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J Virol. 2019 Jun 14;93(13). pii: e00528-19. doi: 10.1128/JVI.00528-19. Print 2019 Jul 1.

Phosphoproteome Analysis of Cells Infected with Adapted and Nonadapted Influenza A Virus Reveals Novel Pro- and Antiviral Signaling Networks.

Author information

1
Rudolf Buchheim Institute of Pharmacology, Justus Liebig University Giessen, Giessen, Germany.
2
Institute of Biochemistry, Justus Liebig University, Giessen, Germany.
3
Institute of Medical Virology, Justus Liebig University Giessen, Giessen, Germany.
4
Max Planck Institute for Medical Research, Heidelberg, Germany.
5
Biochemistry and Molecular Biology, Justus Liebig University, Giessen, Germany.
6
Mass spectrometry facility of the Department of Chemistry, Philipps-Universität Marburg, Marburg, Germany.
7
Cell Signaling Technology, Danvers, Massachusetts, USA.
8
Rudolf Buchheim Institute of Pharmacology, Justus Liebig University Giessen, Giessen, Germany Michael.Kracht@pharma.med.uni-giessen.de Lienhard.Schmitz@biochemie.med.uni-giessen.de.
9
Member of the German Center for Lung Research.
10
Institute of Biochemistry, Justus Liebig University, Giessen, Germany Michael.Kracht@pharma.med.uni-giessen.de Lienhard.Schmitz@biochemie.med.uni-giessen.de.
#
Contributed equally

Abstract

Influenza A viruses (IAVs) quickly adapt to new environments and are well known to cross species barriers. To reveal a molecular basis for these phenomena, we compared the Ser/Thr and Tyr phosphoproteomes of murine lung epithelial cells early and late after infection with mouse-adapted SC35M virus or its nonadapted SC35 counterpart. With this analysis we identified a large set of upregulated Ser/Thr phosphorylations common to both viral genotypes, while Tyr phosphorylations showed little overlap. Most of the proteins undergoing massive changes of phosphorylation in response to both viruses regulate chromatin structure, RNA metabolism, and cell adhesion, including a focal adhesion kinase (FAK)-regulated network mediating the regulation of actin dynamics. IAV also affected phosphorylation of activation loops of 37 protein kinases, including FAK and several phosphatases, many of which were not previously implicated in influenza virus infection. Inhibition of FAK proved its contribution to IAV infection. Novel phosphorylation sites were found on IAV-encoded proteins, and the functional analysis of selected phosphorylation sites showed that they either support (NA Ser178) or inhibit (PB1 Thr223) virus propagation. Together, these data allow novel insights into IAV-triggered regulatory phosphorylation circuits and signaling networks.IMPORTANCE Infection with IAVs leads to the induction of complex signaling cascades, which apparently serve two opposing functions. On the one hand, the virus highjacks cellular signaling cascades in order to support its propagation; on the other hand, the host cell triggers antiviral signaling networks. Here we focused on IAV-triggered phosphorylation events in a systematic fashion by deep sequencing of the phosphoproteomes. This study revealed a plethora of newly phosphorylated proteins. We also identified 37 protein kinases and a range of phosphatases that are activated or inactivated following IAV infection. Moreover, we identified new phosphorylation sites on IAV-encoded proteins. Some of these phosphorylations support the enzymatic function of viral components, while other phosphorylations are inhibitory, as exemplified by PB1 Thr223 modification. Our global characterization of IAV-triggered patterns of phospho-proteins provides a rich resource to further understand host responses to infection at the level of phosphorylation-dependent signaling networks.

KEYWORDS:

influenza; kinase; phosphoproteome; signaling network

PMID:
30996098
DOI:
10.1128/JVI.00528-19

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