Ramadevi N, Burroughs N J, Mertens P P, Jones I M, Roy P
Natural Environment Research Council Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, United Kingdom.
Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13537-42. doi: 10.1073/pnas.95.23.13537.
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 core 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 for 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 m7GpppGm 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.
蓝舌病毒（BTV）的核心是一种多酶复合物，除病毒基因组外，它由两种主要蛋白质（VP7和VP3）和三种次要蛋白质（VP1、VP4和VP6）组成。该核心具有转录活性，能产生带帽的mRNA，所有BTV蛋白质都由此翻译而来，但每个核心成分在整个反应过程中的相对作用仍不清楚。此前我们表明，76 kDa的VP4蛋白具有鸟苷酸转移酶活性，这是RNA加帽反应的必要组成部分。在此，通过使用高度纯化（>95%）的VP4和含有VP4的合成核心样颗粒，我们研究了该蛋白在多大程度上还负责与帽形成相关的其他活性。我们发现，在S-腺苷-L-甲硫氨酸存在的情况下，VP4能催化未甲基化的GpppG或体外产生的无帽BTV RNA转录本转化为m7GpppGm。通过HPLC对反应的甲基化产物进行分析，确定了与VP4相关的1型和2型甲基转移酶活性，表明完整的BTV加帽反应与这一种蛋白质有关。
