DISULFIDE BONDS OF HERPES-SIMPLEX VIRUS TYPE-2 GLYCOPROTEIN GB

Glycoprotein B (gB) is the most highly conserved envelope glycoprotein of herpesviruses. The gB protein is required for virus infectivity an d cell penetration. Recombinant forms of gB being used for the develop ment of subunit vaccines are able to induce virus-neutralizing antibod ies and protective efficacy in animal models. To gain structural infor mation about the protein, we have determined the location of the disul fide bonds of a 696-amino-acid residue truncated, recombinant form of herpes simplex virus type 2 glycoprotein gB (HSV gB2t) produced by exp ression in Chinese hamster ovary cells. The purified protein, which co ntains virtually the entire extracellular domain of herpes simplex vir us type 2 gB, was digested with trypsin under nonreducing conditions, and peptides were isolated by reversed-phase high-performance liquid c hromatography (HPLC). The peptides were characterized by using mass sp ectrometry and amino acid sequence analysis. The conditions of cleavag e (4 pH urea, pH 7) induced partial carbamylation of the N termini of the peptides, and each disulfide peptide was found with two or three d ifferent HPLC retention times (peptides with and without carbamylation of either one or both N termini). The 10 cysteines of the molecule we re found to be involved in disulfide bridges. These bonds were located between Cys-89 (C1) and Cys-548 (C8), Cys-106 (C2) and Cys-504 (C7), Cys-180 (C3) and Cys-244 (C4), Cys-337 (C5) and Cys-385 (C6), and Cys- 571 (C9) and Cys-608 (C10). These disulfide bonds are anticipated to b e similar in the corresponding gBs from other herpesviruses because th e 10 cysteines listed above are always conserved in the corresponding protein sequences.