Identification and characterization of interactions between Influenza A Virus NS1 protein and the human ubiquitin proteasome system

As a key player involved in various cellular pathways, including innate immune response activation, the human ubiquitin-proteasome system (UPS) is particularly targeted by viral proteins upon infection. Indeed, most viruses have evolved to counteract and hijack this system, as it is the case for the influenza A virus (IAV). The non-structural protein 1 (NS1) is described as the main IAV virulence factor, which is known to interact with several cellular proteins, including some UPS factors that are important for the viral escape of the immune cell response. In this study, we profiled the overall interplay between the NS1 proteins of multiple IAV strains and the human UPS. We identified 98 UPS factors directly interacting with NS1 of all or a subset of the studied strains, and we functionally studied 18 of them. We highlighted the involvement of these UPS factors in the IAV life cycle by performing viral titrations, minigenome replicon assays and an ISRE-luc (IFN pathway) assays. Furthermore, we analyzed the expression and subcellular localizations of FZR1, MKRN3, RC3H2 and SHKBP1 upon IAV infection. This interactomics approach allows for an improved understanding of the interplay between NS1 and UPS pathway in the context of an IAV-mediated inhibition of cellular anti-viral responses. ImportanceInfluenza A viruses (IAV) are pathogens responsible for annual flu epidemics causing up to 650,000 deaths each year, resulting in a significant impact in public health and global economy. IAV are also responsible of occasionally pandemic outbreaks in human population, such as in 1918 that caused the death of 50-100 million people. Non-structural protein 1 (NS1) is the main IAV virulence factor; it acts by direct interactions with several cellular proteins, leading to the host shut-off and to the inhibition of the host cell innate immune response. Since the ubiquitin-proteasome system (UPS) plays a crucial role in the innate immune response activation, it is a designated target for NS1 upon infection. Our research thus focused on the identification of interactions between NS1 of 6 different IAV strains and the UPS, to better understand the interplay between this viral protein and the UPS upon viral infection.