-HABP antibodies inhibited EBV-induced IL-6 protein in a manner similar to anti-EBV antibodies (EBV). On the contrary, anti-nonrelevant peptide antibodies (C(-)) did not have a detectable effect on IL-6 protein synthesis.

 



View larger version (61K):
[in this window]
[in a new window]
  		FIGURE 5.
PCR-amplification of an EBV DNA fragment using DNA obtained from CBLs treated with EBV-containing supernatant. CBLs exposed to EBV, in the presence of HABPs or anti-HABP antibodies, were used to obtain DNA to amplify a specific fragment, using the previously reported EBV primers. This fragment was separated on agarose gels. MWM, molecular weight markers (shown by arrows on left); C(+) and C(-), EBV PCR amplification using DNA obtained from non-EBV-exposed and EBV-exposed CBLs, respectively (see "Materials and Methods"). EBV PCR amplification using DNA obtained from EBV exposed to CBLs in the presence of HABPs (PEPTIDE), antibodies isolated from preimmune sera, and anti-HABP antibodies are shown. R1 and R2 represent the immunized rabbit numbers. The number of the peptide used in rabbit immunization appears on the right.

 
It is known that gp350/220 binding to CR2 increases IL-6 protein synthesis on PBLs; mAb 72A1 inhibits gp350/220 binding and therefore inhibits EBV-induced IL-6 protein synthesis. In our work, HABPs 11382, 11389, and 11416 bound to mAb 72A1, inhibited mAb 72A1 binding to EBV, and blocked the effect of mAb 72A1 in inhibiting EBV-induced IL-6 protein synthesis. HABPs 11382, 1389, and 11416 bound to mAb 72A1, despite there being no apparent homology among them. This finding agrees with other reports that have revealed that highly specific antibodies frequently show cross-reactivity even in unrelated epitopes (53, 54). It was also found that HABPs 11382 and 11416 induced IL-6 synthesis in PBLs in the same way as EBV, and this induction was inhibited by mAb 72A1.



View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 6.
C3dg-CR2 chimerical complex structure. The C3dg-CR2 chimerical complex was constructed based on the resolved co-crystal structure of the C3dg-CR2 complex (Protein Data Bank code 1GHQ [PDB] ), in which C3dg regions presenting homology with gp350 HABP-11382, -11389, and -11416 sequences were changed by these HABP sequences. In yellow and ochre are the ribbon structures of the original resolved cocrystal structure of the CR2-C3dg complex, except for the C3dg regions that are similar to HABP-11382 (white), -11389 (purple), and -11416 (ice blue). In green and orange is the CR2-C3dg chimerical complex, except for the C3dg regions changed by HABP sequences (HABP-11382 in gray, -11389 in red, and -11416 in blue).

 
CBLs exposed to EBV in the presence of nonrelevant peptide were invaded by EBV, because EBV DNA was detected in these cells (Fig. 6). On the contrary, CBLs exposed to EBV in the presence of CR2(+) cell HABPs 11382, 11389, and 11416 were not significantly invaded by this virus, because EBV DNA was not detected by PCR amplification in these cells (Fig. 6).

All of these data indicate that HABPs 11382, 11389, and 11416 specifically inhibited EBV invasion of B-lymphocytes, probably by blocking EBV binding to host cells. HABPs 11382, 11389, and 11416 not only contain gp350 regions involved in EBV binding to host cells but also regions recognized by mAb 72A1, supporting the previously reported hypothesis that mAb 72A1 probably binds to the WCHHAEMQNPVYLIPETVPYIKW sequence (HABP-11382 is underlined) according to the results obtained with gp350/220 recombinant fragments (5).

Interestingly, LALING and GETAREA software (55) have revealed that these HABPs have homology with the C3d regions coming into contact with CR2 in the resolved co-crystal structure (C3d residues coming into contact with CR2 are shown in bold; identical or similar amino acids are underlined) (10): 11382 (HHAEMQNPVYLIPETVPYIK), C3d (LILEKQKPDGVFQEDAPVIH); 11389 (YVFYSGNGPKASGGDYCIQS), C3d (EQVNSLPGSITKAGDFLEAN); 11416 (PSTSSKLRPRWTFTSPPVTTY), C3d (PSSAFAAFVKRAPSTW LTAY).

