title: Mechanism of neutralization of herpes simplex virus by antibodies directed at the fusion domain of glycoprotein B.
abstract: Glycoprotein B (gB), the fusogen of herpes simplex virus (HSV), is a class III fusion protein with a trimeric ectodomain of known structure for the postfusion state. Seen by negative-staining electron microscopy, it presents as a rod with three lobes (base, middle, and crown). gB has four functional regions (FR), defined by the physical location of epitopes recognized by anti-gB neutralizing monoclonal antibodies (MAbs). Located in the base, FR1 contains two internal fusion loops (FLs) and is the site of gB-lipid interaction (the fusion domain). Many of the MAbs to FR1 are neutralizing, block cell-cell fusion, and prevent the association of gB with lipid, suggesting that these MAbs affect FL function. Here we characterize FR1 epitopes by using electron microscopy to visualize purified Fab-gB ectodomain complexes, thus confirming the locations of several epitopes and localizing those of MAbs DL16 and SS63. We also generated MAb-resistant viruses in order to localize the SS55 epitope precisely. Because none of the epitopes of our anti-FR1 MAbs mapped to the FLs, we hyperimmunized rabbits with FL1 or FL2 peptides to generate polyclonal antibodies (PAbs). While the anti-FL1 PAb failed to bind gB, the anti-FL2 PAb had neutralizing activity, implying that the FLs become exposed during virus entry. Unexpectedly, the anti-FL2 PAb (and the anti-FR1 MAbs) bound to liposome-associated gB, suggesting that their epitopes are accessible even when the FLs engage lipid. These studies provide possible mechanisms of action for HSV neutralization and insight into how gB FR1 contributes to viral fusion.
  
  
  OBJECTIVE
  For herpesviruses, such as HSV, entry into a target cell involves transfer of the capsid-encased genome of the virus to the target cell after fusion of the lipid envelope of the virus with a lipid membrane of the host. Virus-encoded glycoproteins in the envelope are responsible for fusion. Antibodies to these glycoproteins are important biological tools, providing a way of examining how fusion works. Here we used electron microscopy and other techniques to study a panel of anti-gB antibodies. Some, with virus-neutralizing activity, impair gB-lipid association. We also generated a peptide antibody against one of the gB fusion loops; its properties provide insight into the way the fusion loops function as gB transits from its prefusion form to an active fusogen.
authors.affiliation: Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Tina M
authors.initials: TM
authors.lastname: Cairns
authors.forename: Juan
authors.initials: J
authors.lastname: Fontana
authors.forename: Zhen-Yu
authors.initials: ZY
authors.lastname: Huang
authors.forename: J Charles
authors.initials: JC
authors.lastname: Whitbeck
authors.forename: Doina
authors.initials: D
authors.lastname: Atanasiu
authors.forename: Samhita
authors.initials: S
authors.lastname: Rao
authors.forename: Spencer S
authors.initials: SS
authors.lastname: Shelly
authors.forename: Huan
authors.initials: H
authors.lastname: Lou
authors.forename: Manuel
authors.initials: M
authors.lastname: Ponce de Leon
authors.forename: Alasdair C
authors.initials: AC
authors.lastname: Steven
authors.forename: Roselyn J
authors.initials: RJ
authors.lastname: Eisenberg
authors.forename: Gary H
authors.initials: GH
authors.lastname: Cohen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D057134:Antibodies, Neutralizing / Q000276:immunology* / Q000378:metabolism; D000914:Antibodies, Viral / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000378:metabolism; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009500:Neutralization Tests; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs / Q000276:immunology*; D018139:Simplexvirus / Q000235:genetics / Q000276:immunology*; D014709:Vero Cells; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008081:Liposomes; D014760:Viral Fusion Proteins