Localization of a binding site for herpes simplex virus glycoprotein D on herpesvirus entry mediator C by using antireceptor monoclonal antibodies.
The human herpesvirus entry mediator C (HveC), also known as the poliovirus receptor-related protein 1 (PRR1) and as nectin-1, allows the entry of herpes simplex virus type 1 (HSV-1) and HSV-2 into mammalian cells. The interaction of virus envelope glycoprotein D (gD) with such a receptor is an essential step in the process leading to membrane fusion. HveC is a member of the immunoglobulin (Ig) superfamily and contains three Ig-like domains in its extracellular portion. The gD binding site is located within the first Ig-like domain (V domain) of HveC. We generated a panel of monoclonal antibodies (MAbs) against the ectodomain of HveC. Eleven of these, which detect linear or conformational epitopes within the V domain, were used to map a gD binding site. They allowed the detection of HveC by enzyme-linked immunosorbent assay, Western blotting, and biosensor analysis or directly on the surface of HeLa cells and human neuroblastoma cell lines, as well as simian Vero cells. The anti-HveC V-domain MAbs CK6, CK8, and CK41, as well as the previously described MAb R1.302, blocked HSV entry. Their binding to soluble HveC was blocked by the association of gD with the receptor, indicating that their epitopes overlap a gD binding site. Competition assays on an optical biosensor showed that CK6 and CK8 (linear epitopes) inhibited the binding of CK41 and R1.302 (conformational epitopes) to HveC and vice versa. Epitope mapping showed that CK6 and CK8 bound between residues 80 and 104 of HveC, suggesting that part of the gD binding site colocalizes in the same region.
Krummenacher
C
C
Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. krumm@biochem.dental.upenn.edu
Baribaud
I
I
Ponce de Leon
M
M
Whitbeck
J C
JC
Lou
H
H
Cohen
G H
GH
Eisenberg
R J
RJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001665:Binding Sites; D015374:Biosensing Techniques; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000276:immunology; D018604:Epitope Mapping; D006801:Humans; D008969:Molecular Sequence Data; D000075983:Nectins; D011991:Receptors, Virus / Q000032:analysis*; D014407:Tumor Cells, Cultured; D014759:Viral Envelope Proteins / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; C000619010:NECTIN1 protein, human; D000075983:Nectins; D011991:Receptors, Virus; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1