These three binding regions could be held together in the gp350 structure, assembling a single binding site, as seen in the C3dg-CR2 resolved co-crystal structure of the complex. In fact, a chimera was built by homology modeling based on the C3dg-CR2 complex structure (Protein Data Bank code 1GHQ [PDB] ), in which C3dg CR2-contact regions were changed (i.e. replacements were made in C3dg but not in CR2) by HABP-11382, -11389, and -11416 sequences using Insight II (2000) Biopolymer module software (Accelrys Inc. software) run on an Indigo 2 Station (Silicon Graphics). The minimized chimerical and C3dg-CR2 resolved co-crystal structure of the complex were superimposed, revealing that the structures were very similar; contact residues were conserved (including secondary structure elements), and 2.8 Å root mean square deviation values were presented for the overall structure and 1.25 Å for the contact regions in both proteins (Fig. 6). This was due in part to the interaction between CR2 and C3dg being mediated mainly through backbone atom interaction (10). This suggests that in addition to these HABP sequences not disturbing the resolved co-crystal structure of the C3dg-CR2 complex contact regions, HABP sequences could also supply the C3dg protein function for binding to CR2 in the chimerical complex.

Anti-gp350/220 antibodies, inhibiting EBV binding to B-lymphocytes, are involved in protection against EBV infection. In fact, gp350/220 induces anti-EBV-neutralizing antibodies, restricting viral pathogenesis in vivo and virus infection in vitro (46, 56). It has also been reported that gp350-immunized marmosets are protected from EBV infection, developing gp350-reacting antibodies, several of which exhibit virus-neutralizing activity (57). Neutralizing antibody epitopes on gp350/220 are not generally glycosylation-dependent (58); some of them are located on the amino acid backbone (59), suggesting that the amino acid sequence of this protein induces protective antibodies. The gp350/220 B-lymphocyte-binding regions are thus suitable targets for inducing protective immunity against EBV infection.

Each rabbit immunized with one of the CR2(+) cell HABPs induced specific anti-HABP antibodies, which also recognized EBV (Fig. 3A), with anti-HABP antibody titers higher than anti-EBV antibody titers except in serum from rabbit 423 (Fig. 3A). These HABPs were recognized by anti-EBV antibodies but with lower anti-HABP titers than anti-EBV titers (Fig. 3A). This suggests that there are differences between peptide and protein structure and/or differences in epitope accessibility in these sequences.

Anti-HABP antibodies specifically recognized around 10% of EBV-infected cells (B95-8 cells) by flow cytometry analysis (Fig. 3B). Interestingly, a similar percentage of 12-O-tetradecanoylphorbol-13-acetate-treated B95-8 cells enters the viral lytic cycle (60). Anti-EBV antibodies recognized a high percentage of B95-8 cells, suggesting that some EBV antigen inducing these antibodies was expressed in the majority of B95-8 cells and not only in viral lytic cycle cells. These results showed that these HABPs are immunogenic and antigenic, that anti-HABP antibodies recognize some EBV native antigen in EBV-infected cells (probably gp350), and that the whole HABP sequence (or part of it) is exposed on native protein in EBV-infected cells.

Anti-HABP 11382, 11389, or 11416 antibodies significantly inhibited the IL-6 protein synthesis induced by EBV and EBV invasion of CBLs (Fig. 5), probably because these antibodies inhibited gp350 binding to CR2 (taking into account that these HABPs contain gp350 regions involved in EBV binding to host cells). These results suggest that HABPs 11382, 11389, and 11416 are capable of inducing antibodies inhibiting EBV binding of host cells in which EBV invasion is mediated by gp350/220. These results agree with other reports showing that monoclonal and human neutralizing antibodies recognize epitopes between gp350 residues 65-174 and 236-327 in which HABPs 11382 and 11389, respectively, are located (5, 45, 49, 50).

HABPs 11382, 11389, and 11416 clearly bound with high affinity to EBV-susceptible cells, inhibited EBV binding to B-lymphocytes, were recognized by mAb 72A1, and elicited antibodies not only interacting with EBV-infected cells but also inhibiting EBV binding and invasion of B-lymphocytes. These data thus suggest that HABP-11382, -11389, and -11416 sequences are involved in EBV binding and invasion of B-cell lymphocytes and could be used not only for designing strategies against EBV-induced diseases (i.e. EBV-induced post-transplant lymphoproliferative disorder), but also for designing prophylactic EBV vaccine candidates.


   FOOTNOTES
 
* This research was supported by the Office of the President of the Republic of Colombia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Virology Group, Fundación Instituto de Inmunología de Colombia, Cra 50, 26-00, Bogotá, Colombia. Tel.: 57-1-4815269 or 57-1-4815219; Fax: 57-1-3244672 (ext. 108); E-mail: mauricio_urquiza{at}fidic.org.co.

