Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification


Broad-spectrum antiviral drugs targeting host processes could potentially treat a wide range of viruses while reducing the likelihood of emergent resistance. Despite great promise as therapeutics, such drugs remain largely elusive. Here we used parallel genome-wide high-coverage short hairpin RNA (shRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad-spectrum antiviral with unexplained cytotoxicity. We found that GSK983 blocked cell proliferation and dengue virus replication by inhibiting the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). Guided by mechanistic insights from both genomic screens, we found that exogenous deoxycytidine markedly reduced GSK983 cytotoxicity but not antiviral activity, providing an attractive new approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Our results highlight the distinct advantages and limitations of each screening method for identifying drug targets, and demonstrate the utility of parallel knockdown and knockout screens for comprehensive probing of drug activity.

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Figure 1: shRNA and CRISPR-Cas9 screens to identify the cellular target and mechanism of action of GSK983.
Figure 2: GSK983 inhibits DHODH to block virus replication and cell proliferation.
Figure 3: Deoxycytidine reverses the antiproliferative effect of GSK983 but not antiviral activity.


  1. 1

    Lou, Z., Sun, Y. & Rao, Z. Current progress in antiviral strategies. Trends Pharmacol. Sci. 35, 86–102 (2014).

    CAS  Article  Google Scholar 

  2. 2

    Debing, Y., Jochmans, D. & Neyts, J. Intervention strategies for emerging viruses: use of antivirals. Curr. Opin. Virol. 3, 217–224 (2013).

    CAS  Article  Google Scholar 

  3. 3

    Harvey, R. et al. GSK983: a novel compound with broad-spectrum antiviral activity. Antiviral Res. 82, 1–11 (2009).

    CAS  Article  Google Scholar 

  4. 4

    Aman, M.J. et al. Development of a broad-spectrum antiviral with activity against Ebola virus. Antiviral Res. 83, 245–251 (2009).

    CAS  Article  Google Scholar 

  5. 5

    Panchal, R.G. et al. Identification of an antioxidant small-molecule with broad-spectrum antiviral activity. Antiviral Res. 93, 23–29 (2012).

    CAS  Article  Google Scholar 

  6. 6

    Kinch, M.S. et al. FGI-104: a broad-spectrum small molecule inhibitor of viral infection. Am. J. Transl. Res. 1, 87–98 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Giaever, G. et al. Chemogenomic profiling: identifying the functional interactions of small molecules in yeast. Proc. Natl. Acad. Sci. USA 101, 793–798 (2004).

    CAS  Article  Google Scholar 

  8. 8

    Gregori-Puigjané, E. et al. Identifying mechanism-of-action targets for drugs and probes. Proc. Natl. Acad. Sci. USA 109, 11178–11183 (2012).

    Article  Google Scholar 

  9. 9

    Lamb, J. et al. The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease. Science 313, 1929–1935 (2006).

    CAS  Article  Google Scholar 

  10. 10

    Parsons, A.B. et al. Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast. Cell 126, 611–625 (2006).

    CAS  Article  Google Scholar 

  11. 11

    Petrone, P.M. et al. Rethinking molecular similarity: comparing compounds on the basis of biological activity. ACS Chem. Biol. 7, 1399–1409 (2012).

    CAS  Article  Google Scholar 

  12. 12

    Echeverri, C.J. & Perrimon, N. High-throughput RNAi screening in cultured cells: a user's guide. Nat. Rev. Genet. 7, 373–384 (2006).

    CAS  Article  Google Scholar 

  13. 13

    Schenone, M., Dančík, V., Wagner, B.K. & Clemons, P.A. Target identification and mechanism of action in chemical biology and drug discovery. Nat. Chem. Biol. 9, 232–240 (2013).

    CAS  Article  Google Scholar 

  14. 14

    Bassik, M.C. et al. A systematic mammalian genetic interaction map reveals pathways underlying ricin susceptibility. Cell 152, 909–922 (2013).

    CAS  Article  Google Scholar 

  15. 15

    Bassik, M.C. et al. Rapid creation and quantitative monitoring of high coverage shRNA libraries. Nat. Methods 6, 443–445 (2009).

    CAS  Article  Google Scholar 

  16. 16

    Kampmann, M., Bassik, M.C. & Weissman, J.S. Integrated platform for genome-wide screening and construction of high-density genetic interaction maps in mammalian cells. Proc. Natl. Acad. Sci. USA 110, E2317–E2326 (2013).

