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An RNA-centric dissection of host complexes controlling flavivirus infection

Nature Microbiology volume 4, pages 2369–2382 (2019)Cite this article

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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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Fig. 1: ChIRP-MS reveals the protein interactome of the DENV and ZIKV RNA genomes.
Fig. 2: Intersection of ChIRP-MS with genome-wide CRISPR screens nominates functionally relevant pro-viral host proteins.
Fig. 3: RRBP1 and vigilin interact at the ER.
Fig. 4: DENV and ZIKV co-opt the RNA-binding properties of RRBP1 and vigilin in human cells.
Fig. 5: RRBP1 and vigilin modulate DENV translation and replication.
Fig. 6: RRBP1 and vigilin promote DENV infection and vRNA stability.

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Data availability

The raw and processed sequencing data can be found at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE109194.

References

  1. Bhatt, S. et al. The global distribution and burden of dengue. Nature 496, 504–507 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Shepard, D. S., Undurraga, E. A., Halasa, Y. A. & Stanaway, J. D. The global economic burden of dengue: a systematic analysis. Lancet Infect. Dis. 16, 935–941 (2016).

    PubMed  Google Scholar 

  3. Kaufmann, S. H. E., Dorhoi, A., Hotchkiss, R. S. & Bartenschlager, R. Host-directed therapies for bacterial and viral infections. Nat. Rev. Drug Discov. 17, 35–56 (2018).

    CAS  PubMed  Google Scholar 

  4. Puschnik, A. S. et al. A small-molecule oligosaccharyltransferase inhibitor with pan-flaviviral activity. Cell Rep. 21, 3032–3039 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Apte-Sengupta, S., Sirohi, D. & Kuhn, R. J. Coupling of replication and assembly in flaviviruses. Curr. Opin. Virol. 9, 134–142 (2014).

    CAS  PubMed  Google Scholar 

  6. Fernandez-Garcia, M. D., Mazzon, M., Jacobs, M. & Amara, A. Pathogenesis of flavivirus infections: using and abusing the host cell. Cell Host Microbe 5, 318–328 (2009).

    CAS  PubMed  Google Scholar 

  7. Walsh, D. & Mohr, I. Viral subversion of the host protein synthesis machinery. Nat. Rev. Microbiol. 9, 860–875 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Garcia-Blanco, M. A., Vasudevan, S. G., Bradrick, S. S. & Nicchitta, C. Flavivirus RNA transactions from viral entry to genome replication. Antivir. Res. 134, 244–249 (2016).

  9. Chu, C. et al. Systematic discovery of Xist RNA binding proteins. Cell 161, 404–416 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Chen, C. K. et al. Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing. Science 354, 468–472 (2016).

    CAS  PubMed  Google Scholar 

  11. Balinsky, C. A. et al. IRAV (FLJ11286), an interferon-stimulated gene with antiviral activity against dengue virus, interacts with MOV10. J. Virol. 91, e01606-16 (2017).

  12. Paranjape, S. M. & Harris, E. Y box-binding protein-1 binds to the dengue virus 3′-untranslated region and mediates antiviral effects. J. Biol. Chem. 282, 30497–30508 (2007).

    CAS  PubMed  Google Scholar 

  13. Umareddy, I. et al. Dengue virus regulates type I interferon signalling in a strain-dependent manner in human cell lines. J. Gen. Virol. 89, 3052–3062 (2008).

    CAS  PubMed  Google Scholar 

  14. Lei, Y. et al. Functional interaction between cellular p100 and the dengue virus 3′ UTR. J. Gen. Virol. 92, 796–806 (2011).

    CAS  PubMed  Google Scholar 

  15. Hentze, M. W., Castello, A., Schwarzl, T. & Preiss, T. A brave new world of RNA-binding proteins. Nat. Rev. Mol. Cell Biol. 19, 327–341 (2018).

    CAS  PubMed  Google Scholar 

  16. Marceau, C. D. et al. Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens. Nature 535, 159–163 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Savidis, G. et al. Identification of Zika virus and dengue virus dependency factors using functional genomics. Cell Rep. 16, 232–246 (2016).

    CAS  PubMed  Google Scholar 

  18. Zhang, R. et al. A CRISPR screen defines a signal peptide processing pathway required by flaviviruses. Nature 535, 164–168 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Lin, D. L. et al. Dengue virus hijacks a noncanonical oxidoreductase function of a cellular oligosaccharyltransferase complex. mBio 8, e00939-17 (2017).

