RNA polymerase of vesicular stomatitis virus specifically associates with translation elongation factor-1 αβγ for its activity
RNA polymerase of vesicular stomatitis virus specifically associates with translation elongation factor-1 αβγ for its activity
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DOI：
10.1073/pnas.95.4.1449
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发表时间：
1998-02-17
影响因子：
11.1
通讯作者：
Banerjee, AK
中科院分区：
文献类型：
--
作者：
Das, T;Mathur, M;Banerjee, AK
依赖于RNA的RNA聚合酶被包装在纯化的水泡性口炎病毒的病毒粒子中，水泡性口炎病毒是一种非节段负链RNA病毒，它在体外和体内都能将基因组RNA转录成mRNAs。该聚合酶由两个病毒编码的多肽组成：一个是大蛋白L(240kDa)，另一个是磷酸蛋白P(29kDa)。最近，我们用表达L基因的重组杆状病毒感染昆虫细胞，获得了具有生物活性的L蛋白。在从Sf21细胞中纯化L蛋白的过程中，除了获得一个具有活性的L组分外，我们还获得了一个需要未感染的昆虫细胞提取液来恢复其活性的非活性组分。细胞因子现在已经被提纯、鉴定，并被证明是蛋白质合成延伸因子EF-1的β和伽马亚基。我们还证明了EF-1的α亚基与非活性部分中的L蛋白紧密结合，而β伽马亚基与L(α)复合体有关。对L(α)的进一步纯化表明，该复合体具有部分活性，并被从Sf21细胞中纯化的β-伽马亚基显著刺激。一种可能的抑制物(S)似乎与阻止L(阿尔法)活性的非活性部分一起洗脱。纯化的病毒粒子还包装了EF-1的所有三个亚基。这些发现与QβRNA噬菌体有惊人的相似性，QβRNA噬菌体也与EF-1的细菌同源物具有复制酶功能，暗示这些宿主蛋白与RNA病毒依赖RNA的RNA聚合酶之间可能存在进化关系。
An RNA-dependent RNA polymerase is packaged within the virions of purified vesicular stomatitis virus, a nonsegmented negative-strand RNA virus, which carries out transcription of the genome RNA into mRNAs both in vitro and in vivo. The RNA polymerase is composed of two virally encoded polypeptides: a large protein L (240 kDa) and a phosphoprotein P (29 kDa). Recently, we obtained biologically active L protein from insect cells following infection by a recombinant baculovirus expressing L gene. During purification of the L protein from Sf21 cells, we obtained in addition to an active L fraction an inactive fraction that required uninfected insect cell extract to restore its activity. The cellular factors have now been purified, characterized, and shown to be beta and gamma subunits of the protein synthesis elongation factor EF-1. We also demonstrate that the alpha subunit of EF-1 remains tightly bound to the L protein in the inactive fraction and beta gamma subunits associate with the L(alpha) complex. Further purification of L(alpha) from the inactive fraction revealed that the complex is partially active and is significantly stimulated by the addition of beta gamma subunits purified from Sf21 cells. A putative inhibitor(s) appears to co-elute in the inactive fraction that blocked the L(alpha) activity. The purified virions also package all three subunits of EF-1. These findings have a striking similarity with Q beta RNA phage, which also associates with the bacterial homologue of EF-1 for its replicase function, implicating a possible evolutionary relationship between these host proteins and the RNA-dependent RNA polymerase of RNA viruses.