Epstein-Barr virus requires glycoproteins gH/gL, gB, and gp42 to fuse its lipid envelope with B cells. Gp42 is a type II membrane protein consisting of a flexible N-terminal region, which binds gH/gL, and a C-terminal lectin-like domain that binds to the B cell entry receptor human leukocyte antigen (HLA) class II. Gp42 triggers membrane fusion after HLA binding, a process that requires simultaneous binding to gH/gL and a functional hydrophobic pocket in the lectin domain adjacent to the HLA binding site. Here we present the structure of gp42 in its unbound form. Comparisons to the previously determined structure of a gp42:HLA complex reveals additional N-terminal residues forming part of the gH/gL binding site and structural changes in the receptor-binding domain. While the core of the lectin domain remains similar, significant shifts in two loops and a α-helix bordering the essential hydrophobic pocket suggest a structural mechanism for triggering fusion.

Epstein-Barr virus (EBV) membrane glycoprotein 42 (gp42) is required for viral entry into B lymphocytes through binding to human leukocyte antigen (HLA) class II on the B-cell surface. EBV gp42 plays multiple roles during infection, including acting as a coreceptor for viral entry into B cells, binding to EBV glycoprotein H (gH) and gL during the process of membrane fusion, and blocking T-cell recognition of HLA class II-peptide complexes through steric hindrance. EBV gp42 occurs in two forms in infected cells, a full-length membranebound form and a soluble form generated by proteolytic cleavage that is secreted from infected cells due to loss of the N-terminal transmembrane domain. Both the full-length and the secreted gp42 forms bind to gH/gL and HLA class II, and the functional significance of gp42 cleavage is currently unclear. We found that in a virus-free cell-cell fusion assay, enhanced secretion of gp42 promoted fusion with B lymphocytes, and mutation of the site of gp42 cleavage inhibited membrane fusion activity. The site of gp42 cleavage was found to be physically distinct from the residues of gp42 necessary for binding to gH/gL. These results suggest that cleavage and secretion of gp42 are necessary for the process of membrane fusion with B lymphocytes, providing the first indicated functional difference between full-length and cleaved, secreted gp42.

Glycoprotein B (gB) homologues within the herpesvirus family display high sequence conservation, and a number of gB homologues contain a cleavage motif R-X-K/R-R recognized by the cellular protease furin. Epstein-Barr virus (EBV) gB contains this motif and cleaved gB is found in EBV virions. To determine the functional significance of this cleavage motif in EBV gB, a deletion mutant (gB Dfurin) was created lacking the motif. This cleavage mutant was expressed well in cell culture but was not cleaved. Experiments examining gB Dfurin in a cellfusion assay revealed that fusion was reduced by 52 % in epithelial and 28 % in B cells when compared with wild-type EBV gB. This decrease in cell-cell fusion is similar to that observed with multiple alphaherpesvirus gB cleavage mutants and supports a conserved function for cleaved gB. Epstein-Barr virus (EBV)is an orally transmitted human gammaherpesvirus that establishes a persistent infection in greater than 90 % of the world's adult population. EBV is spread through saliva, and following initial infection the virus establishes life-long latency in B cells of its human host . EBV is recognized to infect epithelial cells as well as B lymphocytes during its normal cycle of persistence (HuttFletcher, 2007). EBV is the causative agent of infectious mononucleosis, and has also been associated with a number of human malignancies of epithelial and B-cell origin, including Burkitt's lymphoma and nasopharyngeal carcinoma . Similar to other herpesvirus family members, EBV encodes a number of membrane glycoproteins. Membrane glycoproteins are important in a variety of viral processes including entry of herpesviruses into target cells. Along with membrane glycoproteins H (gH) and gL, herpesvirus gB has been shown to be essential for herpesvirus fusion, and together they form the core virus fusion machinery (Pereira, 1994;Spear & Longnecker, 2003).Herpesvirus gB homologues are highly conserved, and a number of gB homologues across all three of the herpesvirus subfamilies (alpha-, beta-and gammaherpesviruses) possess a known cleavage motif R-X-K/R-R recognized by the cellular protease furin and are cleaved (Backovic et al., 2007;Baghian et al., 2000;Britt & Vugler, 1989;Fleckenstein et al., 1982;Hampl et al., 1984;Johannsen et al., 2004;Loh, 1991; Meredith et al., 1989;Okazaki, 2007; Ross et al., 1989;Sullivan et al., 1989;van Drunen Littel-van den Hurk & Babiuk, 1986;Vey et al., 1995;Whealy et al., 1990; Wolfer et al., 1990). EBV has been shown to possess the defined cleavage motif and is cleaved at this defined site by a cellular protease (Backovic et al., 2007). In EBV virions, most gB present is in the cleaved form, with only a fraction of total gB present in the uncleaved form (Johannsen et al., 2004). The physiological relevance of this proteolytic processing of gB to its function in infection is not well understood and loss of cleavage has no effect on viral growth of bovine herpes virus 1 (BoHV-1), pseudorabies virus (PRV) or human cytomegalovirus gB (Kopp et al., 1994;Okazaki,...