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. 2007 Jun 20;363(1):148-55.
doi: 10.1016/j.virol.2007.01.025. Epub 2007 Feb 20.

Point mutations in EBV gH that abrogate or differentially affect B cell and epithelial cell fusion

Liguo Wu  1 , Lindsey M Hutt-Fletcher
Affiliations

Point mutations in EBV gH that abrogate or differentially affect B cell and epithelial cell fusion

Liguo Wu et al. Virology. .

Abstract

Cell fusion mediated by Epstein-Barr virus requires three conserved glycoproteins, gB and gHgL, but activation is cell type specific. B cell fusion requires interaction between MHC class II and a fourth virus glycoprotein, gp42, which complexes non-covalently with gHgL. Epithelial cell fusion requires interaction between gHgL and a novel epithelial cell coreceptor and is blocked by excess gp42. We show here that gp42 interacts directly with gH and that point mutations in the region of gH recognized by an antibody that differentially inhibits epithelial and B cell fusion significantly impact both the core fusion machinery and cell-specific events. Substitution of alanine for glycine at residue 594 completely abrogates fusion with either B cells or epithelial cells. Substitution of alanine for glutamic acid at residue 595 reduces fusion with epithelial cells, greatly enhances fusion with B cells and allows low levels of B cell fusion even in the absence of gL.

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Figures

Fig. 1
Fig. 1
Linear map of EBV gH indicating the positions of the predicted signal sequence, the predicted transmembrane domain and the Flag tag. The sequence of amino acids surrounding the insertion mutants M10 and M11 and the positions of the alanine substitutions are indicated.
Fig. 2
Fig. 2. Surface expression of point mutants measured by flow cytometry
AGS cells were transfected with plasmids expressing gB and gL together with wild type gH or mutant gH and stained with Mab anti-Flag and fluorescein-conjugated sheep anti-mouse antibody. The mean level of fluorescence for cells transfected with gB, gL and wild type gH and stained with fluorescein-conjugated sheep anti-mouse antibody alone was subtracted from the mean levels of cells transfected with wild type or mutant gH. The remaining value for wild type gH was set at 100 and the remaining values for each mutant were expressed as a percent of this value. Vertical lines equal the standard deviation of 3 experiments
Fig 3
Fig 3. Fusion of AGS cells supported by point mutants
AGS cells were transfected with plasmids expressing gB and gL together with wild type gH, mutant gH or empty vector. After 24 h cells were stained with Mab CL55 to gB and fusion events were counted by fluorescence microscopy. Numbers of cells containing four or more nuclei were considered as having undergone fusion and the percent of cells that had undergone fusion with each mutant is expressed as a percentage of those that had undergone fusion with wild type gH. In the absence of gH no fusion events were seen. Vertical lines equal the standard deviation of 6 experiments.
Fig 4
Fig 4
Fusion of Daudi B cells supported by point mutants and measured as relative luciferase activity. CHO-K1 cells were transfected with plasmids expressing gB and gL together with wild type gH, mutant gH or empty vector and a plasmid expressing luciferase under control of the T7 promoter. Transfected cells were overlayed with Daudi 29 cells expressing T7 RNA polymerase. Luciferase activity in the presence of empty vector was subtracted from activity in the presence of wild type or mutant gH. The remaining value for wild type gH was set at 100 and luciferase activity in the presence of each mutant was expressed as a percentage of this value. Vertical lines indicate the standard deviation of 8 experiments.
Fig. 5
Fig. 5. Interaction of wild type gH and G495A with gL and gp42
SDS-PAGE analysis of proteins immunoprecipitated by antibody to gp42, antibody to gL or antibody to gH from CV1 cells labeled with [35S] methionine and transfected as indicated with pTM1-EBV gH, pTM1-EBV gL and pTM1-EBV gp42, pTM1-EBV gL and pTM1-EBV gp42, pTM1-EBV G594A, pTM1-EBV gL and pTM1-EBV gp42, pTM1 gL and pTM1 empty vector (vect), pTM1 gL and pTM1-EBV G594A or pTM1 alone. The band indicated with an arrow is gH.
Fig 6
Fig 6. Complex formation by wild type gH and E595A
SDS-PAGE analysis of proteins immunoprecipitated by antibody to gH, antibody to gL or antibody to gp42 from CV1 cells labeled with [35S] methionine and transfected as indicated with pTM1-EBV gH, pTM1-EBV gL and pTM1-EBV gp42, pTM1-EBV gH and pTM1-EBV gp42, pTM1-EBV E595A, pTM1-EBV gL and pTM1-EBV gp42 or pTM1-EBV E595A and pTM1-EBV gp42. The band indicated with an arrow is gH.
Fig 7
Fig 7
Effect of wild type gH, E595A and gL on surface expression of gp42 measured by flow cytometry. AGS cells were transfected with plasmids expressing gB and gp42 together with combinations of gL, gH, E595A or empty vector (vect) as indicated and stained with Mab to gp42 and fluorescein-conjugated sheep anti-mouse antibody. The mean level of fluorescence for cells transfected with gB, gL gH and gp42 and stained with fluorescein-conjugated sheep anti-mouse antibody alone was subtracted from the values obtained for each combination. The remaining value for expression of gp42 with gB, gL and wild type gH was set at 100 and the values for each of the other combinations were expressed as a percent of this value. Vertical lines indicate the standard deviation of 3 experiments.
Fig 8
Fig 8
Surface expression of wild type gH and E595A in the absence of gL and the ability to mediate fusion of Daudi B cells. Black bars: AGS cells were transfected with combinations of plasmids expressing gH or E595A, gB, gp42 and gL or empty vector (vect) as indicated, stained with Mab anti-Flag and fluorescein-conjugated sheep anti-mouse antibody and analyzed by flow cytometry. The mean level of fluorescence for cells transfected with gB, gL gH and gp42 and stained with fluorescein-conjugated sheep anti-mouse antibody alone was subtracted from the values obtained for each combination. The remaining value for cells transfected with plasmids expressing gH, gB, gp42 and gL was set at 100 and values for other combinations were expressed as a percent of this value. Vertical lines equal the standard deviation of 3 experiments. Grey bars: CHO-K1 cells were transfected with a plasmid expressing luciferase under control of the T7 promoter and the indicated combinations of plasmids expressing gB, gL, gp42, gH, and E595A. Transfected cells were overlayed with Daudi 29 cells expressing T7 RNA polymerase. Luciferase activity in the presence of gB, gL, gp42 and empty vector was subtracted from activity in the presence of gH or E595A. The remaining value for gB, gL, gp42 and gH was set at 100 and luciferase activity in the presence of other combinations was expressed as a percentage of this value. Vertical lines indicate the standard deviation of 4 experiments.
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