Capping and methylation of mRNA by purified recombinant VP4 protein of bluetongue virus
Capping and methylation of mRNA by purified recombinant VP4 protein of bluetongue virus
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DOI：
10.1073/pnas.95.23.13537
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发表时间：
1998-11-10
影响因子：
11.1
通讯作者：
Roy, P
中科院分区：
文献类型：
--
作者：
Ramadevi, N;Burroughs, NJ;Roy, P
蓝舌病病毒（BTV）的核心是除病毒基因组外还由两种主要蛋白（VP7和VP3）和三种次要蛋白（VP1、VP4和VP6）组成的多酶复合物。核心具有转录活性，并产生加帽的 mRNA，所有 BTV 蛋白均由该 mRNA 翻译，但每个护理成分在整个反应过程中的相对作用仍不清楚。之前我们表明 76-kDa VP4 蛋白具有鸟苷基转移酶活性，这是 RNA 加帽反应的必要部分。在这里，通过使用高纯度 (>95%) VP4 和含有 VP4 的合成核状颗粒，我们研究了该蛋白质在多大程度上也负责与帽子形成相关的其他活动。我们发现，在 S-腺苷-L-甲硫氨酸存在的情况下，VP4 催化未甲基化的 GpppG 或体外产生的未加帽的 BTV RNA 转录物转化为 m(7)GpppGm。通过 HPLC 对反应的甲基化产物进行分析，鉴定出与 VP4 相关的 1 型和 2 型甲基转移酶活性，证明完整的 BTV 加帽反应与该蛋白质相关。
The core of bluetongue virus (BTV) is a multienzyme complex composed of two major proteins (VP7 and VP3) and three minor proteins (VP1, VP4, and VP6) in addition to the viral genome. The core is transcriptionally active and produces capped mRNA from which all BTV proteins are translated, but the relative role of each care component in the overall reaction process remains unclear, Previously we showed that the 76-kDa VP4 protein possesses guanylyltransferase activity, a necessary part of the RNA capping reaction. Here, through the use of highly purified (>95%) VP4 and synthetic core-like particles containing VP4, we have investigated the extent to which this protein is also responsible fur other activities associated with cap formation. We show that VP4 catalyzes the conversion of unmethylated GpppG or in vitro-produced uncapped BTV RNA transcripts to m(7)GpppGm in the presence of S-adenosyl-L-methionine. Analysis of the methylated products of the reaction by HPLC identified both methyltransferase type 1 and type 2 activities associated with VP4, demonstrating that the complete BTV capping reaction is associated with this one protein.