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

疱疹病毒 mRNP 形成：细胞辅助因子和靶标特异性的鉴定

• 批准号: 403670311
• 负责人: Privatdozent Dr. Jens Bohne
• 金额: --
• 依托单位: Institut für Virologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/403670311?language=en
• 关键词: Herpesviral; mRNP; formation; identification; cellular

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.

病毒是专性细胞寄生虫，因此它们的基因表达与细胞机制密切相关。疱疹病毒是复杂的DNA病毒，其在其宿主中建立终身持久性，其特征在于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，裂解期的特征在于病毒基因表达的大量上调和子代病毒的形成。卡波西肉瘤相关疱疹病毒（KSHV）的基因组长度为165 kb，只有25%的基因含有内含子。然而，内含子的剪接增强了mRNA生命中的所有后续步骤。所有的疱疹病毒都编码一个转录后调节因子来代替剪接。在KSHV中，ORF 57确保形成具有输出能力的mRNA-蛋白质复合物（mRNP）。虽然已经描述了与细胞因子的各种相互作用，但病毒RNA的ORF 57靶向机制仍然不清楚。ORF 57与RNA的低亲和力和大部分无序区域使得分析困难。我们采取了不同的方法，并询问：导致ORF 57依赖性的病毒RNA上的决定因素是什么？我们根据KSHV裂解RNA的序列组成提出了不同病毒mRNP编码的概念。我们假设特定细胞RNA结合蛋白（RBP）的基序编码在病毒RNA序列中，可能嵌入二级结构中。为了鉴定ORF 57的推定细胞伴侣，我们将使用最近描述的称为“LNA mRNP捕获”的mRNP沉淀技术。该方法能够基于经由寡核苷酸探针的选择来纯化特异性mRNP。作为另一种假设，我们将ORF 57的RNA识别与细菌RBP进行了比较，后者依赖于“隐藏的特异性”而不是保守的基序。与此同时，Mandel-Gutfreund实验室将开发新的算法和机器学习方法，以找到ORF 57依赖与非依赖基因的序列属性。此外，一个新的非结构化蛋白质结构域的预测工具将被开发，以检索更多的信息，对N-末端区域的ORF 57最可能参与RNA识别。最后，我们计划通过DMSseq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组合。结构信息将被集成到算法中。该输出也是体外探测推定的ORF 57识别位点的基础。最后，使用推定的靶位点的邻位连接测定将证明ORF 57的体内结合。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，我们的报告系统之一也被激活的ORF 57同源的爱泼斯坦-巴尔病毒和水痘带状疱疹病毒。这些数据还将有助于进一步破译细胞mRNP密码，这是后基因组时代的一项重要任务。