Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), Aix-Marseille Université, UM63 IRD 198, IHU-Méditerranée Infection, Marseille, France.
Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), Aix-Marseille Université, UM63 IRD 198, IHU-Méditerranée Infection, Marseille, France
J Virol. 2018 Apr 27;92(10). doi: 10.1128/JVI.00337-18. Print 2018 May 15.
The mimivirus is the first giant virus ever described, with a 1.2-Mb genome which encodes 979 proteins, including central components of the translation apparatus. One of these proteins, R458, was predicted to initiate translation, although its specific role remains unknown. We silenced the R458 gene using small interfering RNA (siRNA) and compared levels of viral fitness and protein expression in silenced versus wild-type mimivirus. Silencing decreased the growth rate, but viral particle production at the end of the viral cycle was unaffected. A comparative proteomic approach using two-dimensional difference-in-gel electrophoresis (2D-DIGE) revealed deregulation of the expression of 32 proteins in silenced mimivirus, which were defined as up- or downregulated. Besides revealing proteins with unknown functions, silencing R458 also revealed deregulation in proteins associated with viral particle structures, transcriptional machinery, oxidative pathways, modification of proteins/lipids, and DNA topology/repair. Most of these proteins belong to genes transcribed at the end of the viral cycle. Overall, our data suggest that the R458 protein regulates the expression of mimivirus proteins and, thus, that mimivirus translational proteins may not be strictly redundant in relation to those from the amoeba host. As is the case for eukaryotic initiation factor 4a (eIF4a), the R458 protein is the prototypical member of the ATP-dependent DEAD box RNA helicase mechanism. We suggest that the R458 protein is required to unwind the secondary structures at the 5' ends of mRNAs and to bind the mRNA to the ribosome, making it possible to scan for the start codon. These data are the first experimental evidence of mimivirus translation-related genes, predicted to initiate protein biosynthesis. The presence in the genome of a mimivirus of genes coding for many translational processes, with the exception of ribosome constituents, has been the subject of debate since its discovery in 2003. In this work, we focused on the R458 mimivirus gene, predicted to initiate protein biosynthesis. After silencing was performed, we observed that it has no major effect on mimivirus multiplication but that it affects protein expression and fitness. This suggests that it is effectively used by mimivirus during its developmental cycle. Until large-scale genetic manipulation of giant viruses becomes possible, the silencing strategy used here on mimivirus translation-related factors will open the way to understanding the functions of these translational genes.
拟菌病毒是首个被描述的巨型病毒，其基因组大小为 1.2Mb，编码 979 种蛋白质，其中包括翻译装置的核心组件。这些蛋白质之一的 R458 被预测为起始翻译，尽管其具体作用仍不清楚。我们使用小干扰 RNA（siRNA）沉默了 R458 基因，并比较了沉默型和野生型拟菌病毒的病毒适应性和蛋白质表达水平。沉默降低了生长速度，但病毒周期结束时的病毒粒子产量不受影响。使用二维差异凝胶电泳（2D-DIGE）的比较蛋白质组学方法揭示了沉默型拟菌病毒中 32 种蛋白质的表达失调，这些蛋白质被定义为上调或下调。除了揭示具有未知功能的蛋白质外，沉默 R458 还揭示了与病毒粒子结构、转录机制、氧化途径、蛋白质/脂质修饰以及 DNA 拓扑/修复相关的蛋白质的失调。这些蛋白质大多数属于在病毒周期结束时转录的基因。总的来说，我们的数据表明 R458 蛋白调节拟菌病毒蛋白的表达，因此，拟菌病毒的翻译蛋白与变形虫宿主的翻译蛋白可能不是严格冗余的。与真核起始因子 4a（eIF4a）一样，R458 蛋白是 ATP 依赖性 DEAD 盒 RNA 解旋酶机制的典型成员。我们认为，R458 蛋白需要解开 mRNA 5' 端的二级结构，并将 mRNA 与核糖体结合，从而能够扫描起始密码子。这些数据是首次证明拟菌病毒翻译相关基因参与蛋白质生物合成的实验证据。自 2003 年发现以来，拟菌病毒基因组中存在许多翻译过程的编码基因，而核糖体成分除外，这一直是争论的主题。在这项工作中，我们专注于 R458 拟菌病毒基因，该基因预测为蛋白质生物合成的起始。沉默后，我们观察到它对拟菌病毒的繁殖没有重大影响，但它会影响蛋白质表达和适应性。这表明它在拟菌病毒的发育周期中被有效利用。在大规模对巨型病毒进行遗传操作成为可能之前，我们在此对拟菌病毒翻译相关因子进行的沉默策略将为理解这些翻译基因的功能开辟道路。
