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. 2006 Oct;80(19):9444-54.
doi: 10.1128/JVI.00572-06.

Soluble Epstein-Barr virus glycoproteins gH, gL, and gp42 form a 1:1:1 stable complex that acts like soluble gp42 in B-cell fusion but not in epithelial cell fusion

Austin N Kirschner  1 , Jasmina Omerovic, Boris Popov, Richard Longnecker, Theodore S Jardetzky
Affiliations

Soluble Epstein-Barr virus glycoproteins gH, gL, and gp42 form a 1:1:1 stable complex that acts like soluble gp42 in B-cell fusion but not in epithelial cell fusion

Austin N Kirschner et al. J Virol. 2006 Oct.

Abstract

Epstein-Barr virus (EBV) is a herpesvirus that infects cells by fusing its lipid envelope with the target cell membrane. The fusion process requires the actions of viral glycoproteins gH, gL, and gB for entry into epithelial cells and additionally requires gp42 for entry into B cells. To further study the roles of these membrane-associated glycoproteins, purified soluble forms of gp42, gH, and gL were expressed that lack the membrane-spanning regions. The soluble gH/gL protein complex binds to soluble gp42 with high affinity, forming a stable heterotrimer with 1:1:1 stoichiometry, and this complex is not formed by an N-terminally truncated variant of gp42. The effects of adding soluble gp42, gH/gL, and gH/gL/gp42 were examined with a virus-free cell-cell fusion assay. The results demonstrate that, in contrast to gp42, membrane fusion does not proceed with secreted gH/gL. The addition of soluble gH/gL does not inhibit or enhance B-cell or epithelial cell fusion when membrane-bound gH/gL, gB, and gp42 are present. However, the soluble gH/gL/gp42 complex does activate membrane fusion with B cells, similarly to soluble gp42, but it does not inhibit fusion with epithelial cells, as observed for gp42 alone. A gp42 peptide, derived from an N-terminal segment involved in gH/gL interactions, binds to soluble gH/gL and inhibits EBV-mediated epithelial cell fusion, mimicking gp42. These observations reveal distinct functional requirements for gH/gL and gp42 complexes in EBV-mediated membrane fusion.

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Figures

FIG. 1.
FIG. 1.
Soluble gH/gL and gH/gL/gp42. (A) Schematic of soluble protein constructs for EBV gp42, EBV gH, and EBV gL. SS is the signal sequence from gp64. His/S represents six-His and S tags. (B) SDS-PAGE showing gel filtration-purified samples of soluble gH/gL purified by E1D1 affinity column (left side) and soluble gH/gL/gp42 purified by Talon resin (right side). The gL protein has no N-terminal tags. (C) SDS-PAGE and Western blotting of the single gel filtration peak containing the purified soluble gH/gL/gp42 complex. Anti-gH/gL antibody HL-800 detects gH and gL (left side), while the tag-specific S protein detects gp42 (right side). The values on the left are molecular sizes in kilodaltons.
FIG. 2.
FIG. 2.
Demonstration of the soluble gH/gL/gp42 complex. (A) Gel filtration traces (overlay) showing the soluble gH/gL/gp42 complex in a 1:1:1 ratio as determined by size. (B) Gel filtration traces (overlay) showing soluble gH/gL unable to bind soluble gp42-ΔN86. For panels A and B, peak positions for sizing standard proteins are 200 kDa at 9.99 ml, 150 kDa at 10.87 ml, 66 kDa at 11.98 ml, and 29 kDa at 14.5 ml. (C) Native PAGE showing the soluble gH/gL/gp42 complex and lack of gH/gL binding to soluble gp42-ΔN86. S is the prestained standard proteins used to guide the gel running time.
FIG. 3.
FIG. 3.
Dose-dependent effects of soluble gp42 on membrane fusion. Fusion assay graphs showing concentration-dependent effects of added soluble gp42 on B-cell fusion (A) and epithelial cell fusion (B); CHO cells were transfected with gH, gL, and gB. The data shown are representative examples of at least three independent experiments, and error bars indicate standard deviations.
FIG. 4.
FIG. 4.
Effects of soluble gH/gL and soluble gH/gL/gp42 on membrane fusion. Fusion assay graphs showing concentration-dependent effects of added soluble gH/gL on B-cell fusion (A) and epithelial cell fusion (B) and of added soluble gH/gL/gp42 on B-cell fusion (C) and epithelial cell fusion (D). The data shown are the average of at least three independent experiments, and error bars represent standard deviations. CHO cells were transfected with gH, gL, and gB (B, C, D); transfected gp42 was additionally present in the B-cell fusion with soluble gH/gL (A).
FIG. 5.
FIG. 5.
Peptide from gp42 residues 36 to 65 binds to soluble gH/gL and competes with soluble gp42 for binding to gH/gL. (A) Gel filtration traces (overlay) on a peptide column monitored at 280 nm showing the free gp42-36-65 peptide peak (10.77 ml) as gH/gL was increasingly titrated from 0.099 μM to 9.86 μM. (B) Percent peptide binding derived from area-under-the-curve analysis of gel filtration experiments; control data (red square) show peptide unable to efficiently bind gH/gL/gp42 at 9.86 μM (6.9% binds). In all experiments, the gp42-36-65 peptide concentration was constant at 7.55 μM.
FIG. 6.
FIG. 6.
Effect of peptide from gp42 residues 36 to 65 on membrane fusion. Epithelial cell fusion assay graphs showing that incubation with the gp42-36-65 peptide can inhibit fusion in a dose-dependent manner (A) and a control peptide does not inhibition fusion (B). The data shown are representative examples of at least three independent experiments, and error bars represent standard deviations. The wild-type fusion was normalized to 100%, and CHO cells were transfected with gH, gL, and gB.
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