Herpesviral mRNP formation: identification of cellular co-factors and target specificity

病毒是专性的细胞寄生虫，因此它们的基因表达与细胞机制密切相关。疱疹病毒是一种复杂的DNA病毒，它在宿主中建立了终身的持久性，其特征是被称为潜伏期和裂解期的振荡状态。在潜伏期，病毒是沉默的。相反，裂解期的特点是病毒基因表达大量上调，并形成后代病毒。卡波西肉瘤相关疱疹病毒(KSHV)基因组全长165kb，只有25%的基因含有内含子。然而，内含子的剪接增强了mRNAs生命中的所有后续步骤。为了代替所有疱疹病毒的剪接，编码一个转录后调控因子。在KSHV中，ORF57确保形成具有出口能力的信使核糖核酸-蛋白质复合体(MRNP)。虽然已经描述了与细胞因子的各种相互作用，但ORF57靶向病毒RNA的机制仍然不清楚。ORF57对RNA的低亲和力和大部分无序区使分析变得困难。我们采取了一种不同的方法，并问道：病毒RNA上导致ORF57依赖的决定因素是什么？我们根据KSHV裂解RNA的序列组成开发了一个独特的病毒mRNP编码的概念。我们假设，特定细胞RNA结合蛋白(RBPs)的基序编码在病毒RNA序列中，可能嵌入在二级结构中。为了确定ORF57的假定细胞伙伴，我们将使用最近描述的一种名为“LNA mRNP捕获”的mRNP沉淀技术。该方法能够通过寡核苷酸探针的选择来纯化特定的mRNPs。作为另一种假设，我们将ORF57对RNA的识别与细菌的RBP进行比较，后者依赖于“隐藏的特异性”，而不是保守的基序。同时，Mandel-Gutfreund实验室将开发新的算法和机器学习方法，以在依赖于ORF57和不依赖于ORF57的基因中寻找序列属性。此外，还将开发一种新的非结构蛋白结构域预测工具，以检索有关ORF57最可能参与RNA识别的N-末端区域的更多信息。最后，我们计划通过DMSseq来确定KSHV裂解RNA的体内结构，DMSseq是结构探测和深度测序的全基因组适用的组合。结构信息将被整合到算法中。这一结果也是在体外探测假定的ORF57识别位点的基础。最后，使用假定的靶点的邻近连接试验将证明ORF57在体内结合。所获得的结果将扩展到其他疱疹病毒。到目前为止，我们已经可以证明我们的一个报告系统也被Epstein-Barr病毒和水痘带状疱疹病毒的ORF57同源物激活。这些数据还将有助于进一步破译细胞mRNP代码，这是后基因组时代的一项重要任务。

Viruses are obligate cellular parasites and thus their gene expression is intimately linked to cellular machineries. Herpesviruses are complex DNA viruses, which establish a lifelong persistence in their hosts characterized by oscillating states named latent and lytic phases. During the latent phase the virus is silent. In contrast, the lytic phase is characterized by a massive up-regulation of viral gene expression and the formation of progeny virus. The genome of Kaposi’s sarcoma-associated herpesvirus (KSHV) is 165 kb in length and only 25% of its genes contain introns. However, splicing of introns enhances all later steps in the life of mRNAs. To substitute for splicing all herpesviruses encode a post-transcriptional regulator. In KSHV ORF57 ensures the formation of an export-competent mRNA-protein complex (mRNP). While various interactions with cellular factors have been described, the ORF57 targeting mechanism for viral RNA remains obscure. The low affinity of ORF57 to RNA and the large portion of disordered regions made analyses difficult. We took a different approach and asked: what are the determinants on the viral RNA leading to ORF57-dependency? We developed the concept of a distinct viral mRNP code based on the sequence composition of KSHV lytic RNAs. We hypothesize that motifs for specific cellular RNA-binding proteins (RBPs) are encoded in the viral RNA sequence possibly embedded in secondary structures. To identify the putative cellular partner of ORF57, we will use a recently described mRNP precipitation technique called 'LNA mRNP capture'. The method is able to purify specific mRNPs based on selection via oligonucleotides probes. As an alternative hypothesis we compare the RNA recognition by ORF57 to a bacterial RBP, which relies on a 'hidden specificity' rather than conserved motifs. In parallel, the Mandel-Gutfreund laboratory will develop new algorithms and machine learning approaches to find sequence attributes in ORF57-dependent vs. non-dependent genes. In addition, a new prediction tool for unstructured protein domains will be developed to retrieve more information on the N-terminal region of ORF57 most probably involved in RNA recognition. Finally, we plan to determine the in-vivo structure of KSHV lytic RNAs by DMSseq, a genome-wide applicable combination of structural probing and deep sequencing. The structural information will be integrated into the algorithms. This output is also the basis for probing putative ORF57 recognition sites in-vitro. In the end, a proximity ligation assay using putative target sites will demonstrate in-vivo binding of ORF57. The results obtained will be expanded to other herpesviruses. So far we could already demonstrate that one of our reporter systems is also activated by the ORF57 homologues of Epstein-Barr virus and Varicella Zoster Virus. The data will also help to further decipher the cellular mRNP code, an important task in the post-genome era.