  20. Li, J.et al. A short hairpin RNA screen of interferon-stimulated genes identifies a novel negative regulator of the cellular antiviral response. mBio 4, e00385-13 (2013).

  21. Cui, X. A., Zhang, H. & Palazzo, A. F. p180 promotes the ribosome-independent localization of a subset of mRNA to the endoplasmic reticulum. PLoS Biol. 10, e1001336 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Mobin, M. B. et al. The RNA-binding protein vigilin regulates VLDL secretion through modulation of Apob mRNA translation. Nat. Commun. 7, 12848 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Losfeld, M. E., Soncin, F., Ng, B. G., Singec, I. & Freeze, H. H. A sensitive green fluorescent protein biomarker of N-glycosylation site occupancy. FASEB J. 26, 4210–4217 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Reid, D. W. et al. Dengue virus selectively annexes endoplasmic reticulum-associated translation machinery as a strategy for co-opting host cell protein synthesis. J. Virol. 92, e01766-17 (2018).

  25. Zarnegar, B. J. et al. irCLIP platform for efficient characterization of protein–RNA interactions. Nat. Methods 13, 489–492 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Savitz, A. J. & Meyer, D. I. Identification of a ribosome receptor in the rough endoplasmic reticulum. Nature 346, 540–544 (1990).

    CAS  PubMed  Google Scholar 

  27. Eliseev, B. et al. Structure of a human cap-dependent 48S translation pre-initiation complex. Nucleic Acids Res. 46, 2678–2689 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Belanger, L., Roy, S. & Allard, D. New albumin gene 3′ adjacent to the α1-fetoprotein locus. J. Biol. Chem. 269, 5481–5484 (1994).

    CAS  PubMed  Google Scholar 

  29. Shelness, G. S., Ingram, M. F., Huang, X. F. & DeLozier, J. A. Apolipoprotein B in the rough endoplasmic reticulum: translation, translocation and the initiation of lipoprotein assembly. J. Nutr. 129, 456S–462S (1999).

    CAS  PubMed  Google Scholar 

  30. Darnell, J. C. et al. FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell 146, 247–261 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Chen, Y. L., Yokokawa, F. & Shi, P. Y. The search for nucleoside/nucleotide analog inhibitors of dengue virus. Antivir. Res. 122, 12–19 (2015).

    CAS  PubMed  Google Scholar 

  32. Cheng, M. H. & Jansen, R. P. A jack of all trades: the RNA-binding protein vigilin. WIRES RNA 8, e1448 (2017).

    Google Scholar 

  33. Cui, X. A. & Palazzo, A. F. Localization of mRNAs to the endoplasmic reticulum. WIRES RNA 5, 481–492 (2014).

    CAS  PubMed  Google Scholar 

  34. Cunningham, K. S., Dodson, R. E., Nagel, M. A., Shapiro, D. J. & Schoenberg, D. R. Vigilin binding selectively inhibits cleavage of the vitellogenin mRNA 3′-untranslated region by the mRNA endonuclease polysomal ribonuclease 1. Proc. Natl Acad. Sci. USA 97, 12498–12502 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Hyde, M., Block-Alper, L., Felix, J., Webster, P. & Meyer, D. I. Induction of secretory pathway components in yeast is associated with increased stability of their mRNA. J. Cell Biol. 156, 993–1001 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Reid, D. W. & Nicchitta, C. V. Diversity and selectivity in mRNA translation on the endoplasmic reticulum. Nat. Rev. Mol. Cell Biol. 16, 221–231 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Ueno, T., Kaneko, K., Sata, T., Hattori, S. & Ogawa-Goto, K. Regulation of polysome assembly on the endoplasmic reticulum by a coiled-coil protein, p180. Nucleic Acids Res. 40, 3006–3017 (2012).

    CAS  PubMed  Google Scholar 

  38. Kruse, C. et al. The multi-KH protein vigilin associates with free and membrane-bound ribosomes. Cell. Mol. Life Sci. 60, 2219–2227 (2003).

    CAS  PubMed  Google Scholar 

  39. Batlle, M., Marsellach, F. X., Huertas, D. & Azorin, F. Drosophila vigilin, DDP1, localises to the cytoplasm and associates to the rough endoplasmic reticulum. Biochim. Biophys. Acta 1809, 46–55 (2011).

