Epstein-Barr virus (EBV) is a herpesvirus that infects cells by fusing its lipid envelope with the target cellmembrane. The fusion process requires the actions of viral glycoproteins gH, gL, and gB for entry into epithelial cells and additionally requires gp42 for entry into B cells. To further study the roles of these membrane-associated glycoproteins, purified soluble forms of gp42, gH, and gL were expressed that lack the membrane-spanning regions. The soluble gH/gL protein complex binds to soluble gp42 with high affinity, forming a stable heterotrimer with 1:1:1 stoichiometry, and this complex is not formed by an N-terminally truncated variant of gp42. The effects of adding soluble gp42, gH/gL, and gH/gL/gp42 were examined with a virus-free cell-cell fusion assay. The results demonstrate that, in contrast to gp42, membrane fusion does not proceed with secreted gH/gL. The addition of soluble gH/gL does not inhibit or enhance B-cell or epithelial cell fusion when membrane-bound gH/gL, gB, and gp42 are present. However, the soluble gH/gL/gp42 complex does activate membrane fusion with B cells, similarly to soluble gp42, but it does not inhibit fusion with epithelial cells, as observed for gp42 alone. A gp42 peptide, derived from an N-terminal segment involved in gH/gL interactions, binds to soluble gH/gL and inhibits EBV-mediated epithelial cell fusion, mimicking gp42. These observations reveal distinct functional requirements for gH/gL and gp42 complexes in EBV-mediated membrane fusion.

Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with malignancies of both epithelial and lymphoid origin. Efficient infection of the latent host reservoir B lymphocytes involves the binding of glycoproteins gp350/220 for initial attachment, followed by the concerted action of gH, gL, gB, and gp42 for membrane fusion. The type II membrane protein gp42 is required for infection of B cells and assembles into a complex with gH and gL. The cellular host receptor for gp42, class II human leukocyte antigen (HLA), has been structurally verified by crystallization analyses of gp42 bound to HLA-DR1. Interestingly, the crystal structure revealed a hydrophobic pocket consisting of many aromatic and aliphatic residues from the predicted C-type lectin domain of gp42 that in other members of the C-type lectin family binds major histocompatibility complex class I or other diverse ligands. Although the hydrophobic pocket does not bind HLA class II, mutational analyses presented here indicate that this domain is essential for EBV-induced membrane fusion. In addition, mutational analysis of the region of gp42 contacting HLA class II in the gp42-HLA-DR1 cocrystal confirms that this region interacts with HLA class II and that this interaction is also important for EBVinduced membrane fusion.Epstein-Barr virus (EBV) is a ubiquitous member of the human gammaherpesvirus subfamily that is able to establish lifelong latency in host B cells. The double-stranded enveloped virus has been associated with diseases of both lymphoid and epithelial origin (3, 9). Infection in infancy is generally asymptomatic but during adolescence can cause infectious mononucleosis. EBV infection is associated with Burkitt's lymphoma, Hodgkin's disease, and lymphoproliferative diseases in AIDS patients (for reviews, see references 25 and 32). Epithelial diseases associated with EBV infection include oral hairy leukoplakia and nasopharyngeal carcinoma (for a review, see reference 32). Although EBV encodes as many as 11 glycoproteins, efficient entry of EBV into B cells requires only 5 glycoproteins: gp350/220, gH, gL, gB, and gp42 (for reviews, see references 16, 33, and 34). The type II membrane protein gp42 of the BZLF2 open reading frame is required for entry into B lymphocytes but not epithelial cells and has two alternately processed forms: a 42-and a 38-kDa protein (1, 24, 37). The receptor of gp350 has been identified as complement receptor 2 (CR2) or CD21 (10,29,36). Expression of gp350 is not required for membrane fusion, but as a binding factor, it greatly enhances infection efficiency, similar to herpes simplex virus (HSV) gC (17,20). Transmembrane gH dimerizes with gL, which exists as membrane-bound and soluble forms and is required for correct folding and transport of gH (14,24,40). These dimers have been demonstrated to bind gp42 (38). The minimal glycoproteins required for membrane fusion of B cells using experimentally transfected cells are gB, gH, gL, and gp42 (12). It was previously demonstrated that gB is required for lytic rep...

