title: Human herpesvirus-6 glycoprotein H and L homologs are components of the gp100 complex and the gH external domain is the target for neutralizing monoclonal antibodies.
abstract: Previous studies have shown that monoclonal antibody (MAb) 2E4 neutralizes infectivity of human herpesvirus-6 (HHV-6) and also inhibits virus-induced T-lymphocyte syncytia formation. Here we characterize two additional MAbs, 1D3 and 5E7, which have similar properties, and identify the glycoprotein targets. The MAbs could immunoprecipitate and immunofluorescence glycoprotein from both A and B variant strain groups of HHV-6. In reactions with infected cells the MAbs immunoprecipitated a complex of glycoproteins, the "gp100" complex, composed of a major glycoprotein species of 100,000 M(r) and minor components of 80,000 M(r) and 32,000 M(r). We show that the 100,000 M(r) product and most likely the 80,000 M(r) correspond to the HHV-6 homologue of herpes simplex virus-1 (HSV-1) glycoprotein H (gH) while the 32,000 M(r) species corresponds to the glycoprotein L (gL) equivalent. All three MAbs could specifically immunoprecipitate either gH expressed on its own in fibroblasts or a complex of gH and gL co-expressed, but could not immunoprecipitate gL expressed on its own. Consistent with these results, the MAbs could recognize gH in an immunofluorescence assay but not gL. Therefore although the MAbs recognized the complex of glycoproteins, they appeared specific for gH. The HHV-6 glycoproteins were produced in a transient expression system induced by T7-vaccinia virus. Immunoprecipitations were carried out in comparisons with an "epitope-tagged" gH, a recombinant glycoprotein designed to contain at the N-terminus the linear epitope for MAb LP14, raised originally against HSV-1 glycoprotein gD. The epitope-tagged gH was also used as a positive control in determining the domain of HHV-6 gH to which MAbs 2E4, 1D3 and 5E7 were directed. Two gH deletions were constructed, one deleting sequences which may serve as a transmembrane and cytoplasmic anchor domains, the second deleting also part of the external domain. MAb LP14 could immunoprecipitate both HHV-6 gH deletions but the gp100 MAbs recognized only the full-length product or the intact external domain minus the transmembrane and cytoplasmic domains. This indicated the epitopes for these MAbs are contained in the external domain of gH, consistent with the MAbs action in neutralization of virion infectivity and inhibition of virus to cell spread by T-lymphocyte fusion.
authors.affiliation: Department of Medicine, University of Cambridge, United Kingdom.
authors.forename: D X
authors.initials: DX
authors.lastname: Liu
authors.forename: U A
authors.initials: UA
authors.lastname: Gompels
authors.forename: L
authors.initials: L
authors.lastname: Foa-Tomasi
authors.forename: G
authors.initials: G
authors.lastname: Campadelli-Fiume
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D015654:Herpesvirus 6, Human / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D016133:Polymerase Chain Reaction; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014759:Viral Envelope Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D014764:Viral Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; D000939:Epitopes; D006023:Glycoproteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C070917:glycoprotein H, herpesvirus 6; C074553:p100 protein, Human herpesvirus 6