2 The abbreviations used are: EBV, Epstein-Barr virus; HABP, high activity binding peptide; PBL, peripheral blood lymphocyte; CBL, cord blood lymphocyte; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; IL-6, interleukin 6; mAb, monoclonal antibody. Back


   ACKNOWLEDGMENTS
 
We greatly appreciate the collaboration of Manuel A. Patarroyo and Edith Hernandez in PCR amplification, Jhan Arturo in flow cytometry analysis experiments, Mateo Obregon and Fabiola Espejo in computer software data analysis, and Erika Vega in cell culturing. We thank Jason Garry for patiently reading and correcting the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Maruo, S., Yang, L., and Takada, K. (2001). J Gen Virol. 82, 2373-2383[Abstract/Free Full Text]
  2. Moore, M. D., DiScipio R. G., Cooper N. R., and Nemerow, G. R. (1989) J. Biol. Chem. 264, 20576-20582[Abstract/Free Full Text]
  3. Ahearn, J. M., Hayward, S. D., Hickey, J. C., and Fearon, D. T. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 9307-9311[Abstract/Free Full Text]
  4. Tanner, J., Weis, J., Fearon, D., Whang, Y., and Kieff, E. (1987) Cell 50, 203-213[CrossRef][Medline] [Order article via Infotrieve]
  5. Tanner, J., Whang, Y., Sample, J., Sears, A., and Kieff, E. (1988) J. Virol. 62, 4452-4464[Abstract/Free Full Text]
  6. Nemerow, G. R., Mold, C., Schwend, V. K., and Cooper, N. R. (1987) J. Virol. 61, 1416-1420[Abstract/Free Full Text]
  7. Nemerow, G. R., Houghten, R. A., Moore, M. D., and Cooper, N. R. (1989) Cell 56, 369-377[CrossRef][Medline] [Order article via Infotrieve]
  8. Servis, C., and Lambris, J. D. (1989) J. Immunol. 142, 2207-2212[Abstract]
  9. Sarrias, M. R., Franchini, S., Canziani, G., Argyropoulos, E., Mooer, W. T., Sahu, A., and Lambris, J. D. (2001) J. Immunol. 167, 1490-1499[Abstract/Free Full Text]
  10. Szakonyi, G., Guthridge, J. M., Li D, Young, K., Holers, V. M., and Chen, X. S. (2001) Science 292, 1725-1728[Abstract/Free Full Text]
  11. Stocco, R., Sauvageau, G., Stefanescu, I., and Menezes, J. (1990) Intervirology 31, 295-300[Medline] [Order article via Infotrieve]
  12. Salek-Ardakani, S., Lyons, S. A., and Arrand, J. R. (2004) J. Immunol. 173, 321-331[Abstract/Free Full Text]
  13. Roberge, C. J., Poubelle, P. E., Beaulieu, A. D., Heitz, D., and Gosselin, J. (1996) J. Immunol. 156, 4884-4891[Abstract]
  14. Hedrick, J. A., Lao, Z., Lipps, S. G., Wang, Y., Todd, S. C., Lambris, J. D., and Tsoukas, C. D. (1994) J. Immunol. 153, 4418-4426[Abstract]
  15. Tanner, J. E., Alfieri, C., Chatila, T. A., Diaz-and Mitoma, F. (1996) J. Virol. 70, 570-575[Abstract]
  16. Baer, R. J., Bankier, A. T., Biggin, M. D., Deininger, P. L., Farrell, P. J., Gibson, T. J., Hatfull, G. F., Hudson, G. S., Satchwell, S. C., Seguin, C., and Tuffnell, P. S. (1984) Nature 310, 207-211[CrossRef][Medline] [Order article via Infotrieve]
  17. Houghten, R. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 5131-5135[Abstract/Free Full Text]
  18. Tam, J. P., Heath, W. F., and Merrifield, R. B. (1983) Int. J. Pept. Protein Res. 21, 57-65[Medline] [Order article via Infotrieve]
  19. Epstein, M. A., Achong, B. G., Barr, Y. M., Zajac, B., Henle, G., and Henle, W. (1966) J. Natl. Cancer Inst. 37, 547-559
  20. Freeman, C. B., Magrath, I. T., Benjamin, D., Makuch, R., Douglass, E. C., and Santaella, M. L. (1982) Clin. Immunol. Immunopathol. 25, 103-113[CrossRef][Medline] [Order article via Infotrieve]
  21. Hinuma, Y., Konn, M., Yamaguchi, J., and Grace, J. T. (1967) J. Virol. 1, 1203-1206[Abstract/Free Full Text]
