Hecker C, Weise C, Schneider-Schaulies J, Holmes H C, ter Meulen V
Institut für Virologie und Immunbiologie, Würzburg, Germany.
Virus Res. 1997 Jun;49(2):215-23. doi: 10.1016/s0168-1702(97)00039-7.
The observation that HIV in vitro can infect CD4- and Gal-C-negative brain cell lines has stimulated this study to identify alternative gp120-binding proteins on brain cells. HIV-1 gp120 binding proteins of the CD4-negative and Gal-C-negative, non-productively infectable human glioblastoma cell line D54 were purified by affinity chromatography over a gp120-conjugated sepharose column and identified by peptide microsequencing. The binding capacity and specificity of this column was controlled using extracts of CD4-positive cells. Two of seven prominent proteins eluted from the gp120 affinity column specifically bound gp120 in Western blot overlay experiments and were identified by subsequent immunoblotting and microsequencing as ezrin and moesin, members of the ERM (ezrin, radixin, moesin) family of cellular structural membrane proteins. Antibodies to ezrin and moesin specifically recognized the eluted gp120 binding proteins confirming their identification. Ezrin and moesin are structural proteins binding to the cellular membrane and to several cytoskeletal and transmembrane proteins. Our results suggest that ezrin and moesin might play a role as gp160/gp120 binding proteins during the uptake, the assembly or the budding of HIV.
体外实验观察到HIV可感染CD4和半乳糖脑苷脂（Gal-C）均呈阴性的脑细胞系，这激发了本研究去鉴定脑细胞上其他的gp120结合蛋白。通过在gp120偶联的琼脂糖柱上进行亲和层析，对CD4阴性且Gal-C阴性、不能被有效感染的人胶质母细胞瘤细胞系D54的HIV-1 gp120结合蛋白进行纯化，并通过肽微测序进行鉴定。使用CD4阳性细胞提取物来控制该柱的结合能力和特异性。从gp120亲和柱上洗脱的七种主要蛋白质中的两种，在蛋白质印迹覆盖实验中能特异性结合gp120，并通过随后的免疫印迹和微测序鉴定为埃兹蛋白（ezrin）和膜突蛋白（moesin），它们是细胞结构膜蛋白ERM（埃兹蛋白、根蛋白、膜突蛋白）家族的成员。针对埃兹蛋白和膜突蛋白的抗体能特异性识别洗脱的gp120结合蛋白，从而证实了它们的身份。埃兹蛋白和膜突蛋白是与细胞膜以及几种细胞骨架和跨膜蛋白结合的结构蛋白。我们的结果表明，埃兹蛋白和膜突蛋白可能在HIV的摄取、组装或出芽过程中作为gp160/gp120结合蛋白发挥作用。
