Structural and Functional Analysis of Methylation and 5′-RNA Sequence Requirements of Short Capped RNAs by the Methyltransferase Domain of Dengue Virus NS5

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• 作者：Marie-Pierre Egloff ; Etienne Decroly ; Hé ; lè ; ne Malet ; Barbara Selisko ; Delphine Benarroch ; Franç ; ois Ferron ; Bruno Canard
• 关键词：methyltransferase ; capping ; flavivirus ; dengue ; guanylyltransferase
• 刊名：Journal of Molecular Biology
• 出版年：2007
• 出版时间：21 September 2007
• 年：2007
• 卷：372
• 期：3
• 页码：723-736
• 全文大小：1640 K

文摘

The N-terminal 33 kDa domain of non-structural protein 5 (NS5) of dengue virus (DV), named NS5MTase_DV, is involved in two of four steps required for the formation of the viral mRNA cap ^7MeGpppA_2′OMe, the guanine-N7 and the adenosine-2′O methylation. Its S-adenosyl-l-methionine (AdoMet) dependent 2′O-methyltransferase (MTase) activity has been shown on capped ^7Me±GpppAC_n RNAs. Here we report structural and binding studies using cap analogues and capped RNAs. We have solved five crystal structures at 1.8 Å to 2.8 Å resolution of NS5MTase_DV in complex with cap analogues and the co-product of methylation S-adenosyl-l-homocysteine (AdoHcy). The cap analogues can adopt several conformations. The guanosine moiety of all cap analogues occupies a GTP–binding site identified earlier, indicating that GTP and cap share the same binding site. Accordingly, we show that binding of ^7MeGpppAC₄ and ^7MeGpppAC₅ RNAs is inhibited in the presence of GTP, ^7MeGTP and ^7MeGpppA but not by ATP. This particular position of the cap is in accordance with the 2′O-methylation step. A model was generated of a ternary 2′O-methylation complex of NS5MTase_DV, ^7MeGpppA and AdoMet. RNA-binding increased when ^7Me±GpppAGC_n-1 starting with the consensus sequence GpppAG, was used instead of ^7Me±GpppAC_n. In the NS5MTase_DV–GpppA complex the cap analogue adopts a folded, stacked conformation uniquely possible when adenine is the first transcribed nucleotide at the 5′ end of nascent RNA, as it is the case in all flaviviruses. This conformation cannot be a functional intermediate of methylation, since both the guanine-N7 and adenosine-2′O positions are too far away from AdoMet. We hypothesize that this conformation mimics the reaction product of a yet-to-be-demonstrated guanylyltransferase activity. A putative Flavivirus RNA capping pathway is proposed combining the different steps where the NS5MTase domain is involved.