Also, all group III MAbs tested, including ABD, 99-1, and 11S, also coprecipitated HVEMt and gDt (not shown). other anti-gD MAbs coprecipitated HVEMt and gDt. Previous mapping data indicated that site VII includes amino acids 11 to 19 and site Ib includes 222 to 252. The current experiments indicate that these sites contain residues important for HSV binding to HVEM. Group Ib and VII MAbs also blocked HSV entry into HVEM-expressing CHO cells. These results suggest that the mechanism of neutralization by these MAbs is usually via interference with the conversation between gD in the virus and HVEM around the cell. Group Ia and II MAbs failed to block HSV binding to HVEM yet still neutralized HVEM-mediated entry, suggesting that these MAbs block entry at a step other than HVEM binding. The envelope of herpes simplex virus (HSV) contains at least 10 virus-encoded glycoproteins (52). The initial adsorption of HSV to glycosaminoglycan chains (GAGs) of cell surface proteoglycans is usually mediated by glycoprotein C (gC) and/or gB (23,24,58). This Hyperforin (solution in Ethanol) is followed by the conversation of gD with cellular receptors (5,28,29,31,55). Then, gB, gD, and the complex of gH and gL (gH/gL) act individually or Hyperforin (solution in Ethanol) in combination to trigger pH-independent fusion of the viral envelope with the host cell plasma membrane (52). Recently, expression cloning was used to isolate and identify a HeLa cell gene product, which when expressed in normally nonpermissive Chinese hamster ovary (CHO) cells, allows for entry of many HSV strains (35). The gene product, called HVEM (for herpes virus entry mediator) is usually FLJ20285 a 283-amino-acid type I integral membrane protein and is a member of the tumor necrosis factor receptor superfamily (1,26,30,33,35,50). HSV-1 variants rid1 and ANG have changes in gD sequence (11) and infect HVEM-expressing cells with markedly reduced efficiency, suggesting that HVEM might interact directly with gD (35). Subsequently, we exhibited that soluble, truncated gD (gDt) from the KOS strain binds directly to a soluble, truncated form of HVEM (HVEMt) in vitro (55). This binding is dependent on the native conformation of gD but is usually impartial of its asparagine-linked oligosaccharides. Soluble gDt from the rid1 or ANG strains was unable to bind to HVEMt. Thus, the inability of rid1 and ANG to infect HVEM-expressing cells may be due to the failure of these virion gDs to bind to HVEM around the cell. A variant gD protein, gD-1(290299t), showed enhanced binding to HVEMt relative to the binding of wild-type gDt (55,57). Also, gD-1(290299t) and wild-type gDt competed for binding to HVEMt. It is well established that gD can induce potent virus-neutralizing antibodies (Abs) (7,9,25,34,40,43,49). Monoclonal Abs (MAbs) against gD Hyperforin (solution in Ethanol) have been demonstrated to block a postadsorption step in virus entry prior to virus-cell fusion (20,21,25,52). Although the role of gD as a receptor binding protein has been well documented (3,5,28,29,31,55), blocking of virion gD binding to a specific cellular receptor has not been demonstrated as a mechanism of neutralization. The purpose of this study was to characterize the conversation between Hyperforin (solution in Ethanol) the intact virus and HVEM and to begin to identify regions Hyperforin (solution in Ethanol) of gD which are important for binding to HVEM. Here we demonstrate that (i) HSV virions bind specifically and directly to HVEMt, providing formal proof that HVEM is usually a viral receptor; (ii) anti-gD.