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

为了快速和有效地对环境和需求做出反应，细胞已经进化到在转录后水平上局部和时间上调节基因表达。这可以通过对信使RNA的剪接、运输、稳定性和翻译的联合调控来实现。然而，细胞如何通过可调节的分子开关在时间和空间上协调RNA新陈代谢仍然知之甚少。正如生物史上多次证明的那样，基础病毒学往往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(包括丙型肝炎病毒(HCV)、登革病毒(DENV)和西尼罗河病毒(WNV))构成了研究RNA细胞质代谢时空调控的最新原型模型。事实上，由于没有核步骤和DNA中间体，黄病毒的生命周期完全取决于单一RNA物种的命运，即基因组RNA(GRNA)。值得注意的是，最佳的病毒复制取决于gRNA进入后靶向内质网(ER)、翻译成病毒蛋白、复制和随后封装成组装病毒之间的微调平衡。这些过程不能同时发生，因此，必须通过被误解的监管机制在时间和空间上紧密协调。 这项为期5年的发现拨款的第一项建议包括对在时间和空间上控制黄病毒科RNA命运的新的调节宿主机制和病毒因子进行鉴定、功能解剖和全病毒比较。首先，使用基于高分辨率显微镜的方法，我们建议识别宿主机制和参与病毒进入后将黄病毒基因组定位到ER的初始靶向的顺式RNA决定因素。其次，我们将利用和扩展我们以前对丙型肝炎病毒的工作，以评估包括DENV和WNV在内的黄毒科是否保守gRNA复制/包膜平衡的微调调节，这些黄毒科通过与丙型肝炎病毒不同的策略利用ER膜。第三，也是最后一点，我们将更多地利用质谱学来探索黄病毒核蛋白组学，以确定病毒进入、复制或组装的特定机制。这可能会揭开分子开关的面纱，控制生命周期不同阶段之间的转换。 最终，拟议的项目将确定被黄病毒科劫持的新宿主机器以及控制生命周期不同阶段之间微调平衡的病毒RNA调控元件。这些发现将突出RNA命运时空控制的新的重要方面，这些方面对于我们对细胞分子生物学的基本理解具有很高的相关性。

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.