Abstract
Influenza A virus (IAV) still poses a major threat to global health with seasonal epidemics and its omnipresent pandemic potential [1, 2]. As a virus, IAV is an obligate intracellular parasite and therefore highly dependent on cellular functions for successful infection [3, 4]. However, little is known about cellular signaling networks that orchestrate viral infections. In this project, we developed and applied a label free quantification (LFQ) based phosphoproteomics method to quantitatively measure phosphorylation changes induced after 10 min infection of human respiratory epithelial cells by different human and avian IAV subtypes. The majority of residues undergoing IAV-induced changes were serines with 9144, followed by threonine with 1335 and tyrosine with 136 phospho-changes. In total, our method allowed the measurement of 7930 unique phoshopeptides belonging to 2991 phosphoproteins. Surprisingly, host factors undergoing phosphorylation changes upon IAV infection were highly similar between human and avian IAV subtypes. By bioinformatically assigning these changes to molecular functions, we revealed an overproportionate number of cytoskeletal associated factors and cellular adhesion molecules to be IAV responsive with regard to their phosphorylation status. With selected hits of the overrepresented group, we conducted a small interfering RNA (siRNA) mediated entry efficiency screen. We used four different siRNAs to knock down expression levels of the selected hits and monitored their effect on viral NP production. Interestingly, actin cytoskeleton associated proteins exhibited the most pro- and antiviral effects highlighting the two-sided role of actin networks during IAV entry. Further characterization of cortactin (CTTN), that showed the strongest restrictive effect on early stages of IAV infection, revealed that IAV binding induces the formation of actin-rich lamellipodia at IAV attachment sites. CTTN trans-locates to these structures upon multiple serine phosphorylations in its regulatory domain. Our data show that the small Rho GTPase Cdc42 mediates CTTN’s translocation to the cell cortex as well as the formation of lamellipodia at IAV attachment sites. Cdc42 activity alongside with active actin polymerization are necessary for CTTN’s restrictive effect on IAV entry. Cdc42 mediates actin polymerization by directly activating the nucleation promoting factor N-WASP associated with the formation of finger like membrane protrusions called filopodia. Our data indicate that CTTN lowers the abundance of N-WASP in IAV-induced lamellipodia and therefore restricts actin polymerization and the formation of filopodia and ultimately IAV uptake into endosomal vesicles.
Hunziker, Annika