Localization of the gD-binding region of the human herpes simplex virus receptor, HveA

During virus entry, herpes simplex virus (HSV) glycoprotein D (gD) binds to one of several human cellular receptors. One of these, herpesvirus entry m ediator A (HveA), is a member of the tumor necrosis factor receptor (TNFR) superfamily, and its ectodomain contains four characteristic cysteine-rich pseudorepeat (CRP) elements. We previously showed that go binds the ectodom ain of HveA expressed as a truncated, soluble protein [HveA(200t)]. To loca lize the go-binding domain of HveA, we expressed three additional soluble f orms of HveA consisting of the first CRP [HveA(76t)], the second CRP [HveA( 77-120t)], or the first and second CRPs [HveA(120t)]. Biosensor and enzyme- linked immunosorbent assay studies showed that go bound to HveA(120t) and H veA(200t) with the same affinity. However, go did not bind to HveA(76t) or HveA(77-120t). Furthermore, HveA(200t) and HveA(120t), but not HveA(76t) or HveA(77-120t), blocked herpes simplex virus (HSV) entry into CHO cells exp ressing HveA. We also generated six monoclonal antibodies (MAbs) against Hv eA(200t). MAbs CW1, -2, and -4 bound linear epitopes within the second CRP, while CW7 and -8 bound linear epitopes within the third or fourth CRPs. No ne of these MAbs blocked the binding of go to HveA. In contrast, MAb CW3 re cognized a discontinuous epitope within the first CRP of HveA, blocked the binding of go to HveA, and exhibited a limited ability to block virus entry into cells expressing HveA, suggesting that the first domain of HveA conta ins at least a portion of the go binding site. The inability of go to bind HveA(76t) suggests that additional amino acid residues of the go binding si te may reside within the second CRP.