Whitbeck J C, Peng C, Lou H, Xu R, Willis S H, Ponce de Leon M, Peng T, Nicola A V, Montgomery R I, Warner M S, Soulika A M, Spruce L A, Moore W T, Lambris J D, Spear P G, Cohen G H, Eisenberg R J
School of Dental Medicine, Center for Oral Health Research, University of Pennsylvania, Philadelphia 19104, USA.
J Virol. 1997 Aug;71(8):6083-93. doi: 10.1128/JVI.71.8.6083-6093.1997.
Glycoprotein D (gD) is a structural component of the herpes simplex virus (HSV) envelope which is essential for virus entry into host cells. Chinese hamster ovary (CHO-K1) cells are one of the few cell types which are nonpermissive for the entry of many HSV strains. However, when these cells are transformed with the gene for the herpesvirus entry mediator (HVEM), the resulting cells, CHO-HVEM12, are permissive for many HSV strains, such as HSV-1(KOS). By virtue of its four cysteine-rich pseudorepeats, HVEM is a member of the tumor necrosis factor receptor superfamily of proteins. Recombinant forms of gD and HVEM, gD-1(306t) and HVEM(200t), respectively, were used to demonstrate a specific physical interaction between these two proteins. This interaction was dependent on native gD conformation but independent of its N-linked oligosaccharides, as expected from previous structure-function studies. Recombinant forms of gD derived from HSV-1(KOS)rid1 and HSV-1(ANG) did not bind to HVEM(200t), explaining the inability of these viruses to infect CHO-HVEM12 cells. A variant gD protein, gD-1(delta290-299t), showed enhanced binding to HVEM(200t) relative to the binding of gD-1(306t). Competition studies showed that gD-1(delta290-299t) and gD-1(306t) bound to the same region of HVEM(200t), suggesting that the differences in binding to HVEM are due to differences in affinity. These differences were also reflected in the ability of gD-1(delta290-299t) but not gD-1(306t) to block HSV type 1 infection of CHO-HVEM12 cells. By gel filtration chromatography, the complex between gD-1(delta290-299t) and HVEM(200t) had a molecular mass of 113 kDa and a molar ratio of 1:2. We conclude that HVEM interacts directly with gD, suggesting that HVEM is a receptor for virion gD and that the interaction between these proteins is a step in HSV entry into HVEM-expressing cells.
糖蛋白D（gD）是单纯疱疹病毒（HSV）包膜的一种结构成分，对病毒进入宿主细胞至关重要。中国仓鼠卵巢（CHO-K1）细胞是少数几种对许多HSV毒株的进入不敏感的细胞类型之一。然而，当这些细胞用疱疹病毒进入介质（HVEM）基因进行转化时，产生的细胞CHO-HVEM12对许多HSV毒株，如HSV-1（KOS）敏感。凭借其富含四个半胱氨酸的假重复序列，HVEM是肿瘤坏死因子受体超家族蛋白的成员。分别使用重组形式的gD和HVEM，即gD-1（306t）和HVEM（200t），来证明这两种蛋白之间存在特异性的物理相互作用。正如先前结构功能研究所预期的那样，这种相互作用依赖于天然gD构象，但不依赖于其N-连接寡糖。源自HSV-1（KOS）rid1和HSV-1（ANG）的gD重组形式不与HVEM（200t）结合，这解释了这些病毒无法感染CHO-HVEM12细胞的原因。一种变异的gD蛋白gD-1（delta290 - 299t）相对于gD-1（306t）与HVEM（200t）的结合增强。竞争研究表明，gD-1（delta290 - 299t）和gD-1（306t）与HVEM（200t）的同一区域结合，这表明与HVEM结合的差异是由于亲和力不同。这些差异也反映在gD-1（delta290 - 299t）而非gD-1（306t）阻断CHO-HVEM12细胞中1型HSV感染的能力上。通过凝胶过滤色谱法，gD-1（delta290 - 299t）和HVEM（200t）之间的复合物分子量为113 kDa，摩尔比为1:2。我们得出结论，HVEM直接与gD相互作用，这表明HVEM是病毒粒子gD的受体，并且这些蛋白之间的相互作用是HSV进入表达HVEM的细胞的一个步骤。
