The giant Mimivirus is a member of the nucleocytoplasmic large DNA viruses (NCLDV), a group of diverse viruses that contain double-stranded DNA (dsDNA) genomes that replicate primarily in eukaryotic hosts. Two members of the NCLDV, Vaccinia Virus (VACV) and African Swine Fever Virus (ASFV), both synthesize Nudix enzymes that have been shown to decap mRNA, a process thought to accelerate viral and host mRNA turnover and promote the shutoff of host protein synthesis. Mimivirus encodes two Nudix enzymes in its genome, denoted as L375 and L534. Importantly, L375 exhibits sequence similarity to ASFV-DP and eukaryotic Dcp2, two Nudix enzymes shown to possess mRNA decapping activity. In this work, we demonstrate that recombinant Mimivirus L375 cleaves the 5’ m 7 GpppN mRNA cap, releasing m 7 GDP as a product. L375 did not significantly cleave mRNAs containing an unmethylated 5’GpppN cap, indicating that this enzyme specifically hydrolyzes methylated-capped transcripts. A point mutation in the L375 Nudix motif completely eliminated cap hydrolysis, showing that decapping activity is dependent on this motif. Addition of methylated cap derivatives or uncapped RNA inhibited L375 decapping activity, suggesting that L375 recognizes its substrate through interaction with both the mRNA cap and RNA body.

中文翻译：

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Mimivirus L375 Nudix酶水解5'mRNA帽

巨型模仿病毒是核质大DNA病毒（NCLDV）的成员，NCLDV是一组包含主要在真核宿主中复制的双链DNA（dsDNA）基因组的多种病毒。NCLDV的两个成员，牛痘病毒（VACV）和非洲猪瘟病毒（ASFV）都合成了已显示能使mRNA脱帽的Nudix酶，该过程被认为可以加速病毒和宿主mRNA的更新并促进宿主蛋白合成的关闭。 。拟病毒在其基因组中编码两种Nudix酶，分别称为L375和L534。重要的是，L375与ASFV-DP和真核Dcp2（两个显示具有mRNA脱盖活性的Nudix酶）具有序列相似性。在这项工作中，我们证明重组Mimivirus L375裂解5'm 7 GpppN mRNA帽，释放m 7 GDP产品。L375不能显着切割含有未甲基化的5'GpppN帽的mRNA，表明该酶特异性水解了甲基化加帽的转录物。L375 Nudix基序中的点突变完全消除了帽水解，表明脱盖活性取决于该基序。添加甲基化的帽衍生物或未封端的RNA会抑制L375的去盖活性，这表明L375通过与mRNA帽和RNA体的相互作用来识别其底物。