The amino terminus of Epstein-Barr virus glycoprotein gH is important for fusion with epithelial and B cells

Abstract

Epstein-Barr virus (EBV) infects B lymphocytes and epithelial cells. While the glycoproteins required for entry into these two cell types differ, the gH/gL glycoprotein complex is essential for entry into both epithelial and B cells. Analysis of gH protein sequences from three gammaherpesviruses (EBV, marmoset, and rhesus) revealed a potential coiled-coil domain in the N terminus. Four leucines located in this region in EBV gH were replaced by alanines by site-directed mutagenesis and analyzed for cell-cell membrane fusion with B cells and epithelial cells. Reduction in fusion activity was observed for mutants containing L65A and/or L69A mutations, while substitutions in L55 and L74 enhanced the fusion activity of the mutant gH/gL complexes with both cell types. All of the mutants displayed levels of cell surface expression similar to those of wild-type gH and interacted with gL and gp42. The observation that a conservative mutation of leucine to alanine in the N terminus of EBV gH results in fusion-defective mutant gH/gL complexes is striking and points to an important role for this region in EBV-mediated membrane fusion with B lymphocytes and epithelial cells.

FIG. 1.

Schematic diagram of a putative coiled-coil domain in gH of EBV and primate gammaherpesviruses. (A) gH protein sequences from EBV, rhesus, and marmoset (CalHV-3) gammaherpesviruses were analyzed by the COILS program (EMBnet) for the probability to form coiled-coils (http://www.ch.embnet.org/software/COILS_form.html) (23). A region at the N terminus was detected as a potential coiled-coil domain for all three proteins, which in EBV gH is located between amino acids 54 and 74. Conserved leucine residues are highlighted in boldface type. (B) Point mutants containing either a single or a double leucine-to-alanine substitution were generated by site-directed mutagenesis with the QuikChange kit (Stratagene). Mutations were verified by restriction digestion and DNA sequencing.

FIG. 2.

Residue L65 is important for the integrity of the E1D1 epitope. (A and C) CHO-K1 cells were transiently transfected to express gp42, gB, gL, and either a wild-type gH or a mutant gH. (B) CHO-K1 cells were transiently transfected with either gH or gL alone or both proteins together. Posttransfection, cells were transferred to 96 wells in triplicate and CELISA was performed with either E1D1 Ab (A) or a rabbit gH/gL Ab (HL-800) (B and C). Data are averages of three independent experiments with the standard deviations indicated by vertical lines. (D and E) CHO-K1 cells were transfected with gL and either a wild-type or mutant gH plasmid. Cells were harvested 36 h posttransfection and the cell surface proteins were labeled with biotin at 4^°C. Biotinylated lysates were immunoprecipitated with either the E1D1 Ab (D) or the HL-800 Ab (E) and probed with avidin-HRP in a Western blot. Data for some mutants are shown in panel E, but all analyses were performed. gH and gL bands are indicated with an arrow and a bracket, respectively. Asterisks indicate the location of a background band that somewhat overlaps with gL. Molecular masses (in kilodaltons) are noted to the left.

FIG. 3.

gH mutants retain their ability to associate with gp42. CHO-K1 cells were transfected with gp42, gB, gL, and either a wild-type or mutant gH plasmid and biotinylated at 4°C. (A) Biotinylated lysates were immunoprecipitated for gp42 with the F-2-1 Ab and probed with avidin-HRP in a Western blot. Cells transfected with an empty vector were used as a negative control (labeled as vector). The asterisks indicate the locations of two background bands with the lower one overlapping with gL. (B) Lysates that were used in the experiment shown in panel A were immunoprecipitated for gH/gL with the HL-800 Ab and probed with a rabbit gp42 Ab (PB1114) in a Western blot. For a control, CHO-K1 cells transfected with gp42 were immunoprecipitated with the F-2-1 Ab and probed with the PB1114 Ab (lane 1). The two bands observed for gp42 were reported previously (27). Molecular masses (in kilodaltons) are noted to the left of the blots.

FIG. 4.

EBV gH mutants have altered fusion with B cells and epithelial cells. CHO-K1 cells were transfected with EBV glycoproteins gp42, gB, and gL and either a wild-type or mutant gH. The transfected CHO-K1 cells were mixed 1:1 with either a B-cell line, Daudi cells (black bars), or an epithelial cell line, 293 cells (gray bars); 24 h later, the relative luciferase activity was measured. Luciferase activity was normalized to wild-type levels, which was set to 100% for both cell types. (A) Fusion results from mutants containing a single leucine-to-alanine change in residue 55, 65, 69, or 74; (B) data from mutants with a combination of two leucine residues mutated. Data are averages of three independent experiments with the standard deviations indicated by vertical lines.