Identification and molecular dissection of novel host and viral factors for the spatiotemporal control of Flaviviridae RNA metabolism

为了快速和有效地响应其环境和需求，细胞已经进化到在转录后水平上局部和暂时地调节基因表达。这可以通过信使RNA剪接、运输、稳定性和翻译的组合调节来实现。然而，细胞如何通过调节分子开关在时间和空间上协调RNA代谢仍然知之甚少。正如生物学历史上多次证明的那样，基础病毒学通常可以为重要的分子生物学问题提供许多答案。在这方面，RNA病毒黄病毒科（包括丙型肝炎病毒（HCV）、登革病毒（DENV）和西尼罗河病毒（WNV））构成了用于研究RNA细胞质代谢的时空调节的最先进的原型模型。事实上，由于没有核步骤和DNA中间体，黄病毒科的生命周期完全依赖于一个单一的RNA物种，即基因组RNA（gRNA）的命运。值得注意的是，最佳病毒复制取决于gRNA进入后靶向内质网（ER）、其翻译成病毒蛋白、其复制及其随后的组装成病毒之间的微调平衡。这些过程不可能同时发生，因此必须通过被误解的调节机制在时间和空间上紧密协调。 这是第一个5年发现资助的提案，包括鉴定，功能解剖和新的调节宿主机制和病毒因子的泛病毒比较，这些机制和病毒因子在时间和空间上控制黄病毒科RNA的命运。首先，使用高分辨率显微镜为基础的方法，我们建议确定主机和顺式RNA决定簇参与黄病毒基因组的初始靶向ER病毒进入后。第二，我们将利用和扩展我们先前对HCV的工作，以评估gRNA复制/糖苷化平衡的微调调节是否在黄病毒科（包括DENV和WNV）中是保守的，与HCV相比，黄病毒科通过不同的策略利用ER膜。第三也是最后，我们将更大程度地探索使用质谱的黄病毒科核糖蛋白组学，以确定病毒进入，复制或组装的特异性机制。这将潜在地揭示控制生命周期不同步骤之间过渡的分子开关。 最终，拟议的项目将确定被黄病毒科劫持的新宿主机制以及控制生命周期不同步骤之间微调平衡的病毒RNA调控元件。这些发现将突出新的重要方面的时空控制的RNA的命运，这是高度相关的，我们的细胞的分子生物学的基本理解。

In order to rapidly and efficiently respond to their environment and their needs, cells have evolved to both locally and temporally modulate gene expression at the posttranscriptional level. This can be achieved through the combined regulation of messenger RNA splicing, trafficking, stability and translation. However, how the cell orchestrates in time and space RNA metabolism through modulatory molecular switches remains poorly understood. As demonstrated many times in the history of biology, fundamental virology can often provide many answers to important molecular biology questions. In this respect, RNA viruses Flaviviridae (comprising Hepatitis C virus (HCV), Dengue virus (DENV) and West Nile virus (WNV)) constitute a state-of-the-art prototype model for the study of spatiotemporal regulation of RNA cytoplasmic metabolism. Indeed, with no nuclear step and DNA intermediate, Flaviviridae life cycle entirely relies on the fate of one single RNA species, namely the genomic RNA (gRNA). Notably, optimal viral replication depends on a fine-tuned equilibrium between the post-entry targeting of gRNA to the endoplasmic reticulum (ER), its translation into viral proteins, its replication and its subsequent encapsidation into assembling viruses. These processes cannot occur at the same time and hence, must be tightly coordinated in time and space via misunderstood regulation mechanisms. This first proposal for a 5-year Discovery Grant consists in the identification, the functional dissection and the panviral comparison of novel regulatory host machineries and viral factors which control in time and space the fate of Flaviviridae RNA. First, using a high resolution microscopy-based approach, we propose to identify host machineries and cis RNA determinants involved in the initial targeting of Flaviviridae genome to the ER following viral entry. Second, we will exploit and extend our previous work on HCV to evaluate if the fine-tuned regulation of the gRNA replication/encapsidation equilibrium is conserved among Flaviviridae including DENV and WNV which exploit ER membranes through different strategies as compared to HCV. Third and lastly, we will explore more largely Flaviviridae riboproteomics using mass spectrometry in order to identify machineries specific for virus entry, replication or assembly. This will potentially unveil molecular switches controlling the transitions between the different steps of the life cycle. Ultimately, the proposed project will identify new host machineries hijacked by Flaviviridae as well as viral RNA regulatory elements controlling the fine-tuned equilibrium between the different steps of the life cycle. These discoveries will highlight novel important aspects of the spatiotemporal control of the RNA fate which are of high relevance for our fundamental understanding of the molecular biology of the cell.