  22. Duncan, M. R., and Stanish, S. M. (1978) J. Virol. 28, 444-449[Abstract/Free Full Text]
  23. Delves, P. J. (1997) in Antibody Productions: Essential Techniques (Delves, P. J., ed) pp. 64-65, John Wiley and Sons, Chichester, NY
  24. Weiland, G. A., and Molinoff, P. B. (1981). Life Sci. 29, 313-330[CrossRef][Medline] [Order article via Infotrieve]
  25. Prieto, I., Hervas-Stubbs, S., Garcia-Granero, M., Berasain, C., Riezu-Boj, J. I., Lasarte, J. J., Sorobe, P., Prieto, J., and Boras-Cuesta, F. (1995) Eur. J. Immunol. 25, 877-883[Medline] [Order article via Infotrieve]
  26. Koizumi, S., Zhang, X. K., Imai, S., Sugiura, M., Usui, N., and Osato, T. (1992) Virology 188, 859-863[CrossRef][Medline] [Order article via Infotrieve]
  27. McKinney, M. M., and Parkinson, A. (1987) J. Immunol. Methods 96, 271-278[CrossRef][Medline] [Order article via Infotrieve]
  28. Sambrook, J. Fritsch, E., and Maniatis, T. (1989) in Molecular Cloning: A Laboratory Manual (Nolan, C., ed) 2nd Ed., pp. 2.1.1-2.1.7, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  29. Chen, P. C., Pan, C. C., Yang, A. H., Wang, L. S., and Chiang, H. (2002) J. Pathol. 197, 684-688[CrossRef][Medline] [Order article via Infotrieve]
  30. Salimi, B., Alonso, E. M., Cohn, R. A., Mendley, S. R., and Katz, B. Z. (2002) Pediatr. Pathol. Mol. Med. 21, 433-443[CrossRef][Medline] [Order article via Infotrieve]
  31. Klein, E., Di Renzo, L., and Yefenof, E. (1990) Mol. Immunol. 27, 1343-1347[CrossRef][Medline] [Order article via Infotrieve]
  32. Reisert, P. S., Spiro, R. C., Townsend, P. L., Standford, S. A., Sairenji, T., and Humphreys, R. E. (1985) J. Immunol. 134, 3776-3780[Abstract]
  33. Weis, J. J., Tedder, T. F., and Fearon, D. T. (1984). Proc. Natl. Acad. Sci. U. S. A. 81, 881-885[Abstract/Free Full Text]
  34. D'Addario, M., Libermann, T. A., Xu, J., Ahmad, A., and Menezes, J. (2001) J. Mol. Biol. 308, 501-514[CrossRef][Medline] [Order article via Infotrieve]
  35. Tanner, J. E., and Menezes, J. (1994) Blood 84, 3956-3964[Abstract/Free Full Text]
  36. Bertoni, G., Kostyal, D. A., Reisert, P. S., Humphreys, R. E., and Sairenji, T. (1990) Intervirology 31, 290-294[Medline] [Order article via Infotrieve]
  37. Garcia, J. E., Puentes, A., Suarez, J., Lopez, R., Vera, R., Rodriguez, L. E., Ocampo, M., Curtidor, H., Guzman, F., Urquiza, M., and Patarroyo, M. E. (2002) J. Hepatol. 36, 254-262[Medline] [Order article via Infotrieve]
  38. Vera-Bravo, R., Ocampo, M., Urquiza, M., Garcia, J. E., Rodriguez, L. E., Puentes, A., Lopez, R., Curtidor, H., Suarez, J. E., Torres, E., Guzman, F., Diaz, D, Cortes, J., Bravo, M. M., Combita, A. L. Orozco, O., and Patarroyo, M. E. (2003) Int. J. Cancer 107, 416-424[CrossRef][Medline] [Order article via Infotrieve]
  39. Hu, C., Zhang, P., Liu, X., Qi, Y., Zou, T., and Xu, Q. (2004) Biochem. Biophys. Res. Commun. 316, 698-704[CrossRef][Medline] [Order article via Infotrieve]
  40. Grandi P, Wang S, Schuback D, Krasnykh, V., Spear, M., Curiel, D. T., Manservigi, R., and Breakefield, X. O. (2004) Mol. Ther. 9, 419-427[CrossRef][Medline] [Order article via Infotrieve]
  41. Hardy, H., and Skolnik, P. R. (2004) Pharmacotherapy 24, 198-211[CrossRef][Medline] [Order article via Infotrieve]
  42. Egelhofer, M., Brandenburg, G., Martinius, H., Schult-Dietriech, P., Melikyan, G., Kunert, R., Baum, C., Choi, I., Alexandrov, A., and Von-Laer, D. (2004) J. Virol. 78, 568-575[Abstract/Free Full Text]
