Constitutive expression of human cytomegalovirus glycoprotein B (gpUL55) with mutagenized carboxy-terminal hydrophobic domains.
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 (hd1) or segment 2 (hd2) of the carboxy-terminal potential bipartite membrane anchor domain. Comparative analysis of HCMV gB forms from cell lines gB(Mhd1) and gB(Mhd2), expressing mutagenized gB, and those from cells expressing authentic gB showed that deletion of hd1, but not that of hd2, 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 of hd1, 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 hd1, 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.
Reschke
M
M
Institut für Virologie, Philipps-Universität, Marburg, Germany.
Reis
B
B
Nöding
K
K
Rohsiepe
D
D
Richter
A
A
Mockenhaupt
T
T
Garten
W
W
Radsak
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000502:physiology*; D017353:Gene Deletion; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D013329:Structure-Activity Relationship; D014162:Transfection; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus