Clegg J C, Lloyd G
J Gen Virol. 1983 May;64(Pt 5):1127-36. doi: 10.1099/0022-1317-64-5-1127.
Lassa virus was purified from culture fluids of infected CV-1 monkey kidney cells and its structural proteins analysed by polyacrylamide gel electrophoresis. Stained gels showed a typical arenavirus profile, with a prominent protein of molecular weight 60000, corresponding to the nucleocapsid protein N, and two faint broad bands with molecular weights of 45000 and 38000, the envelope glycoproteins G1 and G2. G1 and G2 were shown to be glycosylated by their ability to bind concanavalin A to nitrocellulose transfers of the separated proteins ('Western blots'). N and G2 bound antibody from guinea-pig or human convalescent sera but G1 was inactive, presumably as a result of denaturation. This technique also revealed other apparently virus-specific minor bands with molecular weights of 76000 and 68000. When Western blots of proteins of infected cells which had been lysed in SDS were probed with anti-Lassa virus serum or stained for glycoproteins, four virus-specific bands were apparent: the N, G1 and G2 proteins seen in purified virus, and a glycoprotein of molecular weight 72000 which probably corresponds to the envelope protein precursor (GPC) seen in other arenavirus systems. Immunoprecipitates from infected CV-1 cells labelled with [35S]methionine contained three major virus-specific proteins: the nucleocapsid protein N and proteins of 36000 and 24000 molecular weight (designated fN1 and fN2). Similar immunoprecipitates from Vero cells contained fN1 and fN2 and only very low levels of N. The polypeptides fN1 and fN2 are most probably fragments of N, since Western blots probed with anti-Lassa virus serum showed that lysis of cells in non-ionic detergent rather than SDS results in the appearance of fN2 with concomitant reduction or disappearance of N. These fragments do not exist in the intact cell, but are found as a consequence of rather specific proteolysis upon disruption under non-denaturing conditions. The proteolytic activity responsible was refractory to inhibition by phenylmethylsulphonyl fluoride, aprotinin, pepstatin A or sodium bisulphite, and was more active in Vero than in CV-1 cells.
从感染的CV - 1猴肾细胞培养液中纯化拉沙病毒，并通过聚丙烯酰胺凝胶电泳分析其结构蛋白。染色后的凝胶呈现出典型的沙粒病毒图谱，有一条分子量为60000的突出蛋白带，对应核衣壳蛋白N，还有两条较淡的宽带，分子量分别为45000和38000，即包膜糖蛋白G1和G2。通过它们与伴刀豆球蛋白A结合到分离蛋白的硝酸纤维素转移膜上的能力（“Western印迹法”），证明G1和G2是糖基化的。N和G2能结合豚鼠或人类康复血清中的抗体，但G1无活性，推测是变性所致。该技术还揭示了其他分子量为76000和68000的明显的病毒特异性小条带。当用抗拉沙病毒血清探测在SDS中裂解的感染细胞蛋白的Western印迹，或对糖蛋白进行染色时，出现了四条病毒特异性条带：纯化病毒中可见的N、G1和G2蛋白，以及一条分子量为72000的糖蛋白，它可能对应于其他沙粒病毒系统中见到的包膜蛋白前体（GPC）。用[35S]甲硫氨酸标记的感染CV - 1细胞的免疫沉淀物含有三种主要的病毒特异性蛋白：核衣壳蛋白N以及分子量为36000和24000的蛋白（分别命名为fN1和fN2）。来自Vero细胞的类似免疫沉淀物含有fN1和fN2，而N的含量极低。多肽fN1和fN2很可能是N的片段，因为用抗拉沙病毒血清探测的Western印迹显示，在非离子去污剂而非SDS中裂解细胞会导致fN2出现，同时N减少或消失。这些片段在完整细胞中不存在，而是在非变性条件下细胞破裂时因相当特异性的蛋白水解作用而产生。负责的蛋白水解活性不受苯甲基磺酰氟、抑肽酶、胃蛋白酶抑制剂A或亚硫酸氢钠抑制，且在Vero细胞中比在CV - 1细胞中更活跃。
