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. 2004 Jun;78(11):5946-56.
doi: 10.1128/JVI.78.11.5946-5956.2004.

Mutational analyses of Epstein-Barr virus glycoprotein 42 reveal functional domains not involved in receptor binding but required for membrane fusion

Amanda L Silva  1 , Jasmina Omerovic, Theodore S Jardetzky, Richard Longnecker
Affiliations

Mutational analyses of Epstein-Barr virus glycoprotein 42 reveal functional domains not involved in receptor binding but required for membrane fusion

Amanda L Silva et al. J Virol. 2004 Jun.

Abstract

Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with malignancies of both epithelial and lymphoid origin. Efficient infection of the latent host reservoir B lymphocytes involves the binding of glycoproteins gp350/220 for initial attachment, followed by the concerted action of gH, gL, gB, and gp42 for membrane fusion. The type II membrane protein gp42 is required for infection of B cells and assembles into a complex with gH and gL. The cellular host receptor for gp42, class II human leukocyte antigen (HLA), has been structurally verified by crystallization analyses of gp42 bound to HLA-DR1. Interestingly, the crystal structure revealed a hydrophobic pocket consisting of many aromatic and aliphatic residues from the predicted C-type lectin domain of gp42 that in other members of the C-type lectin family binds major histocompatibility complex class I or other diverse ligands. Although the hydrophobic pocket does not bind HLA class II, mutational analyses presented here indicate that this domain is essential for EBV-induced membrane fusion. In addition, mutational analysis of the region of gp42 contacting HLA class II in the gp42-HLA-DR1 cocrystal confirms that this region interacts with HLA class II and that this interaction is also important for EBV-induced membrane fusion.

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Figures

FIG. 1.
FIG. 1.
Important structural features of EBV gp42 and residue sequence mapping location of mutations. (A) Three-dimensional ribbon model of soluble EBV gp42 (rose) bound to class II HLA-DR1 (beige). The amino terminus of residues 33 to 85 was disordered in the crystal structure. Backbones of residues interacting with HLA class II are yellow, oxygen atoms are red, and nitrogen atoms are blue. The orange and green arrows indicate the regions enlarged in panels B and C, respectively. (B) Close-up depicting key gp42 contact residues at the HLA class II interface. The color scheme is the same as in panel A. (C) Close-up of gp42 hydrophobic pocket highlighting aliphatic and aromatic residues. (D) The residue sequence of gp42 reveals the locations of point mutations above residues and linker insertion mutations containing 5-amino-acid inserts above arrows. Linker insertion mutants are named based on the residue that their mutation precedes, e.g., LI12, and point mutants reveal original and mutation residue, e.g., W44A. The baculovirus-produced soluble gp42 (sgp42) used as a positive control in many experiments spans residues 33 through 223 and was kindly provided by Maureen Mullen. Residues of the potential gH/gL binding site are in green. HLA class II contact sites are indicated as follows: aromatic ring residues in red, α-2-helix in purple, and arginine 220 in blue. Hydrophobic residues in the pocket are in yellow. Mutants are color-coded by their function in the fusion assay: those in green have levels similar to the wild-type levels, those in orange have reduced levels of fusion, and those in red do not mediate fusion.
FIG. 2.
FIG. 2.
Verification of mutant gp42 surface expression by CELISA and of whole-cell expression by Western blotting. (A) CHO cells were transiently transfected to express wild-type or mutant gp42 and transferred to 96 wells, 3 wells per sample. Shown is a representative CELISA experiment with a random sampling of linker insertion mutants followed by point mutants in sequential order from amino terminus to carboxyl terminus. Positive expression is at least a threefold higher average reading of triplicate samples than the vector control shown at left. OD, optical density. (B and C) Mutant gp42 expression was verified as both a transmembrane form in whole lysates (B) and a secreted form in supernatants (C) of transiently transfected CHO cells by Western blotting. Molecular mass (kDa) is noted to the left of blots.
FIG. 3.
FIG. 3.
Mutant gp42s vary in ability to mediate cell-cell fusion. CHO cells transiently transfected to express EBV gH, gL, gB, wild-type or mutant gp42, and luciferase driven by a T7 promoter were overlaid with Daudi cells stably transfected to express T7 RNA polymerase. Mutants are in order of location from the amino terminus to carboxyl terminus, linker insertion mutants are followed by point mutants, and readings are averages of two samples.
FIG. 4.
FIG. 4.
FACS analysis reveals differences among mutants in binding to HLA class II and mediating fusion. Daudi B lymphocytes (5 × 105) expressing HLA class II were incubated with 25 μl of supernatant from transfected CHO cells expressing wild-type or mutant gp42. Cells were stained for gp42 and class II HLA-DQ and analyzed by flow cytometry using a Becton-Dickinson FACS-Sort. Columns represent five groups. Column 1 shows controls; the top two are negative media and vector controls, the bottom are baculovirus-produced sgp42 and transfected CHO cell supernatant gp42 (wild type). Column 2 reveals the mutants that are unaffected in binding to HLA class II and are able to mediate fusion. Column 3 shows those that cannot mediate binding to HLA class II or fusion, but the locations are not in HLA class II contact sites. Column 4 contains mutants unable to bind HLA class II and mediate fusion, but the mutations are localized to HLA class II contact sites. Column 5 contains mutants that are able to bind to HLA class II but are unable to mediate fusion and localize to the hydrophobic pocket.
FIG. 5.
FIG. 5.
Ribbon models identifying locations of all gp42 mutants based on classes identified from Table 2. The classes are inconsequential, core/distant, HLA class II binding, or hydrophobic pocket. In each panel, the sites of the linker insertion (LI) mutants are indicated in blue and the site-specific mutants are indicated in yellow. Abbreviations and color scheme follow those in previous figures.
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