Production and characterization of monoclonal antibodies against the Epstein-Barr virus membrane antigen

107

Four unique glycoproteins or glycoprotein complexes were recognized by a panel of monoclonal antibodies to bovine herpesvirus 1 (BHV-1), i.e., GVP 6/lla/16 (130,000-molecular-weight glycoprotein [130K glycoprotein]/74K/55K), GVP 7 (108K), GVP 3/9 (180K/91K), and GVP llb (71K). The absence of any antigenic or structural relationship between GVP lla and GVP llb, which were previously identified as one glycoprotein, GVP 11, demonstrated that these two GVP 11 species are unique glycoproteins. GVP 3 and GVP 9 showed complete sequence homology, as shown by the identity of their antigenic determinants and by partial peptide mapping. This observation, as well as the ratio of their apparent molecular weights, indicated that GVP 3 (180K) is a dimeric form of GVP 9 (91K). GVP 6 and GVP lla, as well as GVP 6 and GVP 16, showed at least partial sequence homology, since they shared several antigenic determinants and peptides. In addition, GVP 6, GVP lla, and GVP 16 were derived from one primary precursor. These results, as well as the ratio of their apparent molecular weights, indicated that the GVP 6/lla/16 complex consists of two forms: one in which GVP 6 (130K) is uncleaved and the other one in which GVP 6 is cleaved and composed of GVP 11a (74K) and GVP 16 (55K), linked by disulfide bridges. An antigenically distinct precursor to each of the four BHV-1 glycoproteins or glycoprotein complexes was identified by monoclonal antibodies. These precursors, pGVP 6 (117K), pGVP Ila (62K), pGVP 7 (lOOK), pGVP 9 (69K), and pGVP llb (63K) were sensitive to endo-o-N-acetylglucosaminidase H treatment, indicating that they represent the partially glycosylated highmannose-type intermediate forms generated by cotranslational glycosylation of the primary, unglycosylated precursors to GVP 6/lla/16, GVP 7, GVP 3/9, and GVP llb, which were identified as having apparent molecular weights of 105,000, 90,000, 61,000, and 58,000, respectively. A new nomenclature for the BHV-1 glycoproteins, based on roman numerals, is proposed. 401 on July 6, 2020 by guest http://jvi.asm.org/ Downloaded from bated with a 1:1,000 dilution of ascites fluid and then with a 1:1,000 dilution of rabbit anti-mouse IgG (Cooper Biomedical, Inc., West Chester, Pa.) and 5 x 105 to 1 x 106 cpm of 125I-labeled protein A (Pharmacia). Finally, the nitrocellulose strips were dried and analyzed by autoradiography on 3M X-ray film.

Section: Discussionmentioning

confidence: 99%

Synthesis and processing of bovine herpesvirus 1 glycoproteins

Hurk¹,

Babiuk²

1986

107

“…Monoclonal antibodies E1D1 and F-2-1 were gifts from L. Hutt-Fletcher (Louisana State University Health Sciences Center, Shreveport, La.) and recognize the gH/gL complex and gp42, respectively (1,44). A large-scale preparation of the E1D1 and the F-2-1 antibodies was made at the Northwestern University Monoclonal Antibody Facility.…”

Section: Methodsmentioning

confidence: 99%

The Amino Terminus of Epstein-Barr Virus Glycoprotein gH Is Important for Fusion with Epithelial and B Cells

Omerović

Lev

Longnecker

2005

Epstein-Barr virus (EBV) infects B lymphocytes and epithelial cells. While the glycoproteins required forentry into these two cell types differ, the gH/gL glycoprotein complex is essential for entry into both epithelial and B cells. Analysis of gH protein sequences from three gammaherpesviruses (EBV, marmoset, and rhesus) revealed a potential coiled-coil domain in the N terminus. Four leucines located in this region in EBV gH were replaced by alanines by site-directed mutagenesis and analyzed for cell-cell membrane fusion with B cells and epithelial cells. Reduction in fusion activity was observed for mutants containing L65A and/or L69A mutations, while substitutions in L55 and L74 enhanced the fusion activity of the mutant gH/gL complexes with both cell types. All of the mutants displayed levels of cell surface expression similar to those of wild-type gH and interacted with gL and gp42. The observation that a conservative mutation of leucine to alanine in the N terminus of EBV gH results in fusion-defective mutant gH/gL complexes is striking and points to an important role for this region in EBV-mediated membrane fusion with B lymphocytes and epithelial cells. Epstein-Barr virus (EBV) is a gammaherpesvirus that has tropism for both B lymphocytes and epithelial cells (18, 42).Similar to other enveloped viruses, EBV entry into the target cells occurs in two steps: the initial attachment of the virus to the cell surface and the subsequent fusion of the viral envelope and the cell membrane (18, 51). EBV infection can occur by cell-free virus or through cell-cell spread, but fusion is required for both entry pathways (18, 42). The initiation of infection by EBV is driven by interaction of viral glycoproteins with cellsurface receptors (42). EBV encodes as many as 11 glycoproteins, but only a subset is required for efficient EBV entry (15,42). In general, more is known about the mechanism of EBV entry into B lymphocytes than into epithelial cells. Infection of B lymphocytes is initiated by the attachment of glycoprotein gp350/220 to the CD21/CR2 receptor on the B cells (9,43,45). This interaction enhances infection efficiency of B cells, but it is not absolutely required for infection to occur (16). Subsequent to this binding, viral glycoprotein gp42 binds to the B-cell surface protein HLA class II, which triggers fusion mediated by a concerted action of three glycoproteins (gB, gH, and gL) (19,43). The glycoprotein requirement for EBV entry differs between epithelial and B cells; gB, gH, and gL are essential for infection of both epithelial and B cells, while gp42 is required only for B-cell entry (12,20,24,47). Even though gB, gH, and gL are conserved throughout the herpesvirus family, the structure and the exact mechanism of action of these three glycoproteins in the fusion process are still unclear.

“…The gp92 and gp83 species seem to represent the mature virion polypeptides, as deduced by their overrepresentation relative to gp55 in virions. It is noteworthy that monoclonal antibodies to gC detect more than one glycoprotein, an observation that has been made in other members of the herpesvirus family, e.g., herpes simplex virus (4,8,23), Epstein-Barr virus (9,28), and cytomegalovirus (22). Since these cross-reactions do not appear to be nonspecific in nature, the multiple polypeptide forms could be processed from related but distinct precursors, could share pulse-chase relationships, or could represent heterogeneities in glycosylation patterns.…”

mentioning

confidence: 89%

Three major glycoprotein genes of varicella-zoster virus whose products have neutralization epitopes

Keller¹,

Neff²,

Ellis³

1984

Varicella-zoster virus (VZV) codes for approximately eight glycosylated polypeptides in infected cell cultures and in virions. To determine the number of serologically distinct glycoprotein gene products encoded by VZV, we have developed murine monoclonal antibodies to purified virions. Of 10 monoclonal antibodies which can immunoprecipitate intracellular VZV antigens and virion glycoproteins, 1 (termed gA) reacted with gp105, 1 (termed gB) reacted with gp115 (intracellular only), gp62, and gp57, and 8 (termed gC) reacted with gp92, gp83, gp52, and gp45. The anti-gA monoclonal antibody neutralized VZV infectivity in the absence of complement. All eight anti-gC monoclonal antibodies neutralized only in the presence of complement. An anti-gB monoclonal antibody obtained from another laboratory also neutralizes in the absence of complement. Since the above reactivities account for all major detectable VZV glycoprotein species, the data strongly suggest that VZV has three major glycoprotein genes which encode glycosylated polypeptides with neutralization epitopes.