Recognition of RNA Cap in the Wesselsbron Virus NS5 Methyltransferase Domain: Implications for RNA-Capping Mechanisms in Flavivirus
Recognition of RNA Cap in the Wesselsbron Virus NS5 Methyltransferase Domain: Implications for RNA-Capping Mechanisms in Flavivirus
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DOI：
10.1016/j.jmb.2008.10.028
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发表时间：
2009-01-09
影响因子：
5.6
通讯作者：
Bolognesi, Martino
中科院分区：
文献类型：
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作者：
Bollati, Michela;Milani, Mario;Bolognesi, Martino
mRNA 加帽过程首先通过 RNA 三磷酸酶将 5'-三磷酸末端转化为 5'-二磷酸，然后通过鸟苷酸转移酶在 5'-5' 磷酸二酯键中添加单磷酸鸟苷单元。甲基转移酶参与该过程的第三步，将甲基从 S-腺苷-L-甲硫氨酸转移到 N7-鸟嘌呤（帽 0）和第一个 RNA 核苷酸的核糖 2'OH 基团（帽 1）；加帽对于 mRNA 稳定性和正确复制至关重要。在黄病毒属中，N7-甲基转移酶和2'O-甲基转移酶活性最近与病毒NS5蛋白的N末端结构域相关。为了进一步表征支持加帽的一系列酶促反应，我们分析了韦塞尔斯布朗病毒甲基转移酶与 S-腺苷-L-甲硫氨酸辅因子、S-腺苷-L-同型半胱氨酸（甲基化反应的产物）、Sinefungin（酶辅因子的分子类似物）和三种不同帽类似物（GpppG、(N7Me)GpppG 和(N7Me)GpppA)。结构结果以及其他黄病毒甲基转移酶的结构结果表明，加帽的 RNA 类似物均与 RNA 高亲和力结合位点结合。然而，酶和 RNA 链的第一个核苷酸之间缺乏特异性相互作用，这表明在帽后需要最少数量的核苷酸来加强蛋白质/RNA 相互作用。我们的数据还表明，与三磷酸鸟苷一起孵育后，韦塞尔斯布朗病毒甲基转移酶显示出与残基 Lys28 共价结合的鸟苷单磷酸分子，暗示鸟嘌呤基团转移到 ppRNA 的可能影响。在 N7-甲基转移酶和 2'O-甲基转移酶活性模型的背景下讨论了获得的韦塞尔斯布朗病毒甲基转移酶复合物的结构。 (C) 2008 Elsevier Ltd. 保留所有权利。
The mRNA-capping process starts with the conversion of a 5'-triphosphate end into a 5'-diphosphate by an RNA triphosphatase, followed by the addition of a guanosine monophosphate unit in a 5'-5' phosphodiester bond by a guanylyltransferase. Methyltransferases are involved in the third step of the process, transferring a methyl group from S-adenosyl-L-methionine to N7-guanine (cap 0) and to the ribose 2'OH group (cap 1) of the first RNA nucleotide; capping is essential for mRNA stability and proper replication. In the genus Flavivirus, N7-methyltransferase and 2'O-methyltransferase activities have been recently associated with the N-terminal domain of the viral NS5 protein. In order to further characterize the series of enzymatic reactions that support capping, we analyzed the crystal structures of Wesselsbron virus methyltransferase in complex with the S-adenosyl-L-methionine cofactor, S-adenosyl-L-homocysteine (the product of the methylation reaction), Sinefungin (a molecular analogue of the enzyme cofactor), and three different cap analogues (GpppG, (N7Me)GpppG, and (N7Me)GpppA). The structural results, together with those on other flaviviral methyltransferases, show that the capped RNA analogues all bind to an RNA high-affinity binding site. However, lack of specific interactions between the enzyme and the first nucleotide of the RNA chain suggests the requirement of a minimal number of nucleotides following the cap to strengthen protein/RNA interaction. Our data also show that, following incubation with guanosine triphosphate, Wesselsbron virus methyltransferase displays a guanosine monophosphate molecule covalently bound to residue Lys28, hinting at possible implications for the transfer of a guanine group to ppRNA. The structures of the Wesselsbron virus methyltransferase complexes obtained are discussed in the context of a model for N7-methyltransferase and 2'O-methyltransferase activities. (C) 2008 Elsevier Ltd. All rights reserved.