Promoteur de thèse: Monsieur Lionel Tafforeau

Titre de la dissertation: « Identification and characterisation of interactions between Influenza A virus NS1 protein and the human Ubiquitin Proteasome System ».

Résumé de la dissertation: Ubiquitination is a post-translational modification that regulates the fate of target proteins by modifying their stability, localization, and activity. Specific substrate ubiquitination requires a three-step enzymatic cascade involving E1, E2 and E3 enzymes. Target proteins can then either be mono-ubiquitinated or poly-ubiquitinated and can subsequently be recognized by ubiquitin-binding domain containing proteins, of which deubiquitinating enzymes (DUBs). Among ubiquitinated proteins, some can also be recognized and degraded by the 26S proteasome. The whole ubiquitination system, including E1, E2, E3 and DUB enzymes, as well as the 26S proteasome, is commonly referred to as the ubiquitin-proteasome system (UPS).As obligate intracellular parasites, viruses infect host cells and replicate themselves by hijacking cell machineries through interactions between viral and cellular proteins. Because of its crucial role in innate immune response activation, the UPS is particularly targeted by viral proteins. Moreover, this system is intrinsically involved in cell infection by influenza A virus (IAV), regulating entry and replication of the virus. Among IAV proteins, the non-structural protein NS1 is known to interact with a lot of cellular proteins, including within interferon activation pathway, turning NS1 into the main IAV virulence factor.In this work, we wanted to discover and functionally characterize UPS factors and NS1 protein interplays in human cells. By using GPCA (Gaussia princeps Protein Complementation Assay), we screened a human UPS library and identified that NS1 physically interacts with 98 UPS proteins, of which 18 were selected for further characterization. To estimate the involvement of these NS1 interactors in viral replication, we first examined the productive infectious cycle of IAV upon siRNA-mediated depletion of individual interactors. A mini-replicon assay was performed to determine the impact of these UPS factors on IAV polymerase activity, and we also investigated the potential role of these factors on type I interferon pathway upon IAV infection. Since NS1 shuttles between the cytoplasm and the nucleus during infection, we also assessed subcellular localization of some NS1 partners during the course of infection.The results obtained during this thesis provide a first insight into newly identified NS1 interactors within the UPS, while their implications in IAV life cycle need to be further explored.