4C). gammaherpesviruses and is positioned to stabilize a putative syntaxin-like package motif. To analyze the part of these DBs in membrane fusion, gH was mutated by amino acid substitution of the DB cysteines. Mutation of the EBV-specific DB resulted in diminished gH/gL cell surface manifestation that correlated with diminished B cell and epithelial cell fusion. In contrast, mutation of the conserved DB resulted in wild-type-like B cell fusion, whereas epithelial cell fusion was greatly reduced. The gH mutants bound well to gp42 but experienced diminished binding to epithelial cells. Tyrosine 336, located adjacent to cysteine 335 of the conserved DB, also was found to be important for DB stabilization and gH/gL function. We conclude the conserved DB has a cell type-specific function, since it is important for the binding of gH to epithelial cells initiating epithelial cell fusion but not for fusion with B cells and gp42 binding. IMPORTANCE EBV mainly infects epithelial and B cells in humans, which can result in EBV-associated cancers, such PHA-848125 (Milciclib) as Burkitt and Hodgkin lymphoma, as well as nasopharyngeal carcinoma. EBV is also connected with a variety of lymphoproliferative disorders, typically of B cell source, observed in immunosuppressed individuals, such as posttransplant or HIV/AIDS individuals. The gH/gL complex takes on an essential but still poorly characterized part as an important determinant for EBV cell tropism. In the current studies, we found that mutants in the DB C278/C335 and the neighboring tyrosine 336 have cell type-specific practical deficits with selective decreases in epithelial PHA-848125 (Milciclib) cell, but not B cell, binding and fusion. The present study brings fresh insights into the gH function as a determinant for epithelial cell tropism during herpesvirus-induced membrane fusion and shows a specific gH motif required for epithelial cell fusion. Intro Epstein-Barr disease (EBV) causes infectious mononucleosis in young adults or adolescents, resulting in lifelong persistence following primary infection, PHA-848125 (Milciclib) which can lead to the development of various malignancies in B lymphocytes or epithelial cells (1). EBV enters epithelial cells via plasma membrane fusion, requiring the highly conserved core fusion machinery composed of gB and the heterodimeric gH/gL complex (1,C3). In contrast, EBV infects B cells via endocytosis and, in addition to gB and gH/gL, requires gp42, which functions as a tropism switch (1,C3). Fusion of the virion envelope having a cellular membrane is initiated by either binding of gp42 to its B cell receptor human MYCNOT being leukocyte antigen (HLA) class II (4,C7) PHA-848125 (Milciclib) or connection of gH with integrins on epithelial cells (8, 9). The recently determined crystal constructions for the herpesvirus access glycoproteins provide a unique opportunity to further understand herpesvirus-induced membrane fusion. The crystal constructions of herpes simplex virus (HSV) and EBV gB indicate that gB forms spike-like homotrimers (10, 11), which, along with vesicular stomatitis disease (VSV) G and baculovirus gp64, form the group of class III viral fusion proteins (12). Contrary to the highly conserved proteins gH/gL and gB, gp42 is found only in EBV and EBV-related primate herpesviruses (13, 14). The crystal constructions of EBV gp42 (15) and gp42 complexed with the B cell receptor HLA class II (6) indicate that gp42 consists of a C-type lectin domain (CTLD). In addition to the HLA class II binding site, gp42 consists of two additional practical domains, a large hydrophobic pocket contained within the CTLD and an unstructured flexible N terminus of gp42 outside the CTLD that mediates connection with gH/gL (6, 15). In contrast to EBV gB and gp42, the part of the EBV gH/gL complex in fusion is definitely less defined. EBV gL is definitely important for the cell surface expression of the gH/gL complex (16). More recent studies have suggested that gL also functions in the engagement and activation of gB from the gH/gL complex (17). The available crystal constructions of gH/gL of EBV (18) and HSV-2 (19) and the core fragment of gH of the alphaherpesvirus pseudorabies disease (PrV) (20) indicated that gH/gL has no features in common with standard fusion proteins (20). In contrast to the boot-like shape of HSV-2 gH/gL, EBV gH/gL and PrV gH possess a more rod-like overall conformation (18,C20), divided in four domains (Fig. 1A). Furthermore, the crystal constructions verify three disulfide bonds (DBs) for HSV-2 gH, four DBs for PrV gH, and five DBs.