1A and ?andB

1A and ?andB.B. activity and to allow potential interactions across the D-I/D-II groove, TG003 we mutated D-I amino acids V47, P48, and G49 to cysteine, allowing novel intersubunit disulfide bonds to form with the free C153 located in D-II. We found that the G49C mutant, predicted to bridge D-I and D-II with C153 of gH/gL, had normal B cell fusion activity but reduced epithelial cell fusion activity, which was partially restored by treatment with dithiothreitol. We conclude that structural rearrangements and/or interactions across the D-I/D-II groove of gH/gL are required for fusion with epithelial cells but not for fusion with B cells. INTRODUCTION Epstein-Barr virus (EBV), a member of the gammaherpesvirus family, was TG003 first identified in tumor biopsy specimens obtained from young children with Burkitt lymphoma (1C3). Along with this cancer, EBV has been associated with a variety of other cancers, including Hodgkin lymphoma and gastric carcinoma. EBV is also involved in a number of important disorders associated with diminished immunity, including AIDS-related malignancies, posttransplant lymphoproliferative disease (PTLD), and oral hairy leukoplakia (2, 3). EBV replicates in epithelial cells and establishes long-term latency in lymphocytes (2, 3). The binding of EBV and the subsequent fusion of the virion envelope with a host cell is mediated by multiple EBV-encoded glycoproteins and requires multiple steps, culminating with the release of the virus capsid into the cytoplasm. The EBV fusion machinery consists of gB, the heterodimeric gH/gL complex, and glycoprotein 42 (gp42), all of which are required for the fusion of EBV with B cells. The fusion of EBV with epithelial cells requires only gB and the gH/gL complex, which forms the core fusion machinery found in all herpesviruses (4, 5). The presence of gp42 inhibits epithelial cell fusion, thus acting as a switch directing the entry of EBV into B cells or epithelial cells (6). The crystal structure of the EBV gH/gL complex was recently resolved, and a KGD motif was found to be prominently located on the surface of domain II (D-II) of gH. Also found in the gH/gL crystal structure was a large groove between domain I (D-I) and D-II, adjacent to the gH/gL KGD motif (7). We TG003 recently found that the gH/gL KGD motif is bifunctional, orchestrating the infection Rabbit polyclonal to Lamin A-C.The nuclear lamina consists of a two-dimensional matrix of proteins located next to the inner nuclear membrane.The lamin family of proteins make up the matrix and are highly conserved in evolution. of B cells and epithelial cells by interaction with the epithelial cell receptor or gp42 (8). The D-I/D-II groove adjacent to the KGD motif also appears well TG003 suited to participate in B cell and epithelial cell fusion and was investigated in the current study by using a structure-based mutagenesis approach to further define the functional role of this region in EBV fusion. MATERIALS AND METHODS Cell culture. Chinese hamster ovary cells (CHO-K1 cells) were grown in Ham’s F-12 medium (BioWhittaker) containing 10% FetalPlex animal serum complex (Gemini Bio Products) and 1% penicillin-streptomycin (100 U penicillin/ml, 100 g streptomycin/ml; BioWhittaker). The Daudi 29 cell line (for B cell fusion) and human embryonic kidney (HEK) 293 cells (for epithelial cell fusion) stably expressing T7 RNA polymerase (9, 10) were grown in RPMI 1640 medium with 900 g/ml G418 (Sigma) and in Dulbecco’s modified Eagle medium (DMEM) with zeocin, respectively, containing 10% FetalPlex animal serum complex and 1% penicillin-streptomycin. Construction. Mutations of gH (E58A, S154A, Q150A, R152A, C153A, H154A, T174A, D203A, L207A, S212A, T217A, Q220A, V47A, V47C, P48A, P48C, G49A, G49C, and G49S; numbers indicate the wild-type [wt] gH residue mutated to alanine, cysteine, or serine) were generated using the QuikChange site-directed mutagenesis kit (Stratagene). The.