Titre de la dissertation: Deciphering how Influenza virus hijacks the host translation machinery during cell infection, identification of the interplay between NS1 and PA-X and the ribosomal stalk.

Promoteur:  Monsieur Lionel Tafforeau

Résumé de la dissertation: Influenza A virus (IAV) causes seasonal and pandemic infections worldwide. The IAV genome consists of 8 negative-sense single-stranded RNAs requiring host cell translation machinery for the synthesis of the viral proteins. Nonstructural protein 1 (NS1) and PA-X, which are both found in infected cells, play a major role in the virus replication cycle since they are responsible for the host cell shutoff.  In the nucleus, NS1 inhibits the correct processing and the export of cellular mRNA. PA-X, through its interaction with the splicing machinery, specifically cleaves cellular mRNA during their processing. The inhibition of mRNA processing and its nuclear export leads to the enhancement the translation of viral mRNA in the cytoplasm. Furthermore, NS1 inhibits the innate immune system in the infected cells. This finally leads to the inability of the infected cell to respond the viral infection properly. The goal of this research project is to identify the functional role of cellular proteins involved in translation that interacts with NS1 and PA-X during IAV infection. We selected approximately 40 cellular proteins from human-pathogen interaction databases, showing (i) an interaction with NS1 from high-throughput interactome screening and (ii) based on their translational functions. We carried out a screening using the Gaussia Princeps complementation assay (GPCA) to validate the direct physical interactions between the cellular proteins with NS1 and/or PA-X. Among the positive interactions, we further confirmed some of them by co-immunoprecipitation.To characterize the involvement of these interactors in IAV infection cycle, we analyzed an infectious cycle of IAV upon siRNA-mediated depletion of each cellular partner and performed a virus titration assay. Among these NS1/PA-X interactors, we identified that RPLP0 knockdown drastically decreased virus titers upon virus infection. Since RPLP0 interacts with RPLP1/2 heterodimers to form the ribosomal stalk, we also conducted the same demonstration upon RPLP2 knockdown in which the reduction of virus titers was observed. Finally, we further provided the evidence that RPLP0, RPLP1 and RPLP2 knockdown drastically impaired ion channel M2 protein accumulation, implying the role of these ribosomal stalk proteins in viral protein translation. This approach may lead to the development of antiviral drugs against influenza viruses (Flu), by specifically targeting the interplay between NS1/PA-X and the ribosomal stalk.