The herpesvirus entry mediator A (HveA) is a lately characterized member of the tumor necrosis factor receptor family that mediates the entry of most herpes simplex virus type 1 (HSV-1) strains into mammalian cells. tumor necrosis factor receptor-associated factor family lymphotoxin-α (LT-α) and a membrane-associated protein referred to as LIGHT. To study the relationship between HveA its natural ligands and the viral proteins involved in HSV entry into cells we have screened two phage-displayed combinatorial peptide libraries for peptide ligands of a recombinant form of HveA. Affinity selection experiments yielded two peptide ligands BP-1 and BP-2 which could block the interaction between gD and HveA. Of the two peptides only BP-2 inhibited HSV entry into CHO cells transfected with an HveA-expressing plasmid. When we analyzed these peptides for the ability to interfere with HveA binding to its natural ligand LT-α we found that BP-1 inhibited the interaction of cellular LT-α with HveA. Thus we have dissected the sites of interaction between the cell receptor its natural ligand LT-α and gD the virus-specific protein involved in HSV entry into cells. The herpesvirus entry mediator A (HveA; formerly named HVEM) is a member of the tumor necrosis factor receptor (TNFR) superfamily and has been shown to act as a receptor for herpes simplex virus (HSV) (16). Expressed in otherwise nonpermissive CHO cells it rendered these cells susceptible to entry by several HSV strains. This binding was inhibited by recombinant soluble HveA and antibodies to HveA. In addition to the involvement of HveA in the entry of extracellular virus it was found that it Rabbit polyclonal to EGFL6. participates in cell-to-cell transmission of the virus (22 30 The HSV protein mediating its binding with HveA has been shown to be the glycoprotein D (gD) as it binds directly to a soluble form of HveA [HveA(200t)] (34) in a specific and saturable manner and inhibits the binding of HSV to HveA-expressing cells (20 21 27 34 In addition to its involvement in HSV entry several findings suggest that HveA plays a role in KU-55933 the activation of the host immune response. For example HveA predominantly expressed in lymphocyte-rich tissues has been shown to interact with several members of the TNFR-associated factor (TRAF) family of proteins. This interaction leads to the activation of transcriptional regulators such as NF-κB Jun N-terminal kinase and AP-1 (8 14 There are two known ligands for the extracellular domain of HveA lymphotoxin-α (LT-α) and the membrane-associated protein referred to as LIGHT. LIGHT is a newly identified lymphotoxin homolog which is expressed by T cells upon induction with phorbol myristate acetate and Ca2+ ionophore and competes with a soluble form KU-55933 of HSV gD (gDt) for binding to HveA. Thus either LT-α or LIGHT may modulate HSV infection by competing KU-55933 for HveA binding and vice versa which has led to the hypothesis that gD may modify HveA-signaling activities during entry or egress of HSV thus modulating the immune response of the host (15). The mode of HveA interaction with its ligands as well as whether HveA interacts with them via multiple sites or whether these ligands share binding sites is not known. The rich but uncharted molecular diversity that is offered by the surface of the HveA molecule calls for an equally diverse approach to searching for ligands that are complementary and specifically interactive with particular sites. Within the last 10 years random peptide libraries have provided a rich source of structural diversity (10). They have proved to be a useful tool in identifying the peptide epitopes recognized by particular monoclonal antibodies as well as mimetics of ligands for various proteins. In this study our goal was to study the interaction between HveA its natural ligands and HSV gD. To this end we have used recombinant HveA to screen two phage-displayed combinatorial peptide libraries and have selected two peptide ligands that differentially inhibit binding of gDt and LT-α to the receptor. Furthermore one of these peptides was able to block HSV entry into HveA-expressing CHO cells. MATERIALS AND METHODS Chemicals and buffers. All chemicals and reagents used for peptide synthesis were KU-55933 purchased from Applied Biosystems (Foster City Calif.) with the exception of the F-moc (9-fluorenylmethoxycarbonyl) amino.