title: Phosphoproteomics to Characterize Host Response During Influenza A Virus Infection of Human Macrophages.
abstract: Influenza A viruses cause infections in the human respiratory tract and give rise to annual seasonal outbreaks, as well as more rarely dreaded pandemics. Influenza A viruses become quickly resistant to the virus-directed antiviral treatments, which are the current main treatment options. A promising alternative approach is to target host cell factors that are exploited by influenza viruses. To this end, we characterized the phosphoproteome of influenza A virus infected primary human macrophages to elucidate the intracellular signaling pathways and critical host factors activated upon influenza infection. We identified 1675 phosphoproteins, 4004 phosphopeptides and 4146 nonredundant phosphosites. The phosphorylation of 1113 proteins (66%) was regulated upon infection, highlighting the importance of such global phosphoproteomic profiling in primary cells. Notably, 285 of the identified phosphorylation sites have not been previously described in publicly available phosphorylation databases, despite many published large-scale phosphoproteome studies using human and mouse cell lines. Systematic bioinformatics analysis of the phosphoproteome data indicated that the phosphorylation of proteins involved in the ubiquitin/proteasome pathway (such as TRIM22 and TRIM25) and antiviral responses (such as MAVS) changed in infected macrophages. Proteins known to play roles in small GTPase-, mitogen-activated protein kinase-, and cyclin-dependent kinase- signaling were also regulated by phosphorylation upon infection. In particular, the influenza infection had a major influence on the phosphorylation profiles of a large number of cyclin-dependent kinase substrates. Functional studies using cyclin-dependent kinase inhibitors showed that the cyclin-dependent kinase activity is required for efficient viral replication and for activation of the host antiviral responses. In addition, we show that cyclin-dependent kinase inhibitors protect IAV-infected mice from death. In conclusion, we provide the first comprehensive phosphoproteome characterization of influenza A virus infection in primary human macrophages, and provide evidence that cyclin-dependent kinases represent potential therapeutic targets for more effective treatment of influenza infections.
authors.affiliation: From the ‡Institute of Biotechnology, FI-00014 University of Helsinki, Helsinki, Finland; §Unit of Systems Toxicology, Finnish Institute of Occupational Health, FI-00250 Helsinki, Finland.
authors.forename: Sandra
authors.initials: S
authors.lastname: Söderholm
authors.affiliation: ¶Institute for Molecular Medicine Finland (FIMM), FI-00014 University of Helsinki, Helsinki, Finland.
authors.forename: Denis E
authors.initials: DE
authors.lastname: Kainov
authors.affiliation: From the ‡Institute of Biotechnology, FI-00014 University of Helsinki, Helsinki, Finland.
authors.forename: Tiina
authors.initials: T
authors.lastname: Öhman
authors.affiliation: ¶Institute for Molecular Medicine Finland (FIMM), FI-00014 University of Helsinki, Helsinki, Finland.
authors.forename: Oxana V
authors.initials: OV
authors.lastname: Denisova
authors.affiliation: ‖Medical Biotechnology Center, VIB, B-9052 Ghent (Zwijnaarde), Belgium; **Department of Biomedical Molecular Biology, B-9052 Ghent University, Ghent, Belgium.
authors.forename: Bert
authors.initials: B
authors.lastname: Schepens
authors.affiliation: ¶Institute for Molecular Medicine Finland (FIMM), FI-00014 University of Helsinki, Helsinki, Finland.
authors.forename: Evgeny
authors.initials: E
authors.lastname: Kulesskiy
authors.affiliation: ‡‡Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, FI-20520 Turku, Finland.
authors.forename: Susumu Y
authors.initials: SY
authors.lastname: Imanishi
authors.affiliation: ‡‡Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, FI-20520 Turku, Finland.
authors.forename: Garry
authors.initials: G
authors.lastname: Corthals
authors.affiliation: ¶Institute for Molecular Medicine Finland (FIMM), FI-00014 University of Helsinki, Helsinki, Finland.
authors.forename: Petteri
authors.initials: P
authors.lastname: Hintsanen
authors.affiliation: ¶Institute for Molecular Medicine Finland (FIMM), FI-00014 University of Helsinki, Helsinki, Finland.
authors.forename: Tero
authors.initials: T
authors.lastname: Aittokallio
authors.affiliation: ‖Medical Biotechnology Center, VIB, B-9052 Ghent (Zwijnaarde), Belgium; **Department of Biomedical Molecular Biology, B-9052 Ghent University, Ghent, Belgium.
authors.forename: Xavier
authors.initials: X
authors.lastname: Saelens
authors.affiliation: §§Department of Rheumatology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
authors.forename: Sampsa
authors.initials: S
authors.lastname: Matikainen
authors.affiliation: From the ‡Institute of Biotechnology, FI-00014 University of Helsinki, Helsinki, Finland; ¶¶Institute of Clinical Medicine, University of Oslo, Norway tuula.nyman@medisin.uio.no.
authors.forename: Tuula A
authors.initials: TA
authors.lastname: Nyman
mesh_terms: D000818:Animals; D019295:Computational Biology / Q000379:methods; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D005786:Gene Expression Regulation; D054884:Host-Pathogen Interactions; D006801:Humans; D009980:Influenza A virus / Q000472:pathogenicity*; D007251:Influenza, Human / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism / Q000821:virology*; D051379:Mice; D010750:Phosphoproteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction
chemical_list: D010750:Phosphoproteins; D018844:Cyclin-Dependent Kinases