Interactions between Influenza A virus NS1 and the Human ubiquitin-proteasome system

As obligate intracellular parasites, viruses infect host cells and replicate themselves by hijacking cell machineries through interactions between viral and cellular proteins. For example, the human ubiquitin-proteasome system (UPS) is particularly targeted by viral proteins. Ubiquitination is a post-translational modification regulating degradation, localization and activity of targeted proteins. Specific substrate ubiquitination requires a three-step enzymatic cascade involving E1, E2 and E3 enzymes. Ubiquitinated substrates can subsequently be recognized by ubiquitin-binding domain containing proteins such as the 26S proteasome which will degrade targeted proteins. The UPS plays a crucial role in cell infection by Influenza A virus (IAV), regulating entry and replication of the virus, as well as innate immune response in the infected cell.

Among IAV proteins, the non-structural protein NS1 is known to interact with a lot of cellular proteins e.g. within interferon activation pathway. By using GPCA (Gaussia princeps Protein Complementation Assay), we screened a human UPS library and identified that NS1 physically interacts with 98 UPS proteins. These factors were then screened against other IAV and cellular proteins to discard eventual sticky proteins. 18 UPS proteins were finally selected.
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 the individual interactors, by performing the common MDCK-SIAT plaque assay as well as a luciferase assay with a virus encoding for a nano-luciferase. A mini-genome replication assay was performed to determine the impact of these UPS factors on IAV polymerase activity, and we are also investigating the potential role of these factors on type I interferon activation upon IAV infection.
Moreover, a subset of interactors is currently under further characterization. We are performing subcellular localisation of some NS1 partners by immunofluorescence microscopy in presence or absence of NS1 and during the course of IAV infection. In order to know whether these interactors are involved in NS1 ubiquitination we will then perform an ubiquitination assay on NS1. Finally, we will analyze the potential effect of NS1 on few interactors by performing an ubiquitination assay on known targets of the UPS factors.