Influenza A virus is an RNA virus that encodes up to 11 proteins and this small coding capacity demands that the virus use the host cellular machinery for many aspects of its life cycle1. Knowledge of these host cell requirements not only informs us of the molecular pathways exploited by the virus but also provides further targets that could be pursued for antiviral drug development. Here we use an integrative systems approach, based on genome-wide RNA interference screening, to identify 295 cellular cofactors required for early-stage influenza virus replication. Within this group, those involved in kinase-regulated signalling, ubiquitination and phosphatase activity are the most highly enriched, and 181 factors assemble into a highly significant host–pathogen interaction network. Moreover, 219 of the 295 factors were confirmed to be required for efficient wild-type influenza virus growth, and further analysis of a subset of genes showed 23 factors necessary for viral entry, including members of the vacuolar ATPase (vATPase) and COPI-protein families, fibroblast growth factor receptor (FGFR) proteins, and glycogen synthase kinase 3 (GSK3)-β. Furthermore, 10 proteins were confirmed to be involved in post-entry steps of influenza virus replication. These include nuclear import components, proteases, and the calcium/calmodulin-dependent protein kinase (CaM kinase) IIβ (CAMK2B). Notably, growth of swine-origin H1N1 influenza virus is also dependent on the identified host factors, and we show that small molecule inhibitors of several factors, including vATPase and CAMK2B, antagonize influenza virus replication.
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This work was supported in part by National Institutes of Health (NIH) grants U01 AI1074539, U54 AI057158 (Northeast Biodefense Center), HHSN272200900032C, HHSN266200700010C, 1 PO1 AI058113 and 1R21AI083673. S.S. is supported by a fellowship from the German Research Foundation and D.M.T. is supported by NIH fellowship 1F32AI081428. M.B.O. is supported by NIH training grant 1 T32 AI07647 and Mount Sinai Medical Scientist Training Program T32 GM007280. A.I. is supported by a Japan Society for the Promotion of Science Fellowship. The small molecule screen was performed at the National Screening Laboratory for the Regional Centers of Excellence in Biodefense (NSRB), Harvard Medical School, Boston and was supported by NIH grant U54 AI057159. Confocal laser scanning microscopy was performed at the MSSM-Microscopy Shared Resource Facility, supported with funding from NIH-NCI shared resources grant (5R24 CA095823-04), NSF Major Research Instrumentation grant (DBI-9724504) and NIH shared instrumentation grant (1 S10 RR0 9145-01). We thank R. Fouchier for providing influenza A/Netherlands/602/2009 (H1N1) virus.
Author Contributions R.K. and S.S. are equally contributing first authors; M.L.S. and S.K.C. are equally contributing senior authors. R.K., S.S., A.G.-S., J.A.T.Y., P.P., M.L.S. and S.K.C. designed research; R.K., S.S., A.I., H.-H.H., Su.B., S.E.A., J.G.A., D.M.T., M.B.O., Y.L., Q.G., P.D., L.P. and C.S. performed research; R.K., C.S., P.D., B.P.T., L.M. and G.B. performed the screens; A.O. provided siRNAs; R.K., S.S., B.P.T., M.L.S., S.K.C. and P.D. analysed data; Y.Z., G.B., So.B. and T.I. performed bioinformatics analysis; R.K., S.S., A.G.-S., J.A.T.Y., P.P., M.L.S. and S.K.C. co-wrote the manuscript.
The authors declare no competing financial interests.
This file contains Supplementary Methods, Supplementary Data, Supplementary References, Supplementary Figures S1-S14 with Legends and Legends for Supplementary Tables S1-S13. (PDF 14707 kb)
Scores of 295 confirmed genes required for influenza virus replication (see Supplementary Information file for full Legend). (XLS 211 kb)
Overrepresented functional processes and protein domains of proteins required for influenza virus replication (see Supplementary Information file for full Legend). (XLS 60 kb)
Functional classification of biological processes required for influenza virus replication (see Supplementary Information file for full Legend). (XLS 220 kb)
Overrepresented functional pathways required for influenza virus replication (see Supplementary Information file for full Legend). (XLS 44 kb)
References for selected functional categories presented in Table 1 (see Supplementary Information file for full Legend). (XLS 30 kb)
Binary interactions of the host pathogen interaction map for Figure 1C and Supplementary Figure S4 (see Supplementary Information file for full Legend). (XLS 468 kb)
Overlap between viral host factors required by different RNA viruses (see Supplementary Information file for full Legend). (XLS 37 kb)
Biochemical complexes that are required by different RNA viruses (see Supplementary Information file for full Legend). (XLS 172 kb)
Host proteins confirmed to be required for wild-type influenza virus growth and gene expression (see Supplementary Information file for full Legend). (XLS 161 kb)
Examination of interferon induction in siRNAtransfected cells (see Supplementary Information file for full Legend). (XLS 52 kb)
Evaluation of host factors that regulate influenza virus entry (see Supplementary Information file for full Legend). (XLS 33 kb)
Effects of host factor depletion on expression of an influenza virus mini-genome reporter (see Supplementary Information file for full Legend). (XLS 29 kb)
Expression levels of host factor after siRNA silencing (see Supplementary Information file for full Legend). (XLS 28 kb)
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