  43. Urquiza, M., Suarez, J., Lopez, R., Vega, E., Patino, H., Garcia, J., Patarroyo, M. A. Guzman, F., and Patarroyo, M. E. (2004) Biochem. Biophys. Res. Commun. 319, 221-229[CrossRef][Medline] [Order article via Infotrieve]
  44. Patel, R. A., Hutt-Fletcher, L. M., and Crews, F. T. (1993) Virology 195, 121-131[CrossRef][Medline] [Order article via Infotrieve]
  45. Zhang, P. F., Klutch, M., Armstrong, G., Qualtiere, L., Pearson, G., and MarcusSekura, C. J. (1991) J. Gen. Virol. 72, 2747-2755[Abstract/Free Full Text]
  46. Thorley-Lawson, D. A., and Geilinger, K. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5307-5311[Abstract/Free Full Text]
  47. Wells, A., Koide, N., and Klein, G. (1981) Int. J. Cancer 27, 303-309[Medline] [Order article via Infotrieve]
  48. Beisel, C., Tanner, J., Matsuo, T., Thorley-Lawson, D., Kezdy, F., and Kieff, E. (1985) J. Virol. 54, 665-674[Abstract/Free Full Text]
  49. Pither, R. J., Nolan, L., Tarlton, J., Walford, J., and Morgan, A. J. (1992) J. Virol. 6, 1246-1251
  50. Pither, R. J., Zhang, C. X., Tarlton, J., Finerty, S., and Morgan, A. J. (1992) J. Gen. Virol. 73, 1409-1415[Abstract/Free Full Text]
  51. Li Q., Spriggs, M. K., Kovats, S., Turk, S. M., Comeau, M. R., Nepom, B., and Hutt-Fletcher, L. M. (1997) J. Virol. 71, 4657-4662[Abstract]
  52. Khanna, R., Sherrit, M., and Burrows, S. R. (1999) J. Immunol. 162, 3063-3069[Abstract/Free Full Text]
  53. Keitel, T., Kramer, A., Wessner, H., Scholz, C., Schneider-Mergener, J., and Hohne, W. (1997) Cell 91, 811-820[CrossRef][Medline] [Order article via Infotrieve]
  54. Kramer, A., Keitel, T., Winkler, K., Stocklein, W., Hohne, W., and Schneider-Mergener, J. (1997) Cell 91, 799-809[CrossRef][Medline] [Order article via Infotrieve]
  55. Fraczkiewicz, R., and Braun, W. (1998) J. Comput. Chem. 19, 319-333[CrossRef]
  56. Thorley-Lawson, D. A., and Poodry, C. A. (1982) J. Virol. 43, 730-736.[Abstract/Free Full Text]
  57. Emini, E. A., Schleif, W. A., Silberklang, M., Lehman, D., and Ellis, R. W. (1989) J. Med. Virol. 27, 120-123[Medline] [Order article via Infotrieve]
  58. Zhang, P. F., and Marcus-Sekura, C. J. (1993) J. Gen. Virol. 74, 2171-2179[Abstract/Free Full Text]
  59. Nuebling, C. M., Buck, M., Boos, H., Von-Deimling, A., and Mueller-Lantzsch, N. (1992) Virology 191, 443-447[CrossRef][Medline] [Order article via Infotrieve]
  60. Baumann, H., Isseroff, H., Latimer, J. J., and Jahreis, G. P. (1988) J. Biol. Chem. 263, 17390-17396[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Gen. Virol.Home page
A.-D. Gu, Y.-B. Xie, H.-Y. Mo, W.-H. Jia, M.-Y. Li, M. Li, L.-Z. Chen, Q.-S. Feng, Q. Liu, C.-N. Qian, et al.
Antibodies against Epstein-Barr virus gp78 antigen: a novel marker for serological diagnosis of nasopharyngeal carcinoma detected by xMAP technology
J. Gen. Virol., May 1, 2008; 89(5): 1152 - 1158.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. A. Young, X. S. Chen, V. M. Holers, and J. P. Hannan
Isolating the Epstein-Barr Virus gp350/220 Binding Site on Complement Receptor Type 2 (CR2/CD21)
J. Biol. Chem., December 14, 2007; 282(50): 36614 - 36625.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
F. C. Hsiao, M. Lin, A. Tai, G. Chen, and B. T. Huber
Cutting Edge: Epstein-Barr Virus Transactivates the HERV-K18 Superantigen by Docking to the Human Complement Receptor 2 (CD21) on Primary B Cells
J. Immunol., August 15, 2006; 177(4): 2056 - 2060.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/42/35598    most recent
M504544200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow