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

Abstract

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.

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

“…The cross-neutralization activity of monoclonal antibodies directed against these epitopes has not been investigated using different clinical HCMV strains. However, it has been shown that binding of monoclonal antibodies and of human serum antibodies to AD2 differs between laboratory and clinical HCMV strains (Meyer et al, 1992 ;Basgoz et al, 1992). Furthermore, codon changes in AD1 and AD2 between clinical strains have been demonstrated (Roy et al, 1993 ;Meyer-Ko$ nig et al, 1998 b ;MeyerKo$ nig et al, 1998 a).…”

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

“…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).…”

“…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).…”

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

“…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).…”