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. 2006 May 2;103(18):7065-70.
doi: 10.1073/pnas.0510512103. Epub 2006 Apr 10.

Resting B cells as a transfer vehicle for Epstein-Barr virus infection of epithelial cells

C D Shannon-Lowe  1 , B Neuhierl, G Baldwin, A B Rickinson, H-J Delecluse
Affiliations

Resting B cells as a transfer vehicle for Epstein-Barr virus infection of epithelial cells

C D Shannon-Lowe et al. Proc Natl Acad Sci U S A. .

Abstract

Epstein-Barr virus (EBV), an orally transmitted herpesvirus, efficiently targets B lymphocytes through binding of the viral envelope glycoprotein gp350 to the complement receptor CD21. How the virus accesses epithelial cells is less well understood, because such cells are largely resistant to infection with cell-free virus in vitro. Here, we show that, after binding to primary B cells, most Epstein-Barr virions are not internalized but remain on the B cell surface and from there can transfer efficiently to CD21-negative epithelial cells, increasing epithelial infection by 10(3)- to 10(4)-fold compared with cell-free virus. Transfer infection is associated with the formation of B cell-epithelial conjugates with gp350/CD21 complexes focused at the intercellular synapse; transfer involves the gp85 and gp110 viral glycoproteins but is independent of gp42, the HLA class II ligand that is essential for B cell entry. Therefore, through efficient binding to the B cell surface, EBV has developed a means of simultaneously accessing both lymphoid and epithelial compartments; in particular, infection of pharyngeal epithelium by orally transmitted virus becomes independent of initial virus replication in the B cell system.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Characteristics of transfer infection. (A) Photomicrographs show phase contrast and GFP fluorescence images of Ad-AH cultures 72 h after exposure to cell-free recombinant B95.8 EBV at a moi of 100 (direct infection) or 48 h after a 24-h cocultivation with primary B cells preexposed to the same virus (transfer infection). Histograms show efficiencies of direct versus transfer infection of Ad-AH cells in a representative experiment conducted as above but using multiplicities of infection between 1 and 100. Efficiency is expressed on a logarithmic scale as the percentage of GFP-positive cells. Gray bars show corresponding data using a BZLF1-knockout (replication-deficient) EBV. (B) Mean results from repeat assays comparing epithelial cell lines and normal nasopharyngeal epithelial cells with normal fibroblasts, endothelial cells, and primary T cells as acceptors; cells were cocultured for 24 h with primary B cells preexposed to virus at a moi of 100, and transfer infection was expressed as above (linear scale).
Fig. 2.
Fig. 2.
Virus binding versus transfer infection by different donor cells. Primary B cells (with and without prior glutaraldehyde fixation), EBV-negative Akata and BJAB B cell lines, primary T cells, and the Molt4 T cell line were exposed to virus at a moi of 100, and virus binding was assayed independently by Southern blotting (Top) and Q-PCR (Middle); the EBV-producing B95.8 cell line and uninfected primary B cells serve as positive and negative controls. (Bottom) These same cells were then used as donors in 24-h cocultures with Ad-AH cells, and transfer infection was expressed as in Fig. 1B. Mean results from repeat assays are shown.
Fig. 3.
Fig. 3.
Kinetics of transfer infection. (A) Duration of B cell infectivity p.i. Primary B cells were exposed to EBV (moi of 100) for 3 h and then added to Ad-AH acceptor cells either immediately after virus loading or after varying times in culture up to 4 days p.i. In each case, donor cells were left in contact with acceptors for 1 (Upper) or 24 (Lower) h, and transfer infection was assayed 72 h after the first donor–acceptor cell contact. Results are expressed as in Fig. 1B. (B) Duration of gp350 staining on the B cell membrane. Primary B cells were exposed to EBV as above and then stained with anti-gp350 mAb either immediately or up to 6 days p.i.; gp350 staining profiles are shown alongside that of an isotype-matched control mAb (shaded profile). (C) Detection of viral genomes by FISH. Primary B cells were exposed to EBV as above and then fixed either immediately or up to 6 days p.i; extranuclear viral genomes are detected by green fluorescence, and nuclei are identified by DAPI staining.
Fig. 4.
Fig. 4.
Virus binding to B cells, direct B cell infection, transfer infection, and direct epithelial cell infection by different EBV mutants. Primary B cells were exposed to recombinant EBVs (WT, gp110-low, and the glycoprotein knockouts Δgp42, Δgp350, Δgp85, and ΔBILF2) at a moi of 100, and virus binding was assayed by Southern blotting and by Q-PCR (two uppermost panels) as in Fig. 2. B cell infection was assayed by staining for EBNA2 expression after 3 days in culture (17), and the percentage of EBNA2-positive cells are shown (middle panel). Additional aliquots of these virus-loaded B cells were cocultured with Ad-AH cells for 24 h immediately p.i. to assay for transfer infection (second panel from bottom); in parallel, the same recombinant EBV preparations were assayed for direct Ad-AH cell infection at a moi of 100 (bottom panel). Results of transfer and direct epithelial cell infection are expressed as in Fig. 1B.
Fig. 5.
Fig. 5.
Analysis of conjugate formation. (A) FACS profiles of conjugate formation between carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled Ad-AH cells (x axis) and PKH26-labeled primary B cells (y axis) tested without EBV infection, immediately after virus binding at a moi of 100 (0 h), or at 24 h p.i. Conjugates appear in the upper right quadrant, and the percentages of cells in conjugates are shown. (B) Phase contrast and fluorescence images of B cell–epithelial conjugates formed between Ad-AH cells and primary B cells without EBV infection or at 24 h p.i. Before coculture, B cells were stained with red-labeled anti-CD21 mAb or with green-labeled anti-gp350 mAb. (C) Confocal images of 1-μm Z slices through conjugates between epithelial cells and EBV-infected B cells (labeled 24 h p.i.). All gp350 staining (green) colocalizes at the cell–cell junction with CD21 staining (blue, Upper), whereas CD20 staining (blue, Lower) remained diffusely distributed; phalloidin staining (red) shows the cell boundaries. (D) Confocal images of a 1-μm Z slice through an epithelial cell immediately after 24-h coculture with EBV-infected B cells and B cell wash-off. gp350 (green) colocalizes with CD21 (blue) at two points on the epithelial cell membrane. Light-green staining of the epithelial cell indicates GFP expression from EBV infection. DAPI staining for DNA (white) confirms that the infected cell is mononuclear and that B cells have been removed.
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