The role of the influenza A virus genotype on NF-kappaB function and phosphorylation networks

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DamSharmistha_2018_03_05.pdf (5.34 MB)

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2018

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DOI:
http://dx.doi.org/10.22029/jlupub-14541

Abstract

This work is splitted in two parts, which both address the role of the IAV genotype for the function of NF-?B and host cell signaling networks. As a model system for different IAV genotypes I used an avian SC35 virus which infects avian but nu murine cells and its mouse-adapted variant SC35M which infects both avian and murine cells. In the first part the role of NF-?B for IAV infection was investigated, as the literature did not provide a clear picture. Murine MLE-15 cells were used to perturb NF-?Bsignaling either at the level of activating kinases or at the level of transcription activation. CRISPR-Cas9-mediated genome engineering was used to generate cells depleted in either the NEMO scaffold protein (and thus defective in IKK activation) or in the NF-?B DNA-binding and transactivating p65 subunit. While NF-?B was not affecting replication of SC35M, the deletion of NF-?B activity increased replication of the non-adapted SC35 virus. This antiviral effect was most noticeable upon infection of cells with low virus titers as they usually occur during the initiation phase of the IAV infection. The defect in NF-?B signaling resulted in decreased IAV-triggered phosphorylation of IRF3 and expression of the antiviral cytokine IFNß. Reassortant viruses were generated by reverse genetics system to identify the viral proteins responsible for NF-?B dependency. SC35 viruses containing the NA segment from SC35M were completely inert to the inhibitory effect of NF-?B, highlighting the importance of the viral genotype for susceptibility to the antiviral function of NF-?B.The second part of this study was performed to identify new host cell signaling pathways by a phosphoproteomic approach. MLE-15 cells were infected either with SC35 or SC35M and time dependent changes in the phophoproteome of MLE-15 cells were recorded by label-free quantitative phosphoproteome analysis. These experiments revealed thousands of IAV-regulated phosphorylation events. While approximately one-half of the inducedphosphorylations were triggered by both viruses, only one-third of the down-regulatedphosphorylations were co-regulated by SC35 and SC35M. Gene ontology and KEGG analyses revealed the overrepresentation of several pathways including those regulating cell adhesion, actin remodeling, and cytoskeleton organization. IAV-regulated kinases were identified by the occurrence of IAV-dependent changes in the activation loop and also by identification and analysis of common phosphorylation motifs, which allow the prediction of responsible kinases. These approaches disclosed the IAV-mediated regulation of FAK and inhibition of this kinase with pre-clinically used inhibitor Defactinib interfered with IAV replication. In addition, novel phosphorylation sites on IAV-encoded proteins were discovered. The functional analysis of selected phospho-sites showed that some phosphorylations serve to interfere with the enzymatic function of the viral proteins, thus providing an example for a novel antiviral signaling mechanism. Other phosphorylations supported the enzymatic function of viral proteins, opening new avenues for the therapeutic interference with IAV replication.

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