    CAS  Article  Google Scholar 

  17. 17

    Kampmann, M., Bassik, M.C. & Weissman, J.S. Functional genomics platform for pooled screening and generation of mammalian genetic interaction maps. Nat. Protoc. 9, 1825–1847 (2014).

    CAS  Article  Google Scholar 

  18. 18

    Matheny, C.J. et al. Next-generation NAMPT inhibitors identified by sequential high-throughput phenotypic chemical and functional genomic screens. Chem. Biol. 20, 1352–1363 (2013).

    CAS  Article  Google Scholar 

  19. 19

    Sidrauski, C. et al. Pharmacological dimerization and activation of the exchange factor eIF2B antagonizes the integrated stress response. eLife 4, e07314 (2015).

    Article  Google Scholar 

  20. 20

    Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    CAS  Article  Google Scholar 

  21. 21

    Gilbert, L.A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).

    CAS  Article  Google Scholar 

  22. 22

    Gilbert, L.A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

    CAS  Article  Google Scholar 

  23. 23

    Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).

    Article  Google Scholar 

  24. 24

    Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    CAS  Article  Google Scholar 

  25. 25

    Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014).

    CAS  Article  Google Scholar 

  26. 26

    Wang, T., Wei, J.J., Sabatini, D.M. & Lander, E.S. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80–84 (2014).

    CAS  Article  Google Scholar 

  27. 27

    Boggs, S.D. et al. Efficient asymmetric synthesis of N-[(1R)-6-chloro-2,3,4,9-tetrahydro-1H-carbazol-1-yl]-2-pyridinecarboxamide for treatment of human papillomavirus infections. Org. Process Res. Dev. 11, 539–545 (2007).

    CAS  Article  Google Scholar 

  28. 28

    Gudmundsson, K.S. et al. Tetrahydrocarbazole amides with potent activity against human papillomaviruses. Bioorg. Med. Chem. Lett. 19, 4110–4114 (2009).

    CAS  Article  Google Scholar 

  29. 29

    Kampmann, M. et al. Next-generation libraries for robust RNA interference-based genome-wide screens. Proc. Natl. Acad. Sci. USA 112, E3384–E3391 (2015).

    CAS  Article  Google Scholar 

  30. 30

    Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).

    CAS  Article  Google Scholar 

  31. 31

    Bar-Peled, L. et al. A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science 340, 1100–1106 (2013).

    CAS  Article  Google Scholar 

  32. 32

    Bar-Peled, L. & Sabatini, D.M. Regulation of mTORC1 by amino acids. Trends Cell Biol. 24, 400–406 (2014).

    CAS  Article  Google Scholar 

  33. 33

    Liu, S., Neidhardt, E.A., Grossman, T.H., Ocain, T. & Clardy, J. Structures of human dihydroorotate dehydrogenase in complex with antiproliferative agents. Structure 8, 25–33 (2000).

    CAS  Article  Google Scholar 

  34. 34

    Ben-Sahra, I., Howell, J.J., Asara, J.M. & Manning, B.D. Stimulation of de novo pyrimidine synthesis by growth signaling through mTOR and S6K1. Science 339, 1323–1328 (2013).

    CAS  Article  Google Scholar 

  35. 35

    Robitaille, A.M. et al. Quantitative phosphoproteomics reveal mTORC1 activates de novo pyrimidine synthesis. Science 339, 1320–1323 (2013).

    CAS  Article  Google Scholar 

  36. 36

    Chen, E.Y. et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics 14, 128 (2013).

    Article  Google Scholar 

  37. 37

    Davis, J.P., Cain, G.A., Pitts, W.J., Magolda, R.L. & Copeland, R.A. The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase. Biochemistry 35, 1270–1273 (1996).

    CAS  Article  Google Scholar 

  38. 38

    Hoffmann, H.H., Kunz, A., Simon, V.A., Palese, P. & Shaw, M.L. Broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis. Proc. Natl. Acad. Sci. USA 108, 5777–5782 (2011).

    CAS  Article  Google Scholar 

  39. 39

    Wang, Q.Y. et al. Inhibition of dengue virus through suppression of host pyrimidine biosynthesis. J. Virol. 85, 6548–6556 (2011).

