Zakowski J J, Wagner R R
J Virol. 1980 Oct;36(1):93-102. doi: 10.1128/JVI.36.1.93-102.1980.
The location of membrane-associated proteins of vesicular stomatitis virus was investigated by using two monofunctional and three bifunctional probes that differ in the degree to which they partition into membranes and in their specific group reactivity. Two hydrophobic aryl azide probes, [(125)I]5-iodonaphthyl-1-azide and [(3)H]pyrenesulfonylazide, readily partitioned into virion membrane and, when activated to nitrenes by UV irradiation, formed stable covalent adducts to membrane constituents. Both of these monofunctional probes labeled the glyco-protein G and matrix M proteins, but [(125)I]5-iodonaphthyl-1-azide also labeled the nucleocapsid N protein and an unidentified low-molecular-weight component. Protein labeling of intact virions was unaffected by the presence of cytochrome c or glutathione, but disruption of membrane by sodium dodecyl sulfate greatly enhanced the labeling of all viral proteins except G. Labeling of G protein was essentially restricted to the membrane-embedded, thermolysin-resistant tail fragment. Three bifunctional reagents, tartryl diazide, dimethylsuberimidate, and 4,4'-dithiobisphenylazide, were tested for their capacity to cross-link proteins to membrane phospholipids of virions grown in the presence of [(3)H]palmitate. Only G and M proteins of intact virions were labeled with (3)H-phospholipid by these cross-linkers; the reactions were not affected by cytochrome c but were abolished by disruption of virus with sodium dodecyl sulfate. Dimethylsuberimidate, which reacts with free amino groups, cross-linked (3)H-phospholipid to both G and M protein. In contrast, the hydrophilic tartryl diazide cross-linked phospholipid primarily to the M protein, whereas the hydrophobic 4,4'-dithiobisphenylazide cross-linked phospholipid primarily to the intrinsic G protein. These data support the hypothesis that the G protein traverses the virion membrane and that the M protein is membrane associated but does not penetrate very deeply, if at all.
利用两种单功能和三种双功能探针研究了水泡性口炎病毒膜相关蛋白的定位，这些探针在它们分配到膜中的程度以及它们的特异性基团反应性方面存在差异。两种疏水性芳基叠氮化物探针，即[(125)I]5-碘萘基-1-叠氮化物和[(3)H]芘磺酰叠氮化物，很容易分配到病毒粒子膜中，并且当通过紫外线照射激活形成氮烯时，会与膜成分形成稳定的共价加合物。这两种单功能探针都标记了糖蛋白G和基质M蛋白，但[(125)I]5-碘萘基-1-叠氮化物还标记了核衣壳N蛋白和一种未鉴定的低分子量成分。完整病毒粒子的蛋白质标记不受细胞色素c或谷胱甘肽存在的影响，但十二烷基硫酸钠对膜的破坏极大地增强了除G蛋白外所有病毒蛋白的标记。G蛋白的标记基本上仅限于膜嵌入的、耐热胰蛋白酶的尾部片段。测试了三种双功能试剂，酒石酸二叠氮化物、二甲基辛二亚胺和4,4'-二硫代双苯叠氮化物，它们在[(3)H]棕榈酸存在下生长的病毒粒子中，将蛋白质与膜磷脂交联的能力。这些交联剂仅用(3)H-磷脂标记完整病毒粒子的G和M蛋白；这些反应不受细胞色素c的影响，但用十二烷基硫酸钠破坏病毒后反应被消除。与游离氨基反应的二甲基辛二亚胺将(3)H-磷脂与G和M蛋白交联。相比之下，亲水性的酒石酸二叠氮化物主要将磷脂与M蛋白交联，而疏水性的4,4'-二硫代双苯叠氮化物主要将磷脂与内在的G蛋白交联。这些数据支持了以下假设：G蛋白穿过病毒粒子膜，而M蛋白与膜相关，但如果有的话，也不会深入穿透。
