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Journal of Virology, April 2007, p. 3827-3841, Vol. 81, No. 8
0022-538X/07/$08.00+0     doi:10.1128/JVI.02710-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Antigenic and Mutational Analyses of Herpes Simplex Virus Glycoprotein B Reveal Four Functional Regions{triangledown}

Florent C. Bender,1* Minu Samanta,1 Ekaterina E. Heldwein,2,{dagger} Manuel Ponce de Leon,1 Elina Bilman,1 Huan Lou,1 J. Charles Whitbeck,1 Roselyn J. Eisenberg,3 and Gary H. Cohen1

Department of Microbiology, School of Dental Medicine,1 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104,3 Children's Hospital and Harvard Medical School, Boston, Massachusetts 021552

Received 8 December 2006/ Accepted 22 January 2007

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.

* Corresponding author. Mailing address: Department of Microbiology, University of Pennsylvania, School of Dental Medicine, 240 S. 40th Street, Levy Bldg. R217, Philadelphia, PA 19104. Fax: (215) 898-8385. Phone: (215) 898-6558. E-mail: fbender{at}biochem.dental.upenn.edu

{triangledown} Published ahead of print on 31 January 2007.

{dagger} Present address: Department of Molecular Biology and Microbiology Tufts University, School of Medicine, Boston, MA 02111.

Journal of Virology, April 2007, p. 3827-3841, Vol. 81, No. 8
0022-538X/07/$08.00+0     doi:10.1128/JVI.02710-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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