Blocking binding of EBV gHgL to a molecule with characteristics of an integrin also blocks epithelial cell infection

Attachment of EBV to a B cell involves an interaction between glycoprotein gp350 and CD21 and fusion requires gB and a complex of gHgLgp42. It is triggered by an interaction between gp42 and HLA class II. Attachment of EBV to an epithelial cell can also be mediated by gp350 and CD21 and fusion requires only gB and gHgL. However, not all epithelial cells express CD21. In its absence virus uses gHgL for attachment as well as fusion. A soluble truncated form of gHgL (gHtgL) expressed in recombinant baculovirus binds specifically to CD21-negative epithelial cells, but not CD21-positive B cells and a gH-null virus loses the ability to bind. gHtgL binding is reduced by a monoclonal antibody that blocks virus binding to CD21-negative cells. The same antibody blocks entry into CD21-positive epithelial cells suggesting that the molecule that serves as an epithelial gHgL receptor (gHgLR) may also serve to trigger epithelial cell fusion. To characterize gHgLR, gHtgL was purified from the media of baculovirus infected cells by lentil lectin affinity chromatography. Biological activity of gHtgL during purification was evaluated by flow cytometric analysis of protein bound to cells on ice and stained with a monoclonal antibody. Binding was saturable, fitted well to a hyperbolic curve and could inhibit virus infection. Scatchard analysis of binding of radiolabeled protein to three epithelial cell lines indicated that AGS gastric epithelial cells express (286 ± 0.71) x 103 binding sites per cell for gHtgL with a KD of (0.61 ± 0.09) 10-9 M, SVKCR2 keratinocytes express (51±2) x 103 binding sites with a KD of (0.91±0.14) x 10-9 M and 293 cells express (5.6±0.4) x 103 binding sites with a KD of (0.24±0.06) x 10-9 M. Addition of Mn2+, but not Ca2+ or Mg2+ increased the affinity of binding to each cell line by approximately three orders of magnitude without altering the number of binding sites. Vitronectin blocked binding of gHtgL and also blocked infection, even when virus was bound via gp350 to CD21 on SVKCR2 cells. These data are consistent with an essential interaction between EBV gHgL and an integrin.