Abstract
Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum is a shared replication strategy, despite different clinical outcomes. Although the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV genomic RNA in human cells. Genome-scale knockout screens assigned putative functional relevance to the RNA–protein interactions observed by ChIRP-MS. The endoplasmic-reticulum-localized RNA-binding proteins vigilin and ribosome-binding protein 1 directly bound viral RNA and each acted at distinct stages in the life cycle of flaviviruses. Thus, this versatile strategy can elucidate features of human biology that control the pathogenesis of clinically relevant viruses.
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The raw and processed sequencing data can be found at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE109194.
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Acknowledgements
We would like to thank A. Rubin, O. Botvinnik, M. R. Corces and A. Za for their helpful discussions on statistical and computational analyses; J. Ule, J. Zmrzlikar and N. Haberman for help with the iCount peak-calling software; L. Zhang and the Elias lab for assistance running samples for mass spectrometry analysis; J. Coller, X. Ji, D. Wagh and the Stanford Functional Genomics Facility for assistance with running samples for deep sequencing. We also thank the Stanford shared FACS facility and its former director M. Bigos for technical assistance. We acknowledge the NIH Biodefense and Emerging Infections Research Repository (BEI Resources) and the NIAID, NIH for providing the multiple DENV and ZIKV strains mentioned in Methods. We thank S. Braun and the members of the Carette, Kirkegaard and Sarnow labs for their helpful discussions and critical reading of the manuscript. The work was funded in part by the NIH (grant nos DP2 AI104557, R01 AI141970 and U19 AI109662 to J.E.C.; R37 AI047365 and R01 AI069000 to P.S. and R01 AI051622 to C.R.B.), the Burroughs Wellcome Investigators in the Pathogenesis of Infectious Disease (J.E.C.), the David and Lucile Packard Foundation (J.E.C.), the Stanford Dean’s Fellowship (Y.S.O.), the Child Health Research Institute Stanford (K.M.), the Damon Runyon Cancer Research Foundation (R.A.F.), the NSF-GRF (A.G.J. and C.D.M.) and the Howard Hughes Medical Institute (C.R.B.). C.D.M., J.G., L.M. and M.E.B. were supported by the Intramural Research Program of the NIAID.
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Y.S.O., K.M. and R.A.F. were responsible for the design and execution of experiments, data analysis and manuscript preparation. M.A.M. and P.S. performed and analysed the northern blot assays. J.D. carried out the MAGECK analysis for all CRISPR screening results. J.K.L. and N.R. assisted with the proteomic experiments. N.V.B. and K.K. were responsible for the immunofluorescence and fluorescent in situ hybridization targeting assays. A.G.J. assisted with the preparation of manuscript. A.S.P. and C.D.M. helped with the preparation of reagents, cell lines and viruses. L.M., J.M.G. and M.E.B. were responsible for the infection assays and analyses involving POWV. J.E.C. and C.R.B. supervised the research, acquired funding, interpreted data and prepared the manuscript.
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Supplementary Information
Supplementary Figs. 1–9 and legends for Supplementary Tables.
Supplementary Table 1
Oligonucleotides used in this article.
Supplementary Table 2
Proteins identified by ChIRP-MS of DENV or ZIKV.
Supplementary Table 3
Overlap of ChIRP-MS hits with annotated ER-associated proteins.
Supplementary Table 4
RNA-seq of uninfected Huh7.5.1 cells or DENV- or ZIKV-infected cells.
Supplementary Table 5
Proteins identified by ChIRP-MS of RV.
Supplementary Table 6
Host genes identified in genome-scale KO screens for DENV or ZIKV.
Supplementary Table 7
RT stop counts of genes bound to RRBP1 as identified by irCLIP.
Supplementary Table 8
RT stop counts of genes bound to vigilin as identified by irCLIP.
Supplementary Table 9
Comparison of flavivirus host factors with published screens.
Supplementary Table 10
Intersection of ChIRP-MS with published work.
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Ooi, Y.S., Majzoub, K., Flynn, R.A. et al. An RNA-centric dissection of host complexes controlling flavivirus infection. Nat Microbiol 4, 2369–2382 (2019). https://doi.org/10.1038/s41564-019-0518-2
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DOI: https://doi.org/10.1038/s41564-019-0518-2
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