Epstein-Barr Virus Infection of Polarized Epithelial Cells Via the Basolateral Surface By Memory B Cell-Mediated Transfer Infection

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Abstract

Epstein Barr virus (EBV) exhibits a distinct tropism for both B cells and epithelial cells. The virus persists as a latent infection of memory B cells in healthy individuals, but a role for infection of normal epithelial is also likely. Infection of B cells is initiated by the interaction of the major EBV glycoprotein gp350 with CD21 on the B cell surface. Fusion is triggered by the interaction of the EBV glycoprotein, gp42 with HLA class II, and is thereafter mediated by the core fusion complex, gH/gL/gp42. In contrast, direct infection of CD21-negative epithelial cells is inefficient, but efficient infection can be achieved by a process called transfer infection. In this study, we characterise the molecular interactions involved in the three stages of transfer infection of epithelial cells: (i) CD21-mediated co-capping of EBV and integrins on B cells, and activation of the adhesion molecules, (ii) conjugate formation between EBV-loaded B cells and epithelial cells via the capped adhesion molecules, and (iii) interaction of EBV glycoproteins with epithelial cells, with subsequent fusion and uptake of virions. Infection of epithelial cells required the EBV gH and gL glycoproteins, but not gp42. Using an in vitro model of normal polarized epithelia, we demonstrated that polarization of the EBV receptor(s) and adhesion molecules restricted transfer infection to the basolateral surface. Furthermore, the adhesions between EBV-loaded B cells and the basolateral surface of epithelial cells included CD11b on the B cell interacting with heparan sulphate moieties of CD44v3 and LEEP-CAM on epithelial cells. Consequently, transfer infection was efficiently mediated via CD11b-positive memory B cells but not by CD11b-negative naïve B cells. Together, these findings have important implications for understanding the mechanisms of EBV infection of normal and pre-malignant epithelial cells in vivo. © 2011 Shannon-Lowe, Rowe.

Figures

  • Figure 1. EBV induces firm adhesion between B cells and epithelial cells. (A) FACS profiles of conjugate formation between CFSE-labelled primary tonsillar epithelial cells (x-axis) and PKH26-labelled primary B cells (y-axis). The B cells were uninfected, EBV-infected (MOI 100, 24 h p.i.) or incubated with agonist mAb to CD21 (BL13). Conjugates appear in the upper right quadrant and the percentages of cells are shown. (B-C) Electron micrographs of B cell-epithelial cell conjugates. EBV-infected B cells (24 h p.i.) were co-cultured with primary tonsillar epithelial cells for 1 hour and immediately fixed, embedded in Epon 812, stained with uranyl acetate and ultra-thin sectioned. (B) The sites of interaction between the cell types show firm interaction. (C) A second conjugate revealing a firm interaction between the B- and epithelial cell and the fine ultrastructure of the virus particles. doi:10.1371/journal.ppat.1001338.g001
  • Figure 2. Analysis of B cell surface molecules at the virological synapse. (A) Confocal images of 1 mm Z slices through conjugates formed between EBV-infected B cells (24 hr p.i.) and primary tonsillar epithelial cells. Before co-culture, B cells were pre-labeled with nonblocking antibodies to virus (gp350) or cell surface molecules labeled with AlexaFluor 488 (green), 555 (red), 647 (blue) and DAPI (white). Antibodies derived from the same species were pre-labelled using the Zenon antibody-labeling kit. All gp350 staining co-localises at the cellcell junction with the B cell surface molecules CD21, LFA-1, ICAM-1, CD63 and b-1 integrin. B cell surface HLA class II molecules are not restricted to the cell-cell junction, but are also present around the B cell surface. (B) Conjugates labelled with gp350 and ICAM-1 following coculture showed the epithelial cell surface molecules did not relocate to the site of B cell-epithelial cell interaction. doi:10.1371/journal.ppat.1001338.g002
  • Table 1. B cell surface molecules capped following EBV infection and present within the B cell-epithelial cell synapse.
  • Figure 3. Molecules mediating B cell adhesion to epithelial cells. B cell-epithelial cell adhesion was examined by transfer infection for cation, RGD or GAG usage. (A) EBV-infected B cells (24 h p.i.) and tonsillar epithelial cells were washed in Ca2+/Mg2+-free PBS. Co-culture was performed in binding buffer in the absence of cations (EDTA) or the presence of Mg2+, then under optimal cation conditions, B cells and epithelial cells were pre-incubated in blocking antibodies to LFA-1, ICAM-1 and a control avb6. B cells were washed off after 1 hour of coculture and epithelial cell infection (GFP) was examined by flow cytometry after 24 hours. The % GFP +ve epithelial cells were plotted as the mean of triplicates relative to the optimal transfer infection conditions. (B) RGD peptide and a scrambled control peptide were titrated and pre-incubated with the B cells and epithelial cells under optimal cation conditions in binding buffer. Co-culture was performed for 1 hour and epithelial cell infection analysed as above. The mean % GFP +ve of triplicate epithelial cell infections were plotted against the peptide concentration. (C) The glycosaminoglycans (GAGs) heparan sulphate, hyaluronic acid and chondroitin sulphate were titrated as above. The mean % GFP +ve of triplicate epithelial cells infections were plotted against the GAG concentrations. doi:10.1371/journal.ppat.1001338.g003
  • Figure 4. Polarization of primary tonsillar epithelial cells.
  • Figure 6. B cell adhesion to the epithelial cell basolateral surface. Primary tonsillar epithelial cells were plated onto 8 mM poresize Transwells and grown until confluent. The polarized epithelial cells were examined by transfer infection via the basolateral surface for cation, RGD or GAG usage. (A) EBV-infected B cells (24 h p.i.) and epithelial cells were washed in Ca2+/Mg2+-free PBS. As above, co-culture was performed in binding buffer in the absence of cations (EDTA) or the presence of Mg2+, then under optimal cation conditions, B cells and epithelial cells were pre-incubated in blocking antibodies to LFA-1, ICAM-1, RGD peptide or scrambled peptide. Co-culture was performed for 1 hour, then B cells washed off. The epithelial cells were trypsinised off the transwell membrane and plated onto 24 well plates. Infection was monitored by flow cytometry of .10,000 cells for GFP after 24 hours and plotted as % infection (mean of triplicates) relative to the optimal cation conditions. (B) Under optimal conditions, the EBV-infected B cells (24 h p.i.) were preincubated with the GAGs: heparan sulphate, chondroitin sulphate and hyaluronic acid. Transfer infection was performed and analysed as in 6A and plotted as % infection (mean of triplicates) relative to the no GAG control. (C) EBV-infected B cells (24 h p.i.) were pre-incubated with (10– 20 mg/ml) blocking antibodies to CD11b, aVb6 and b1 integrins and LEEP-CAM before co-culture for 1 hour with polarized epithelial cells. The B cells were washed off and epithelial cells re-plated, as above. Epithelial infection was monitored by GFP expression after 24 hours by flow cytometry of .10,000 cells and plotted as % infection (mean of triplicates) relative to the positive control. doi:10.1371/journal.ppat.1001338.g006
  • Figure 5. Transfer infection of polarised epithelial cells.
  • Figure 7. Virus requirements for basolateral surface entry.

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Shannon-Lowe, C., & Rowe, M. (2011). Epstein-Barr Virus Infection of Polarized Epithelial Cells Via the Basolateral Surface By Memory B Cell-Mediated Transfer Infection. PLoS Pathogens, 7(5). https://doi.org/10.1371/journal.ppat.1001338

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