Arginine methylation regulates the function of a key herpesvirus nucleocytoplasmic shuttle protein

MRNA是在细胞核中转录的，必须从细胞核输出到细胞质才能翻译成蛋白质。在细胞核中，RNA在输出之前必须经历一系列处理事件，这些事件由许多多蛋白复合体执行。然而，这些复合体是如何被招募到RNA上的，以及每个多蛋白复合体是如何被调节以在正确的时间执行其特定功能的，目前尚不清楚。在这里，我们的目标是使用病毒模型来研究这些RNA结合多蛋白复合体之一的功能是如何调节的。我们以前已经证明了疱疹病毒ORF57蛋白对于将疱疹病毒mRNA从细胞核输出到翻译是重要的。ORF57的功能是与疱疹病毒RNA结合，特别是将细胞蛋白招募到病毒RNA上，这对病毒的正确处理和出口是必不可少的。该系统唯一地鉴定了称为hTREX的多蛋白质复合体是ORF57需要的唯一细胞蛋白质复合体，用于疱疹病毒mRNA的核输出。此外，我们已经证明，一旦hTREX与ORF57结合到疱疹病毒mRNA上，整个复合体就会通过细胞核内称为核仁的结构域运输，然后从细胞核输出到细胞质。因此，ORF57是一种多功能蛋白，它顺序地与病毒RNA结合，然后将hTREX招募到病毒RNA上，然后将这个复合体运输到核仁，然后允许复合体离开细胞核。但是，是什么告诉ORF57以正确的方式执行其许多功能尚不清楚。蛋白质的这种功能多样性可以通过对蛋白质进行不同的化学修饰来控制，例如在蛋白质上添加或去除磷酸盐、醋酸酯或甲基。有趣的是，我们已经证明疱疹病毒ORF57蛋白经历了一次这样的修饰。我们已经证明ORF57可以在精氨酸残基上甲基化。因此，我们的目标是确定添加或去掉甲基是否会影响ORF57的S功能，从而调节负责从细胞核输出病毒基因的多蛋白复合体。为了评估甲基化的作用，我们将首先确定ORF57的哪些残基是甲基化的。然后我们将突变这些基因，这样ORF57就不会发生甲基化。这将使我们能够评估它对ORF57蛋白功能的影响。其次，我们将确定哪些细胞酶负责对病毒ORF57蛋白进行翻译后修饰。最后，我们有初步数据表明，去除ORF57上的甲基(或去甲基化ORF57)是告知ORF57退出原子核的触发因素。我们认为这发生在核仁中，我们将确定核仁中的哪种酶执行这一功能。这些数据将更好地了解多蛋白复合体如何发挥作用，使RNA能够离开细胞核，也将更好地了解病毒如何发挥作用，以便在长期内识别新的抗病毒靶点。

mRNA is transcribed in the nucleus and must be exported from the nucleus into the cytoplasm to get translated into a protein. In the nucleus, the RNA has to undergo a series of processing events prior to export and these are performed by a number of multi-protein complexes. However, how these complexes are recruited to the RNA and how each multi-protein complex is regulated to perform its specific function at the correct time is unknown. Herein, we aim to investigate how the function of one of these RNA binding multi-protein complexes is regulated using a viral model. We have previously demonstrated that the herpesvirus ORF57 protein is important for exporting herpesvirus mRNA out of the nucleus to get translated. ORF57 functions by binding to the herpesvirus RNA and specifically recruiting cellular proteins onto the viral RNA, which are essential for its correct processing and export. This system has uniquely identified that a multi-protein complex called hTREX is the only cellular protein complex which is required by ORF57 for herpesvirus mRNA nuclear export. Moreover, we have demonstrated that once hTREX is bound by ORF57 to the herpesvirus mRNA, the whole complex is transported through a domain within the nucleus called the nucleolus, before it is exported from the nucleus into the cytoplasm. Therefore, ORF57 is a multifunctional protein which sequentially, binds the viral RNA, then recruits hTREX to the viral RNA, then transports this complex to the nucleolus, then allows the complex to exit the nucleus. But what tells ORF57 to perform its many functions in the correct manner is unknown. This functional diversity of a protein can be controlled by different chemical modifications to the protein, such as adding or taking away a phosphate, acetate or methyl group on the protein. Interestingly, we have demonstrated that the herpesvirus ORF57 protein undergoes one such modification. We have shown that ORF57 can be methylated at arginine residues. Therefore, we aim to determine if adding or taking away methyl groups affects ORF57's function and in turn regulates the multi-protein complex responsible for exporting viral mRNA from the nucleus. To assess the role of methylation we will first identify which residues of ORF57 are methylated. We will then mutate these, so that ORF57 cannnot undergo methylation. This will then allow us to assess what effect it has upon the functioning of the ORF57 protein. Secondly, we will identify which cellular enzymes are responsible for performing this post-translational modification on the viral ORF57 protein. Finally, we have preliminary data to suggest that taking away the methyl group on ORF57 (or demethylating ORF57), is the trigger to tell ORF57 to exit the nucleus. We believe this happens in the nucleolus and we will identify which enzyme in the nucleolus performs this function. These data will provide a better understanding of how multi-protein complexes function to enable RNAs to exit the nucleus and also provide a better understanding of how viruses function in order to identify new antiviral targets in the longer term.