Development of specific dengue virus 2′-O- and N7-methyltransferase assays for antiviral drug screening
Development of specific dengue virus 2′-O- and N7-methyltransferase assays for antiviral drug screening
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DOI：
10.1016/j.antiviral.2013.06.001
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发表时间：
2013-09-01
影响因子：
7.6
通讯作者：
Decroly, E.
中科院分区：
文献类型：
--
作者：
Barral, K.;Sallamand, C.;Decroly, E.
登革热病毒 (DENV) 蛋白 NS5 携带两种 mRNA 帽甲基转移酶 (MTase) 活性，参与病毒 5' 端帽子结构 (7Me)GpppA(2'OMe)-RNA 的合成。 mRNA。帽鸟嘌呤 N7 位的甲基化（N7-MTase，(7Me)GpppA-RNA）对于病毒复制至关重要。大规模合成长帽 RNA 的技术限制阻碍了鉴定 N7-MTase 特异性抑制剂的高通量方法的开发。在这项工作中，我们描述了一种通过连接两个 RNA 片段来生成这种加帽 RNA 的有效方法，GppPA(2'OMe)-RNA(74)。然后，我们使用 GpppA(2'OMe)-RNA(74) 作为底物来评估 DENV N7-MTase 活性并开发稳健且特异的活性测定。我们应用相同的连接程序生成 (7Me)GpppA-RNA(74)，以表征特异于长帽 RNA 的 DENV 2'-O-MTase 活性。接下来，我们比较了 18 个分子的 N7- 和 2'-O-MTase 抑制效果，之前提出会影响 MTase 活性。这些实验可以验证一种快速、灵敏的方法，该方法易于适用于抗黄病毒药物开发中的高通量抑制剂筛选。 (C) 2013 Elsevier B.V. 保留所有权利。
Dengue virus (DENV) protein NS5 carries two mRNA cap methyltransferase (MTase) activities involved in the synthesis of a cap structure, (7Me)GpppA(2'OMe)-RNA, at the 5'-end of the viral. mRNA. The methylation of the cap guanine at its N7-position (N7-MTase, (7Me)GpppA-RNA) is essential for viral replication. The development of high throughput methods to identify specific inhibitors of N7-MTase is hampered by technical limitations in the large scale synthesis of long capped RNAs. In this work, we describe an efficient method to generate such capped RNA, GppPA(2'OMe)-RNA(74), by ligation of two RNA fragments. Then, we use GpppA(2'OMe)-RNA(74) as a substrate to assess DENV N7-MTase activity and to develop a robust and specific activity assay. We applied the same ligation procedure to generate (7Me)GpppA-RNA(74) in order to characterize the DENV 2'-O-MTase activity specifically on long capped RNA.We next compared the N7- and 2'-O-MTase inhibition effect of 18 molecules, previously proposed to affect MTase activities. These experiments allow the validation of a rapid and sensitive method easily adaptable for high-throughput inhibitor screening in anti-flaviviral drug development. (C) 2013 Elsevier B.V. All rights reserved.