Polymorphism of human cytomegalovirus glycoproteins characterized by monoclonal antibodies

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.

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

“…Properties of the panel of MAbs used were published elsewhere (Pereira & Hoffman, 1986;Pereira et al, 1984), but the following properties of antibodies in groups 2 and 4 are relevant to this study. Group 2 antibodies reacted by immunoblot analysis with intact gB and other high Mr forms but failed to react with the carboxy-terminal cleavage fragment, which has an apparent Mr of approximately 58.5K.…”

mentioning

confidence: 99%

“…The CMV gB gene has been mapped in the genomes of strain AD169 (Cranage et al, 1986;Mach et al, 1986) and Towne Spaete et al, 1988). This glycoprotein is produced as a full-length precursor, which is glycosylated (Britt & Auger, 1986;Pereira et al, 1984;Rasmussen et aL, 1985bRasmussen et aL, , 1988 and proteolytically cleaved between residues 460 and 461 after its synthesis (Spaete et al, 1988(Spaete et al, , 1990. It is highly immunogenic in natural infection and elicits a strong humoral immune response in humans (Cremer et al, 1985;Kniess et al, 1991;Pereira et al, 1982Pereira et al, , 1983Rasmussen et al, 1991) and in animals experimentally immunized with the glycoprotein (Britt et al, 1988;Gonczol et al 1986;Rasmussen et al, 1985a).…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Basgoz

Qadri

Navarro

et al. 1992

We mapped three antigenic domains of continuous epitopes on human cytomegalovirus (CMV) glycoprotein B (gB) by reacting a panel of independently derived monoclonal antibodies with deletion mutants expressed transiently in COS-1 cells. One of these antigenic domains, DC2, maps in the last 75 amino acids of the carboxy terminus. These epitopes are conserved in strains Towne and AD169, as well as in 19 clinical CMV isolates. ELISAs of DC2-reactive antibodies with a set of overlapping synthetic oligopeptides from the carboxy terminus showed that the epitopes of antibodies CH405-1 and CH421-5 map between amino acids 833 and 852 and that the epitope of antibody CH28-2 maps between amino acids 878 and 898. These linear epitopes were grouped into domain DC3. The third antigenic domain, DClv, maps at the aminoterminal end of CMV strain AD169 gB but is not contained in strain Towne or in 17 of 19 clinical isolates. Epitopes in this domain are likely to map between amino acids 28 and 67, an area where differences occur in the nucleotide sequence of the gB genes from AD169 and Towne. Analysis of CMVinfected cells by flow cytometry with antibodies to the amino-and carboxy-terminal domains revealed that the amino terminus of gB is extracellular and that the carboxy terminus is not exposed on the cell surface.

“…The HCMV gB gene encodes a 130K precursor protein (gpl30) which is thought to be cleaved by a cellular enzyme, giving rise to a C-terminal 55K glycoprotein, designated gp55 (Spaete et al, 1988). The Mr of the gB complex has been shown to vary (Pereira et al, 1984;Britt, 1984;Britt & Auger, 1986;G6ncz61 et at., 1986;Landini et al, 1987;Mach et al, 1986;Cranage et al, 1986), but its abundance in the virion and immunological importance make it likely that the same glycoprotein was detected in all investigations.…”

Section: Introductionmentioning

confidence: 99%

Characterization of a Major Antigenic Region on gp55 of Human Cytomegalovirus

Silvestri

Sundqvist²,

Rudén³

et al. 1991

A major antigenic region localized to the C-terminal part of the 55K glycoprotein of human cytomegalovirus (HCMV) was mapped using synthetic peptides. Analysis of the region with six sera from healthy anti-HCMV seropositive blood donors showed that the length of the reactive sequence varied between four and eight amino acids (aa). The shortest sequence recognized was VTSG (aa 793 to 801 of the 130K precursor protein), but most sera required the three or four residues C-terminal to this to react, giving a major site ofVTSGSTKD (aa 798 to 805). Using peptides immobilized on polyethylene pins, the importance of both interior (between aa essential for the binding of an antibody) and extension spacer residues was evident. Free peptides containing the reactive sequence were prepared for use in a conventional ELISA and optimal pH conditions for coating were determined. The best results were achieved at pH 2 to 3, which is in agreement with the advantageous net charge of these peptides at this low pH. Anti-HCMV positive sera showed a sensitivity of approximately 50% for both peptides on polyethylene pins and peptides coated onto plates.