E. YÁNGÜEZ¹, M.P. DOBAY², S. STERTZ<sup>1

1</sup>Institute of Medical Virology, University of Zurich, Zurich, Switzerland. , ²Swiss Institute of Bioinformatics, Lausanne, Switzerland.

Influenza A viruses (IAVs) harness the cellular endocytic machinery to enter the cell and traffic through the cytoplasm to reach the replication sites in the nucleus. Coordinated early activation of particular signalling pathways, such as EGFR-mediated signalling, has been shown to be crucial for the entry process and the outcome of viral infection. However, the complexity of these signalling cascades, tightly connected with each other, requires a broader analysis to identify the main routes, the key mediators and the direct effectors involved in the early steps of the viral replication cycle. The aim of the presented work is the identification of signalling and functional pathways that are selectively activated during IAV entry and could play a crucial role in the early steps of the viral infection. We have initially performed an ELISA-based multiplex analysis for the identification of Receptor Tyrosine Kinases (RTKs) that are activated upon IAV entry and observed that the ErbB family, a group of RTKs closely related with EGFR, is consistently activated in response to the virus infection. Moreover, and in order to obtain a more comprehensive view of the signalling events induced during IAV entry, we have conducted a proteome-wide SILAC-based quantitative phosphoproteomic screen of A549 cells within minutes post-infection with IAV. We find that both ErbB and MAPK signalling pathways are activated as early as 5 min after the initial contact of the virus with the cells. This early activation is accompanied by significant changes in the phosphorylation of key proteins of the cytoskeleton, the vesicle-mediated transport and the nuclear import machinery. Currently, we are focusing our efforts on the characterization of the influence of these early events on viral entry and the outcome of viral infection. The key proteins of the identified activated pathways could be used as promising clinical targets for future antiviral interventions.