Constitutive expression of human cytomegalovirus glycoprotein B (gpUL55) with mutagenized carboxy-terminal hydrophobic domains

Abstract

Stable transfectants were selected from human astrocytoma cells (U373) after transfection with recombinant expression vectors carrying the human cytomegalovirus (HCMV) glycoprotein B (gB; gpUL55) gene with alternative deletions of hydrophobic domain segment 1 (hdl) or segment 2 (hd2) of the carboxy-terminal potential bipartite membrane anchor domain. Comparative analysis of HCMV gB forms from cell lines gB(Mhdl) and gB(Mhd2), expressing mutagenized gB, and those from ceils expressing authentic gB showed that deletion ofhdl, but not that ofhd2, interfered with efficient proteolytic cleavage of the gB precursor. Both mutagenized gB forms exhibited correct transport to the cell surface. Deletion of hd2, but not that ofhdl, caused loss of membrane anchoring of the gB molecule, resulting in secretion of the respective gB form into the culture medium. The carboxy-terminal cleavage product of the soluble gB molecule, which migrated more slowly than its authentic counterpart, was modified by complex carbohydrate side chains and formed disulphide-linked complexes. Our observations indicate that hd2 is essential as well as sufficient for membrane anchoring of the HCMV gB molecule. For hdl, a potential fusogenic role is suggested by the conserved positional pattern of glycine residues, which is comparable to that of known fusion peptides of other viruses.

Nuclear translocation of mutagenized forms of human cytomegalovirus glycoprotein B (gpUL55).

“…For this study, stable transfectants of U373 described previously (Reis et al, 1993 ;Bold et al, 1996 ;Reschke et al, 1995), expressing either authentic HCMV gB or derivatives thereof with various deletions, were used. Transfectants gB(Mhd1) and gB(Mhd2) expressed gB derivatives with deletions of hydrophobic domains, hd1 and hd2, in the carboxy-terminal portion of the molecule (Reschke et al, 1995) ; hd2 of HCMV gB has been shown previously to be essential as well as sufficient for membrane anchoring.…”

Structural Domains Involved in Human Cytomegalovirus Glycoprotein B-Mediated Cell-cell Fusion

“…During our recent functional analysis of the prominent hydrophobic domains in the C-terminal portion of HCMV gB (hdI, aa 714-747; hd2, 751-771) we observed that hd2 was essential as well as sufficient for membrane anchoring (Reschke et al, 1995). Sequence alignments of hdl, on the other hand, with established fusogenic domains of envelope glycoproteins of entirely unrelated viruses revealed an intriguing similarity (Reschke et al, 1995) implying that hdI may directly participate in the fusion event.…”

“…Sequence alignments of hdl, on the other hand, with established fusogenic domains of envelope glycoproteins of entirely unrelated viruses revealed an intriguing similarity (Reschke et al, 1995) implying that hdI may directly participate in the fusion event.…”

“…After staining monolayers were microscopically evaluated by counting syncytia containing three or more nuclei. Using this assay, cell line gBI expressing authentic gB was compared with cell line gB(Mhdl) expressing gB with a deletion of hdl (Reschke et al, 1995). The parental cell line U373 served as a control.…”

“…For a routine fusion assay, the previously described stable astrocytoma cell derived transfectants expressing authentic or specifically mutagenized HCMV gB (Reis et al, 1993, Reschke et al, 1995 '.29~ !29 c 8 h later. For inhibition experiments gB-specific MAb or control MAb (final concentrations 0"5--0"8 pg/ml) was added to the culture medium, 30 min before fibroblast seeding.…”