    CAS  PubMed  Google Scholar 

  40. Frey, S., Pool, M. & Seedorf, M. Scp160p, an RNA-binding, polysome-associated protein, localizes to the endoplasmic reticulum of Saccharomyces cerevisiae in a microtubule-dependent manner. J. Biol. Chem. 276, 15905–15912 (2001).

    CAS  PubMed  Google Scholar 

  41. Hirschmann, W. D. et al. Scp160p is required for translational efficiency of codon-optimized mRNAs in yeast. Nucleic Acids Res. 42, 4043–4055 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Phillips, S. L., Soderblom, E. J., Bradrick, S. S. & Garcia-Blanco, M. A. Identification of proteins bound to dengue viral RNA in vivo reveals new host proteins important for virus replication. mBio 7, e01865-15 (2016).

  43. Viktorovskaya, O. V., Greco, T. M., Cristea, I. M. & Thompson, S. R. Identification of RNA binding proteins associated with dengue virus RNA in infected cells reveals temporally distinct host factor requirements. PLoS Negl. Trop. Dis. 10, e0004921 (2016).

    PubMed  PubMed Central  Google Scholar 

  44. Schoggins, J. W. et al. A diverse range of gene products are effectors of the type I interferon antiviral response. Nature 472, 481–485 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Carette, J. E. et al. Ebola virus entry requires the cholesterol transporter Niemann–Pick C1. Nature 477, 340–343 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Mlera, L., Meade-White, K., Saturday, G., Scott, D. & Bloom, M. E. Modeling Powassan virus infection in Peromyscus leucopus, a natural host. PLoS Negl. Trop. Dis. 11, e0005346 (2017).

    PubMed  PubMed Central  Google Scholar 

  47. Shan, C. et al. An infectious cDNA clone of Zika virus to study viral virulence, mosquito transmission, and antiviral inhibitors. Cell Host Microbe 19, 891–900 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Lanke, K. H. et al. GBF1, a guanine nucleotide exchange factor for Arf, is crucial for coxsackievirus B3 RNA replication. J. Virol. 83, 11940–11949 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Metsalu, T. & Vilo, J. ClustVis: a web tool for visualizing clustering of multivariate data using Principal Component Analysis and heatmap. Nucleic Acids Res. 43, W566–W570 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Teo, G. et al. SAINTq: scoring protein-protein interactions in affinity purification - mass spectrometry experiments with fragment or peptide intensity data. Proteomics 16, 2238–2245 (2016).

    CAS  PubMed  Google Scholar 

  51. Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Li, W. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 15, 554 (2014).

    PubMed  PubMed Central  Google Scholar 

  53. Huang da, W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    PubMed  Google Scholar 

  54. Carette, J. E. et al. Global gene disruption in human cells to assign genes to phenotypes by deep sequencing. Nat. Biotechnol. 29, 542–546 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

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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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Author notes

  1. These authors contributed equally: Yaw Shin Ooi, Karim Majzoub, Ryan A. Flynn.

Authors and Affiliations

  1. Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA

    Yaw Shin Ooi, Karim Majzoub, Miguel A. Mata, Jonathan Diep, Andreas S. Puschnik, Caleb D. Marceau, Peter Sarnow & Jan E. Carette

  2. INSERM U1110, Institute of Viral and Liver Diseases, University of Strasbourg, Strasbourg, France

    Karim Majzoub

  3. Department of Chemistry, Stanford University, Stanford, CA, USA

    Ryan A. Flynn, Neil Rumachik & Carolyn R. Bertozzi

  4. Department of Genetics, Stanford University, Stanford, CA, USA

    Jason Kenichi Li, Nicholas van Buuren & Karla Kirkegaard

  5. Department of Chemical and Systems Biology, Stanford University, Stanford, CA, USA

    Alex G. Johnson

  6. Biology of Vector-Borne Viruses Section, Rocky Mountain Laboratories, NIAID/NIH, Hamilton, MT, USA

    Luwanika Mlera, Jeffrey M. Grabowski & Marshall E. Bloom

  7. Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA

    Carolyn R. Bertozzi

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Contributions

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.

Corresponding authors

Correspondence to Ryan A. Flynn or Jan E. Carette.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–9 and legends for Supplementary Tables.

Reporting Summary

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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