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

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. Epstein-Barr virus (EBV) is an important human pathogen that is carried as a latent infection of B cells by most adults worldwide. Infection of epithelial cells is also believed to be important in the normal life-cycle of EBV, and is certainly associated with the pathogenesis of some epithelial tumours. Whilst EBV binds to and infects B cells that express CD21, a receptor for the gp350 viral glycoprotein, binding of EBV to CD21-negative epithelial cells is inefficient. However, we have identified an efficient process of ‘transfer infection’. This process involves EBV first binding to B cells, resulting in CD21-mediated capping of virus and activation of adhesion molecules, which facilitates conjugate formation between B cells and epithelial cells and the subsequent entry of EBV into epithelial cells. We have characterised the molecular processes involved in transfer infection, both in unpolarized cells modelling pre-malignant epithelial cells, and in normal polarized epithelial cells. The details of the molecular interactions in these infection models led us to identify a subset of B cells, memory B cells, as being the primary vehicles for transfer infection. The results reinforce the likely physiological significance of transfer infection of epithelial cells in healthy persistence and in EBV pathogenesis.