Structure of the Epstein-Barr virus major envelope glycoprotein

Abstract

Epstein-Barr virus (EBV) infection of B cells is associated with lymphoma and other human cancers. EBV infection is initiated by the binding of the viral envelope glycoprotein (gp350) to the cell surface receptor CR2. We determined the X-ray structure of the highly glycosylated gp350 and defined the CR2 binding site on gp350. Polyglycans shield all but one surface of the gp350 polypeptide, and we demonstrate that this glycan-free surface is the receptor-binding site. Deglycosylated gp350 bound CR2 similarly to the glycosylated form, suggesting that glycosylation is not important for receptor binding. Structure-guided mutagenesis of the glycan-free surface disrupted receptor binding as well as binding by a gp350 monoclonal antibody, a known inhibitor of virus-receptor interactions. These results provide structural information for developing drugs and vaccines to prevent infection by EBV and related viruses.

Peptides Designed To Spatially Depict the Epstein-Barr Virus Major Virion Glycoprotein gp350 Neutralization Epitope Elicit Antibodies That Block Virus-Neutralizing Antibody 72A1 Interaction with the Native gp350 Molecule

“…Previous epitope mapping of gp350 revealed one dominant neutralizing epitope, which was also shown to be the target of the neutralizing monoclonal antibody 72A1 (13,21,29). Our laboratory cloned and sequenced the heavy-and light-chain variableregion RNA sequences for this antibody and verified by Edman protein sequencing that the cDNA sequences predicted to encode the two antibody chains matched both the mature heavy-and light-chain proteins.…”

“…Following computer modeling of the 72A1 antibody variable-region peptide sequences, in silico identification of the gp350/ 220 interaction with the 72A1 antibody was performed by spatially aligning the 72A1 variable-region peptide sequences with the amino-terminal portion of gp350 using the SnugDock algorithm (13). The SnugDock molecular docking program predicts the optimal alignment for close coupling of the 72A1 variable region with the gp350 molecule by identifying the lowest-energy structure between two molecules and lists the interfacing residues and types of interactions (24).…”

“…Prediction of the gp350 domain that interacts with the 72A1 monoclonal antibody was performed by aligning 72A1 variable-region protein sequences with the previously reported three-dimensional (3D) structure for the gp350 amino-terminal peptide (Molecular Modeling Database accession number 42235) (13). The method used for the identification of the 72A1 complementarity-determining regions (CDRs) was based on previously described methods (http://www.bioinf.org.uk/abs/) (22).…”

“…Experimental evidence indicates, however, that recognition of only one epitope, as represented by the monoclonal antibody 72A1 (10), effectively blocks infection by inhibiting EBV binding to the cellular receptors CD21 and CD35 (11,12). Although an electron density map of the first 440 amino acids of the gp350 molecule localizes the 72A1 epitope to a planar structure devoid of carbohydrates (13), the amino acids within this gp350 surface structure, which interact with the 72A1 antibody and constitute the principal EBV neutralization epitope, are still unidentified.…”

Detection of complement activation using monoclonal antibodies against C3d

“…Recombinant maltose-binding protein-tagged (MBP-tagged) CR2 SCR1-2 (MBP-CR2) comprising residues 1-133 of wild-type CR2 and encompassing the first 2 SCR modules were expressed in E. coli as previously described (41)(42)(43). Briefly, MBP-CR2 SCR1-2-transformed colonies of E. coli BL21 were expanded to 4 liters in LB media and grown at 37°C until an A600 of 0.3 was obtained.…”