Hamaguchi M, Yoshida T, Nishikawa K, Naruse H, Nagai Y
Virology. 1983 Jul 15;128(1):105-17. doi: 10.1016/0042-6822(83)90322-7.
Virions of Newcastle disease virus (NDV) were disrupted with Triton X-100 in the presence of high salt and nucleocapsids were isolated by ultracentrifugation. The nucleocapsids had very low transcriptase activity and contained only NP as a prominent protein constituent, the bulk of L and P proteins not being retained. The L and P proteins were isolated by sequential treatment of the virions with low- and high-salt detergent followed twice by successive chromatography on phosphocellulose column and examined for their effect on RNA synthesis in a standard transcriptase system using the nucleocapsids as template. When both L and P proteins were added to the template, the RNA synthetic activity was greatly stimulated. P protein alone could not enhance but rather suppressed the activity. L protein exhibited stimulation to some extent but due to residual small amount of P protein in both L protein fraction and the template it has not been elucidated whether L protein could function as a polymerase by itself. These results indicate that both L and P proteins are required to reconstitute a fully active transcriptive complex with a functional template. Attempts have been made to isolate intracellular transcriptive complex from NDV-infected MDBK cells and to determine the protein species involved. The active complex has been recovered neither from cytoplasmic extract obtained by hypotonic disruption nor from Triton X-100 soluble fraction of the cells. However, we could isolate the complex from an extract by double detergents (Tween 40 and deoxycholate) solubilization. The complex contained L, P, and NP as virus specific proteins and several cellular proteins. These results support the concept that both L and P proteins are required for NDV-RNA synthesis and suggest further that the intracellular transcriptive complex may be associated with some cellular structure resistant to Triton X-100 but sensitive to the double detergents, presumably cytoskeletal frame work.
新城疫病毒（NDV）的病毒粒子在高盐存在的情况下用Triton X - 100进行裂解，然后通过超速离心分离核衣壳。这些核衣壳具有非常低的转录酶活性，并且仅含有NP作为主要的蛋白质成分，大部分L和P蛋白未被保留。通过用低盐和高盐洗涤剂依次处理病毒粒子，然后在磷酸纤维素柱上连续进行两次色谱法，分离出L和P蛋白，并在以核衣壳为模板的标准转录酶系统中检测它们对RNA合成的影响。当L和P蛋白都添加到模板中时，RNA合成活性受到极大刺激。单独的P蛋白不能增强反而抑制活性。L蛋白在一定程度上表现出刺激作用，但由于L蛋白组分和模板中都残留有少量P蛋白，因此尚未阐明L蛋白自身是否能作为聚合酶发挥作用。这些结果表明，L和P蛋白都需要与功能性模板重新构建一个完全活跃的转录复合物。已尝试从感染NDV的MDBK细胞中分离细胞内转录复合物，并确定其中涉及的蛋白质种类。无论是通过低渗裂解获得的细胞质提取物，还是细胞的Triton X - 100可溶部分，都未回收活性复合物。然而，我们可以通过双去污剂（吐温40和脱氧胆酸盐）溶解从提取物中分离出该复合物。该复合物包含L、P和NP作为病毒特异性蛋白以及几种细胞蛋白。这些结果支持了NDV - RNA合成需要L和P蛋白的观点，并进一步表明细胞内转录复合物可能与一些对Triton X - 100有抗性但对双去污剂敏感的细胞结构相关，推测是细胞骨架框架。
