Browsing School, Graduate by Subject "Glycoproteins"
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Cloning and sequencing of the gene for the 92.5 kDa receptor for glycoprotein H (gH) of human cytomegalovirusHuman cytomegalovirus (HCMV) can cause severe sight-threatening and/or life-threatening disease, particularly in immunocompromised hosts. A 92.5 kDa cell membrane protein was identified as a putative receptor for HCMV envelope glycoprotein H (gH) that mediates virus/cell fusion. Four steps were taken to identify this protein and verify its function: (1) identification of a fusion-negative cell line, (2) isolation of cDNA clones that encode the receptor, (3) isolation of receptor peptides, and (4) transfection of fusion-negative cells with receptor cDNA. Identification of fusion-incompetent cells was performed using immunofluorescence (IFA) and immunoblot assays employing anti-idiotypic antibodies that mimic gH, and a fusion assay with R18-labeled HCMV. CEM, HEL, HFF, CHO and Vero cells showed relatively high receptor densities consistent with their ability to fuse with HCMV, whereas MOLT-4 cells had very low receptor density and were not able to fuse with HCMV. Clones containing cDNA of the receptor were identified from HEL lambdagtl1 expression libraries by screening with the anti-idiotypic antibodies. Inserts from two (131 and 611) of several identified clones were subsequently isolated and fused in frame with the glutathione S-transferase (GST) gene in a pGEX-4T-1 vector, and proteins were expressed in E. coli. The purified proteins (FR131 and FR611) were shown to bind specifically to the antibodies, and inhibit HCMV/cell fusion and viral plaque formation in a specific and dose-dependent manner. The cDNA encoding the partial receptor contained an open reading frame with a predicted 345 amino acids consisting of two potential transmembrane spanning domains and several possible nuclear localization signals. A region of similarity was also identified between the predicted protein and two DNA binding proteins. Transfection of fusion-incompetent MOLT-4 cells with a eucaryotic expression vector containing the partial receptor cDNA rendered these cells susceptible to HCMV/cell fusion. In addition, fusion blocking studies using synthetic receptor peptides indicated the HCMV binding domain is contained within an 18 amino acid segment from the extracellular region in the middle of the protein. This work not only identifies a novel cell membrane protein, but provides additional evidence for the role of the 92.5 kDa cell membrane protein in HCMV/cell fusion.