Use of gHgL for attachment of Epstein-Barr virus to epithelial cells compromises infection

Abstract

Epstein-Barr virus (EBV) is a lymphotropic herpesvirus. However, access to B lymphocytes during primary infection may be facilitated by replication in mucosal epithelial cells. Attachment and penetration of EBV into these two cell types are fundamentally different. Both the distribution of receptors and the cellular origin of the virus impact the efficiency of infection. Epithelial cells potentially offer a wide range of receptors with which virus can interact. We report here on analyses of epithelial cells expressing different combinations of receptors. We find that the stoichiometry of the virus glycoprotein complex that includes gHgL and gp42 affects the use of gHgL not just for entry into epithelial cells but also for attachment. Penetration can be mediated efficiently with either a coreceptor for gp42 or gHgL, but the use of gHgL for attachment as well as penetration greatly compromises its ability to mediate entry.

FIG. 1.

Flow cytometric analysis of gHgLR and α5/β1 integrin expression and effects of chelation on binding. (A) Binding of gHgL to AGS in the presence (arrow 2) or absence (arrow 3) of MAb E1D1. (B) Binding of gHgL to SVKCR2 cells (arrow 2). (C) Binding of gHgL to EBV-negative Akata cells (arrow 2). (D) Binding of MAb 1969 reacting with α5/β1 integrins to EBV-negative Akata cells (arrow 3) or AGS cells (arrow 4). (E) Binding of gHgL to AGS cells in the absence (arrow 3) or presence (arrow 4) of 20 mM EDTA. (F) Binding of B-EBV to AGS cells in the absence (arrow 3) or presence (arrow 4) of 20 mM EDTA. In panels A, B, and C, arrow 1 indicates cells incubated with CL59 in the absence of gHgL. In panel D, arrows 1 and 2 indicate Akata cells (arrow 1) or AGS cells (arrow 2) incubated with second antibody alone. In panels E and F, arrow 1 indicates untreated cells incubated with second antibody alone and arrow 2 indicates EDTA-treated cells incubated with second antibody alone.

FIG. 2.

Infection of SVKCR2 cells (A) or SVKCR2-class II cells (B) with equal amounts of B-EBV (solid circles) or E-EBV (open circles). Infection is measured as the percentage of cells that express GFP at 72 h.

FIG. 3.

Flow cytometric analysis of equal amounts of four different preparations of E-EBV and three different preparations of B-EBV bound to EBV-negative Akata cells (A) or AGS cells (B) and visualized with MAb 72A1 to gp350/220. In panel A, arrow 2 indicates binding of both B-EBV and E-EBV. In panel B, arrow 2 indicates binding of E-EBV and arrow 3 indicates binding of B-EBV. In both panels, arrow 1 indicates binding of MAb 72A1 in the absence of virus.

FIG. 4.

Southern blot of Gardella gel analysis of the amounts of B-EBV bound to gHgLR-positive AGS cells or CR2-positive, gHgLR-negative, EBV-negative Akata cells after preincubation with increasing amounts of soluble gp42. Numbers above the lanes indicate the amount (micrograms) of soluble gp42 used, and numbers below the panel indicate the percent reduction in binding determined by scanning with a Storm PhosphorImager.