The amino terminus of human cytomegalovirus glycoprotein B contains epitopes that vary among strains

Human cytomegalovirus (HCMV) strains can be classified into different glycoprotein B (gB) genotypes. In a previous study, frequent intragenic variation of the gB gene was shown. The aim of this study was to analyse whether gB variation was due to homologous recombination. The gB gene of DNA extracts derived from the peripheral blood leukocytes of 14 immunosuppressed patients was amplified by PCR and cloned. Three variable sites of gB were analysed by restriction fragment analysis and DNA sequencing and compared with published prototypic strains. In three patients doubly infected with two distinct HCMV gB strains, prototypic (60-85 %) and nonprototypic recombinant strains (5-40 %) were detected. To demonstrate that homologous recombination is driving HCMV gB variability, cells were coinfected with plaque-purified prototypic gB strains and recombinant gB genes were selectively amplified by PCR. gB recombinants were detected after 15 days of coculture and cross-over sites were determined by sequencing. These data indicate that homologous recombination contributes to the variability of the gB gene in vitro and in vivo.

Section: Discussionmentioning

confidence: 99%

Variation within the glycoprotein B gene of human cytomegalovirus is due to homologous recombination.

Haberland¹,

Meyer-K√∂nig²,

Hufert³

1999

“…1 (c). Lumenal orientation of the carboxy-terminal portion has been shown to be unlikely (Basgoz et al, 1992;Fig. 1 c, iv and v).…”

Section: Establishment Of Transfectants Expressing Mutagenized Gb Formsmentioning

confidence: 99%

“…The amino-terminal part of gB, with 19 consensus sequences for N-glycosylation, is thought to be lumenal (Basgoz et al, 1992) and the hydrophilic carboxy-terminal portion of about 130 aa represents the cytoplasmic tail (Spaete et al, 1988, Cranage et al, 1986. In addition, HCMV gB exhibits the particular feature of a consensus sequence for intramolecular proteolytic cleavage at aa 459 (Spaete et al, 1988(Spaete et al, , 1990) by cellular furin (Vey et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

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

Reschke

Reis

Nöding

et al. 1995

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.

“…Properties of neutralizing MAbs to HCMV gB (UL55), mapping of their epitopes and analysis of neutralization by blocking fusion of the virion envelope with the plasma membrane have been published (Basgoz et al, 1992;Navarro et al, 1993;Pereira et al, 1984Pereira et al, , 1991Qadri et al, 1992). Immunoglobulin (Ig) was purified from murine ascites fuids by Protein A affinity chromatography according to the manufacturer's instructions (Affi-Gel protein A MAPS II Kit; Bio-Rad).…”

Section: Cells Culture Media Viruses and Propagation Of Cells On Pementioning

confidence: 99%

Role of apical and basolateral membranes in replication of human cytomegalovirus in polarized retinal pigment epithelial cells

Tugizov

Maidji

Pereira

1996

Self Cite

Human retinal pigment epithelial (RPE) cells, which are permissive for human cytomegalovirus (HCMV) replication, were used to evaluate virus infection from apical and basolateral membranes of polarized cells. Tests of HCMV infectivity showed that the apical membrane was 20-30-fold more susceptible to infection than the basolateral membrane; in contrast, both membranes were equally susceptible to infection by herpes simplex virus type 1 (HSV-1). Neutralizing monoclonal antibodies (MAbs) to HCMV glycoprotein B (gB) blocked penetration of virions into polarized RPE cells. This indicated that gB has a function in fusion of the virion envelope with the apical membrane of these cells, as it has with the cell membrane of unpolarized human fibroblasts. In contrast to HSV-l-infected RPE cells, the paracellular permeability of polarized RPE cells changed slowly following infection with HCMV. Confocal microscopy examination of HCMV-infected RPE cells revealed that the pattern of ZO-1 staining was altered at late times. Addition ofgB-specific neutralizing MAbs to the apical and basolateral membranes of HCMV-infected RPE cells failed to inhibit plaque development; this indicated that progeny virions infect adjacent cells before disassembly of tight junctions and are sequestered from neutralization during spread across lateral cell membranes. The finding that progeny HCMV virions cross lateral cell membranes, which differ substantially in protein composition from apical membranes, suggests that polarized RPE cells contain multiple receptors for HCMV.