Theresa Banks

Domain structure of herpes simplex virus 1 glycoprotein B: neutralizing epitopes map in regions of continuous and discontinuous residues

Herpes simplex virus 1 (HSV-1) glycoprotein B (gB) is a multifunctional glycoprotein required for infectivity; it is thought to promote fusion of the viral envelope with the cell membrane and entry of virions into cells. To map the antigenic and functional domains on gB, we constructed amino terminal derivatives lacking the entire carboxyl terminus and internal deletion mutants lacking defined regions of the extracellular and transmembrane domains. Transient expression of the mutants in COS-1 cells revealed that the amino terminal derivatives were released into the medium whereas those with deletions in the extracellular domain were mostly retained within the transfected cells. Analysis of intact gB and the amino terminal derivatives showed that the intact molecule formed dimers whereas the mutant derivatives did not. Reactions of the derivatives with a panel of well-characterized monoclonal antibodies to gB showed that the neutralizing epitopes cluster in two domains. The first maps in the amino terminal 190 residues and contains seven continuous epitopes, five of which are HSV-1-specific. Reactions of antibodies with a set of oligopeptides fine-mapped the epitopes between residues 1 and 47. The second domain is composed of discontinuous epitopes and was expressed by amino terminal derivatives that were at least 457 residues in length or longer. Eleven epitopes map in this region, including those of four potent neutralizing antibodies whose cognitive sites mapped between residues 273 and 298 in mapping studies using antibody-resistant mutants. Results of the present study indicate that the cognitive sites of these antibodies are assembled into the discontinuous domain by juxtaposing residues from the amino-terminal half of gB monomers.

A Major Neutralizing Domain Maps within the Carboxyl-terminal Half of the Cleaved Cytomegalovirus B Glycoprotein

Cytomegalovirus (CMV) encodes several glycoproteins reported to be structural homologues of glycoproteins encoded by herpes simplex virus type 1 (HSV-1). To map the antigenic and functional domains on the 907 amino acid CMV glycoprotein B (gB), we cloned and expressed a subfragment of BamHI fragment R of the CMV (Towne) genome into an expression vector and reacted the resulting gene product with a panel of monoclonal antibodies. Our results showed that the DNA fragment encodes related glycoproteins which we previously designated gA and which others have reported to be homologous to HSV-1 gB in CMV (AD169). Analyses of the processing of CMV gB transiently expressed in eukaryotic cells showed that glycosylation occurred independently of viral infection. Ten antibodies with complement-dependent and independent neutralizing activity reacted with a truncated derivative of gB that contained 619 amino-terminal residues but lacked the transmembrane and intracellular regions of the molecule. Twelve additional antibodies reacted with a CHO cell line expressing a 680 amino-terminal derivative of gB. All of the reactive antibodies precipitated the 447 residue carboxy-terminal cleavage product of gB from extracts of CMV-infected cells. These results showed that the neutralizing epitopes map in at least two domains of gB which are located in a discontinuous segment of 219 amino acids between residues 461 and 680 from the amino terminus of the molecule.

Assessing Cell-Mediated Immune Responses to HSV in Murine Systems.

Protective immunity against a eajority of viral infections is mediated by a combination of both humoral and cell-mediated immune responses. However, in the case of herpesvirus infections, where viral spread is largely cell-to-cell, cell-mediated immune mechanisms (which facilitate the clearance of virally infected cells) are particularly important (1-4). Moreover, cell-mediated immunity (CMI) has also been implicated in the establishment and/or reactivation of latent herpes simplex virus (HSV) infection (5,6). Thus, a major focus of herpesvirus immunology continues to be the identification of those herpesvirus antigens that serve as targets for CMI and the means by which protective responses can be optimally induced. Clearly this information is critical for the rational development of effective vaccine strategies.