1998;72:5552C5558

1998;72:5552C5558. envelope of Epstein-Barr computer virus (EBV), like that of all herpesviruses, includes multiple unique glycoprotein species. Among those mapped to their open reading frames (ORFs) in the virus genome (1) and characterized at least biochemically are gp350/220, the product of the BLLF1 ORF (2); glycoproteins gp85, Sparsentan gp42, and gp25, respective products of the BXLF2 (7, 21), BZLF2 (14), and BKRF2 ORFs (33), which make up the EBV gH-gL-gp42 complex; gp78, the product of the BILF2 ORF (16); gN, the product of the BLRF1 ORF (12); gM, the product of the BBRF3 ORF (12); and gp150, the product of the BDLF3 ORF (11, 20). Functions have been ascribed to gp350/220, which is the viral attachment protein that binds the virus to CR2 or CD21 (19, 30), and to the gH-gL-gp42 complex, which interacts with HLA class II Sparsentan molecules on B cells (27) and is involved in virus penetration through the membranes of both B cells and epithelial cells (6, 13, 17, 31, 32). Little, however, is yet known about the roles played by gN, gM, gp78, and gp150, including whether they are essential for virus replication. Glycoprotein gp150, the largest of the four, is perhaps most remarkable for the extent of its posttranslational modification with sugars (11, 20). The backbone of the molecule has an apparent which digests only cell surface, not soluble, mucin-like molecules (28, 29). The sensitivities of three EBV glycoproteinsgp350, which carries both N- and O-linked sugars; gp78, which carries only N-linked sugars; and gp150were therefore compared by exposing induced Akata cells that had been labeled with [3H]glucosamine to this glycoprotease (a gift of Alan Mellors, University of Guelph) for 1 h at Sparsentan 37C. The cells were pelleted, and medium supernatant and solubilized cells were subjected to immunoprecipitation with MAbs to gp350/220 and gp78, since the potential sites of cleavage relative to the antibody epitopes were unknown for these proteins. Solubilized cells alone were subjected to immunoprecipitation with anti-gp150, since the epitope recognized by this antibody is known to be on the intracellular cytoplasmic tail RAC1 of gp150 (20). No cleavage of gp78 was detected in either cells or medium supernatant (Fig. ?(Fig.6).6). In contrast, both gp350 and gp150 were cleaved. The cleavage site on gp350 was carboxyl terminal to Sparsentan the 72A1 epitope, which has been tentatively mapped to the amino-terminal 162 residues, and was preserved in gp220, which is missing residues 500 to 757. Open in a separate window FIG. Sparsentan 6 Electrophoretic analysis of proteins immunoprecipitated from Akata cells induced with anti-human immunoglobulin, radiolabeled with [3H]glucosamine, and treated with glycoprotease (+) or buffer alone (?). Cells were separated from the supernatant medium by centrifugation, and samples of supernatant and lysed cells were immunoprecipitated with antibodies to gp350, gp78, or gp150 as indicated. Sizes are indicated in kilodaltons. gp150 does not bind E-selectin or P-selectin. To test more directly whether the ability of cells expressing EBV glycoproteins to bind to cells expressing a potential homing receptor, such as E-selectin or P-selectin, had been altered, Akata cells were induced with anti-human immunoglobulin for 24 h and then, along with uninduced cells, tested for their ability to adhere to monolayers of CHO cells (a gift of Karen Bame, University of MissouriKansas City), CHO cells that stably expressed E-selectin or P-selectin (a gift of the Genetics Institute, Cambridge, Mass.), or freshly isolated human platelets prepared according to standard protocols (3). No difference in adherence of induced and uninduced Akata cells was seen, with few cells of either type adhering to monolayers. In contrast, HL60 cells (American Type Culture Collection), used as a positive control, demonstrated.

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