Nicola A V, Ponce de Leon M, Xu R, Hou W, Whitbeck J C, Krummenacher C, Montgomery R I, Spear P G, Eisenberg R J, Cohen G H
Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104-6002, USA.
J Virol. 1998 May;72(5):3595-601. doi: 10.1128/JVI.72.5.3595-3601.1998.
HVEM (for herpesvirus entry mediator) is a member of the tumor necrosis factor receptor superfamily and mediates entry of many strains of herpes simplex virus (HSV) into normally nonpermissive Chinese hamster ovary (CHO) cells. We used sucrose density centrifugation to demonstrate that purified HSV-1 KOS virions bind directly to a soluble, truncated form of HVEM (HVEMt) in the absence of any other cell-associated components. Therefore, HVEM mediates HSV entry by serving as a receptor for the virus. We previously showed that soluble, truncated forms of HSV glycoprotein D (gDt) bind to HVEMt in vitro. Here we show that antibodies specific for gD, but not the other entry glycoproteins gB, gC, or the gH/gL complex, completely block HSV binding to HVEM. Thus, virion gD is the principal mediator of HSV binding to HVEM. To map sites on virion gD which are necessary for its interaction with HVEM, we preincubated virions with gD-specific monoclonal antibodies (MAbs). MAbs that recognize antigenic sites Ib and VII of gD were the only MAbs which blocked the HSV-HVEM interaction. MAbs from these two groups failed to coprecipitate HVEMt in the presence of soluble gDt, whereas the 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 via interference with the interaction between gD in the virus and HVEM on 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.
疱疹病毒进入介质（HVEM）是肿瘤坏死因子受体超家族的成员，介导许多单纯疱疹病毒（HSV）毒株进入通常不允许其进入的中国仓鼠卵巢（CHO）细胞。我们使用蔗糖密度离心法证明，纯化的HSV-1 KOS病毒粒子在没有任何其他细胞相关成分的情况下直接与可溶性截短形式的HVEM（HVEMt）结合。因此，HVEM通过作为病毒的受体介导HSV进入。我们之前表明，可溶性截短形式的HSV糖蛋白D（gDt）在体外与HVEMt结合。在此我们表明，针对gD而非其他进入糖蛋白gB、gC或gH/gL复合物的特异性抗体完全阻断HSV与HVEM的结合。因此，病毒粒子gD是HSV与HVEM结合的主要介导因子。为了定位病毒粒子gD上与其与HVEM相互作用所必需的位点，我们用gD特异性单克隆抗体（MAb）对病毒粒子进行预孵育。识别gD抗原位点Ib和VII的MAb是仅有的阻断HSV-HVEM相互作用的MAb。在可溶性gDt存在的情况下，来自这两组的MAb未能共沉淀HVEMt，而其他抗gD MAb则共沉淀了HVEMt和gDt。先前的定位数据表明，位点VII包括氨基酸11至19，位点Ib包括222至252。当前实验表明，这些位点含有对HSV与HVEM结合很重要的残基。Ib组和VII组MAb也阻断HSV进入表达HVEM的CHO细胞。这些结果表明，这些MAb的中和机制是通过干扰病毒中的gD与细胞上的HVEM之间的相互作用。Ia组和II组MAb未能阻断HSV与HVEM的结合，但仍能中和HVEM介导的进入，这表明这些MAb在HVEM结合以外的步骤阻断进入。
