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

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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.