Antigenic and mutational analyses of herpes simplex virus glycoprotein B reveal four functional regions.
Glycoprotein B (gB), along with gD, gH, and gL, is essential for herpes simplex virus (HSV) entry. The crystal structure of the gB ectodomain revealed it to be an elongated multidomain trimer. We generated and characterized a panel of 67 monoclonal antibodies (MAbs). Eleven of the MAbs had virus-neutralizing activity. To organize gB into functional regions within these domains, we localized the epitopes recognized by the entire panel of MAbs and mapped them onto the crystal structure of gB. Most of the MAbs were directed to continuous or discontinuous epitopes, but several recognized discontinuous epitopes that showed some resistance to denaturation, and we refer to them as pseudo-continuous. Each category contained some MAbs with neutralizing activity. To map continuous epitopes, we used overlapping peptides that spanned the gB ectodomain and measured binding by enzyme-linked immunosorbent assay. To identify discontinuous and pseudocontinuous epitopes, a purified form of the ectodomain of gB, gB(730t), was cleaved by alpha-chymotrypsin into two major fragments comprising amino acids 98 to 472 (domains I and II) and amino acids 473 to 730 (major parts of domains III, IV, and V). We also constructed a series of gB truncations to augment the other mapping strategies. Finally, we used biosensor analysis to assign the MAbs to competition groups. Together, our results identified four functional regions: (i) one formed by residues within domain I and amino acids 697 to 725 of domain V; (ii) a second formed by residues 391 to 410, residues 454 to 475, and a less-defined region within domain II; (iii) a region containing residues of domain IV that lie close to domain III; and (iv) the first 12 residues of the N terminus that were not resolved in the crystal structure. Our data suggest that multiple domains are critical for gB function.
Bender
Florent C
FC
Department of Microbiology, University of Pennsylvania, School of Dental Medicine, 240 S. 40th Street, Levy Bldg. R217, Philadelphia, PA 19104, and Children's Hospital, Boston, MA 02115, USA. fbender@biochem.dental.upenn.edu
Samanta
Minu
M
Heldwein
Ekaterina E
EE
de Leon
Manuel Ponce
MP
Bilman
Elina
E
Lou
Huan
H
Whitbeck
J Charles
JC
Eisenberg
Roselyn J
RJ
Cohen
Gary H
GH
D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D018259:Herpesvirus 1, Human / Q000737:chemistry*; D008958:Models, Molecular; D009500:Neutralization Tests; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000502:physiology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus