Structure-based mutagenesis of herpes simplex virus glycoprotein D defines three critical regions at the gD-HveA/HVEM binding interface
Structure-based mutagenesis of herpes simplex virus glycoprotein D defines three critical regions at the gD-HveA/HVEM binding interface
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DOI：
10.1128/jvi.77.14.8127-8140.2003
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发表时间：
2003-07-01
影响因子：
5.4
通讯作者：
Eisenberg, RJ
中科院分区：
文献类型：
--
作者：
Connolly, SA;Landsburg, DJ;Eisenberg, RJ
单纯疱疹病毒（HSV）进入细胞需要糖蛋白D（gD）与几种细胞表面受体之一结合。与这些受体之一HveA/HVEM结合的gD的晶体结构揭示了gD的核心包含侧接长C末端延伸和N末端发夹环的免疫球蛋白折叠。HveA是肿瘤坏死因子受体家族的成员，包含该家族特有的四个富含半胱氨酸的结构域（CRD）。gD N-末端环内的14个氨基酸构成HveA的整个结合位点。为了确定每个gD接触残基对病毒进入的贡献，我们构建了这些氨基酸突变的gD分子。我们确定了gD突变体结合受体、促进病毒进入和介导细胞-细胞融合的能力。七gD突变体表现出野生型水平的受体结合和gD功能。来自其他七个gD突变体的结果揭示了gD-HveA界面处的三个关键区域。(i)发现参与与HveA的分子间β-折叠的几个gD残基对于HveA结合和进入表达HveA的细胞至关重要。(ii)接触HveA-Y23的两个gD残基有助于HveA结合，但不需要介导进入细胞。HveA-Y23适合gD表面上的裂缝，并且先前显示对于gD结合是必需的。(iii)CRD 2先前显示有助于gD结合，并且该研究显示接触CRD 2的一个gD残基有助于HveA结合。没有gD突变阻止与另一种gD受体nectin-1的相互作用。然而，当与融合所需的其他糖蛋白共转染时，两个gD突变体获得了介导表达nectin-2的细胞融合的能力，nectin-2是一种gD受体，与几种实验室来源的gD突变体相互作用，但不与野生型gD相互作用。因此，来自这组gD突变体的结果以及先前研究的结果（A. Carfi，S. H.威利斯，J.C.惠特贝克角克鲁梅纳赫湾H.科恩河J. Eisenberg和D. C. Wiley，Mol. Cell 8：169-179，2001和S. A. Connolly，D. J. Landsburg，A. Carfi，D. C.威利河J. Eisenberg和G. H. Cohen，J. Virol. 76：10894-10904，2002）提供了gD-HveA界面和功能性相互作用所需的触点的详细图片。结果表明，在构成gD-HveA界面的35个gD和HveA接触残基中，只有少数对于复合物形成是关键的。
Herpes simplex virus (HSV) entry into cells requires the binding of glycoprotein D (gD) to one of several cell surface receptors. The crystal structure of gD bound to one of these receptors, HveA/HVEM, reveals that the core of gD comprises an immunoglobulin fold flanked by a long C-terminal extension and an N-terminal hairpin loop. HveA is a member of the tumor necrosis factor receptor family and contains four cysteine-rich domains (CRDs) characteristic of this family. Fourteen amino acids within the gD N-terminal loop comprise the entire binding site for HveA. To determine the contribution of each gD contact residue to virus entry, we constructed gD molecules mutated in these amino acids. We determined the abilities of the gD mutants to bind receptors, facilitate virus entry, and mediate cell-cell fusion. Seven of the gD mutants exhibited wild-type levels of receptor binding and gD function. Results from the other seven gD mutants revealed three critical regions at the gD-HveA interface. (i) Several gD residues that participate in an intermolecular beta-sheet with HveA were found to be crucial for HveA binding and entry into HveA-expressing cells. (ii) Two gD residues that contact HveA-Y23 contributed to HveA binding but were not required for mediating entry into cells. HveA-Y23 fits into a crevice on the surface of gD and was previously shown to be essential for gD binding. (iii) CRD2 was previously shown to contribute to gD binding, and this study shows that one gD residue that contacts CRD2 contributes to HveA binding. None of the gD mutations prevented interaction with nectin-1, another gD receptor. However, when cotransfected with the other glycoproteins required for fusion, two gD mutants gained the ability to mediate fusion of cells expressing nectin-2, a gD receptor that interacts with several laboratory-derived gD mutants but not with wild-type gD. Thus, results from this panel of gD mutants as well as those of previous studies (A. Carfi, S. H. Willis, J. C. Whitbeck, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and D. C. Wiley, Mol. Cell 8:169-179, 2001, and S. A. Connolly, D. J. Landsburg, A. Carfi, D. C. Wiley, R. J. Eisenberg, and G. H. Cohen, J. Virol. 76:10894-10904, 2002) provide a detailed picture of the gD-HveA interface and the contacts required for functional interaction. The results demonstrate that of the 35 gD and HveA contact residues that comprise the gD-HveA interface, only a handful are critical for complex formation.