Epstein-Barr virus (EBV) infects B lymphocytes and epithelial cells. While the glycoproteins required forentry into these two cell types differ, the gH/gL glycoprotein complex is essential for entry into both epithelial and B cells. Analysis of gH protein sequences from three gammaherpesviruses (EBV, marmoset, and rhesus) revealed a potential coiled-coil domain in the N terminus. Four leucines located in this region in EBV gH were replaced by alanines by site-directed mutagenesis and analyzed for cell-cell membrane fusion with B cells and epithelial cells. Reduction in fusion activity was observed for mutants containing L65A and/or L69A mutations, while substitutions in L55 and L74 enhanced the fusion activity of the mutant gH/gL complexes with both cell types. All of the mutants displayed levels of cell surface expression similar to those of wild-type gH and interacted with gL and gp42. The observation that a conservative mutation of leucine to alanine in the N terminus of EBV gH results in fusion-defective mutant gH/gL complexes is striking and points to an important role for this region in EBV-mediated membrane fusion with B lymphocytes and epithelial cells. Epstein-Barr virus (EBV) is a gammaherpesvirus that has tropism for both B lymphocytes and epithelial cells (18, 42).Similar to other enveloped viruses, EBV entry into the target cells occurs in two steps: the initial attachment of the virus to the cell surface and the subsequent fusion of the viral envelope and the cell membrane (18, 51). EBV infection can occur by cell-free virus or through cell-cell spread, but fusion is required for both entry pathways (18, 42). The initiation of infection by EBV is driven by interaction of viral glycoproteins with cellsurface receptors (42). EBV encodes as many as 11 glycoproteins, but only a subset is required for efficient EBV entry (15,42). In general, more is known about the mechanism of EBV entry into B lymphocytes than into epithelial cells. Infection of B lymphocytes is initiated by the attachment of glycoprotein gp350/220 to the CD21/CR2 receptor on the B cells (9,43,45). This interaction enhances infection efficiency of B cells, but it is not absolutely required for infection to occur (16). Subsequent to this binding, viral glycoprotein gp42 binds to the B-cell surface protein HLA class II, which triggers fusion mediated by a concerted action of three glycoproteins (gB, gH, and gL) (19,43). The glycoprotein requirement for EBV entry differs between epithelial and B cells; gB, gH, and gL are essential for infection of both epithelial and B cells, while gp42 is required only for B-cell entry (12,20,24,47). Even though gB, gH, and gL are conserved throughout the herpesvirus family, the structure and the exact mechanism of action of these three glycoproteins in the fusion process are still unclear.

Epstein-Barr virus (EBV) is a human gamma-herpesvirus that primarily infects B lymphocytes and epithelial cells. Entry of EBV into B cells requires the viral glycoproteins gp42, gH/gL and gB, while gp42 is not necessary for infection of epithelial cells. In EBV, gH and gL form two distinct complexes, a bipartite complex that contains only gH and gL, used for infection of epithelial cells, and a tripartite complex that additionally includes gp42, used for infection of B cells. The gH/gL complex is conserved within the herpesvirus family, but its exact role in entry and mechanism of fusion is not yet known. To understand more about the functionality of EBVgH/gL, we investigated the functional homology of gHs and gLs from human herpesvirus 8 (HHV8) and two primate (rhesus and marmoset) gamma-herpesviruses in EBV-mediated virus-free cell fusion assay. Overall, gHs and gLs from the more homologous primate herpesviruses were better at complementing EBV gH and gL in fusion than HHV8 gH and gL. Interestingly, marmoset gH was able to complement fusion with epithelial cells, but not B cells. Further investigation of this led to the discovery that EBVgH is the binding partner of gp42 in the tripartite complex and the absence of fusion with B cells in the presence of marmoset gH/gL is due to its inability to bind gp42.