Mechanism of transcription of Paramyxovirus genome : Purification and characterization of host factors

仙台病毒（Sendai virus，SeV）是副粘病毒科的一个成员，其RNA基因组为负链，约有15个酶。为了阐明SeV的转录和复制机制，我们使用纯化的病毒颗粒开发了一个有效和可靠的体外转录系统。我们以前已经表明，在体外mRNA合成的SeV几乎完全依赖于细胞蛋白质（宿主因子）的添加。在本研究中，我们对宿主因子的活性进行了进一步的纯化和表征，并获得了以下结果。1)我们纯化的宿主因子的活动，刺激在体外SeV mRNA的合成，从牛脑，并发现该活动由四个互补的蛋白质因子，微管蛋白，磷酸甘油酸激酶，烯醇化酶，和身份不明的p24。2)我们发现，微管蛋白参与了转录起始复合物的形成，通过从RNP中去除病毒M蛋白，并将其自身整合到复合物中。另一方面，磷酸甘油酸激酶、烯醇化酶和p24被发现在RNA链延伸步骤中起作用。此外，3）我们证明了重组病毒L蛋白表现出帽甲基化酶（mRNA鸟嘌呤-7-甲基转移酶）活性。

Sendai virus (SeV), a member of Paramyxovirus family, contains a monopartite negative strand RNA genome of approximately 15 kilobases. To elucidate the mechanism of transcription and replication of SeV, we developed an efficient and faithful in vitro transcription system using purified virus particles. We have previously shown that in vitro mRNA synthesis of SeV is almost entirely dependent on the addition of cellular proteins (host factors). In the present study, we further furified and characterized the host factor activity, and obtained the following results. 1) We purified the host factor activity, which stimulates in vitro SeV mRNA synthesis, from bovine brain, and found that the activity consists of four complementary protein factors, tubulin, phosphoglycerate kinase, enolase, and unidentified p24. 2) We showed that tubulin is involved in a transcription initiation complex formation by removing the viral M protein from the RNP, and by being integrated itself into the complex. On the other hand, phosphoglycerate kinase, enolase, and p24 are found to act at RNA shain elongation step. Furthermore, 3) we demonstrated that a recombinant viral L protein exhibits the cap methylase (mRNA guanine-7-methyltransferase) activity.