MUTATED FORMS OF HUMAN CYTOMEGALOVIRUS GLYCOPROTEIN-B ARE IMPAIRED ININDUCING SYNCYTIUM FORMATION

Human cytomegalovirus (HCMV) glycoprotein B (gB) promotes virion entry into cells by fusing the virion envelope with the cellular membrane. We recently reported that UB cells (U373 glioblastoma cells constructe d to produce HCMV gB constitutively) form multinucleate syncytia that are dependent on the density of gB in the plasma membrane. In this rep ort, we describe the properties of a clonal cell line, UB31-B3, that e xpressed a spontaneously mutated form of gB which lacked the fusion-in ducing function of the wild-type molecule, and three UB cell lines tha t were constructed to investigate the effect of specific mutations in gB on syncytium formation. Flow cytometry analysis with a pool of mono clonal antibodies (mAbs) showed that the UB cells contained a high den sity of gB, which was associated with the cell surface. Immune precipi tation experiments with UB31-B3 cells showed that the mutant gB reacte d with all of the mAbs to the ectodomain of gB but with none of those to the cytoplasmic carboxy terminus, and that it was 35 kDa smaller th an wild-type gB. Nucleotide sequence analysis showed that a terminatio n codon had been introduced after amino acid lysine at position 669 in the ectodomain of UB31-B3 gB, generating a truncated glycoprotein. UB 31-B3 gB was not secreted into the medium and was stably anchored in t he plasma membrane, which suggested that a hydrophobic stretch of amin o acids from 629 to 652 in the ectodomain may serve as a membrane anch or for this truncated form. Analysis of the us cell lines expressing d eleted forms of gB showed that deletion of all or part of the cytoplas mic and transmembrane domains reduced or abolished syncytium formation . In contrast, deletion of a major neutralizing region in the ectodoma in of gB did not alter syncytium formation. Results of these studies i ndicate that different regions of the gB molecule participate in syncy tium formation. (C) 1995 Academic Press, Inc.