    CAS  Article  Google Scholar 

  40. 40

    Bonavia, A. et al. Identification of broad-spectrum antiviral compounds and assessment of the druggability of their target for efficacy against respiratory syncytial virus (RSV). Proc. Natl. Acad. Sci. USA 108, 6739–6744 (2011).

    CAS  Article  Google Scholar 

  41. 41

    Fox, R.I. et al. Mechanism of action for leflunomide in rheumatoid arthritis. Clin. Immunol. 93, 198–208 (1999).

    CAS  Article  Google Scholar 

  42. 42

    Fairbanks, L.D., Bofill, M., Ruckemann, K. & Simmonds, H.A. Importance of ribonucleotide availability to proliferating T-lymphocytes from healthy humans. Disproportionate expansion of pyrimidine pools and contrasting effects of de novo synthesis inhibitors. J. Biol. Chem. 270, 29682–29689 (1995).

    CAS  Article  Google Scholar 

  43. 43

    Qing, M. et al. Characterization of dengue virus resistance to brequinar in cell culture. Antimicrob. Agents Chemother. 54, 3686–3695 (2010).

    CAS  Article  Google Scholar 

  44. 44

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

    CAS  Article  Google Scholar 

  45. 45

    Ullrich, A., Knecht, W., Fries, M. & Löffler, M. Recombinant expression of N-terminal truncated mutants of the membrane bound mouse, rat and human flavoenzyme dihydroorotate dehydrogenase. A versatile tool to rate inhibitor effects? Eur. J. Biochem. 268, 1861–1868 (2001).

    CAS  Article  Google Scholar 

  46. 46

    Bader, B., Knecht, W., Fries, M. & Löffler, M. Expression, purification, and characterization of histidine-tagged rat and human flavoenzyme dihydroorotate dehydrogenase. Protein Expr. Purif. 13, 414–422 (1998).

    CAS  Article  Google Scholar 

  47. 47

    Samsa, M.M. et al. Dengue virus capsid protein usurps lipid droplets for viral particle formation. PLoS Pathog. 5, e1000632 (2009).

    Article  Google Scholar 

  48. 48

    Kinney, R.M. et al. Construction of infectious cDNA clones for dengue 2 virus: strain 16681 and its attenuated vaccine derivative, strain PDK-53. Virology 230, 300–308 (1997).

    CAS  Article  Google Scholar 

  49. 49

    Atasheva, S., Krendelchtchikova, V., Liopo, A., Frolova, E. & Frolov, I. Interplay of acute and persistent infections caused by Venezuelan equine encephalitis virus encoding mutated capsid protein. J. Virol. 84, 10004–10015 (2010).

    CAS  Article  Google Scholar 

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We thank J. Weissman, O. Chen, K. Han, B. Lowry, J. Kuo, N. Plugis and K. Nguyen for helpful discussions, and A. Brunet, J. Sage and D. Vollrath for critical reading of this manuscript. M.K. was supported by NIH/NCI K99/R00 CA181494. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-114747 (R.M.D.). R.M.D. was additionally supported by a Burt and DeeDee McMurtry Stanford Graduate Fellowship. This work was funded by US National Institues of Health grants U19-AI109662 and Director's New Innovator Award Program 1DP2HD084069-01, and a seed grant from Stanford ChEM-H. Any opinion, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

Author information




R.M.D. synthesized and characterized GSK983 and analogs, performed the genome-wide shRNA and CRISPR-Cas9 screens, assisted with statistical analysis of genomic screen results, validated genomic screen hits, designed and conducted GSK983 biological activity assays, pyrimidine supplementation experiments, and cell-cycle analyses, and assisted with DENV and VEEV antiviral assays. D.W.M. designed the maximum likelihood estimator and conducted statistical analysis of genomic screen results. A.Ö. expressed and purified DHODH and performed in vitro enzymatic assays. R.M.D. and A.Ö. expressed and purified CMPK1, and performed in vitro enzymatic assays. S.P. assisted with DENV and VEEV antiviral assays. J.E.C. and R.M. constructed the DENV luciferase reporter. M.K. designed the shRNA library with assistance from M.A.H. and M.C.B. M.A.H. designed the CRISPR-Cas9 sgRNA library with assistance from L.A.G. and M.C.B. A.L. provided technical support. J.E.C. provided guidance for DENV antiviral assays. M.S., J.S.G., C.K. and M.C.B. conceived of the study. R.M.D., C.K. and M.C.B. wrote the manuscript and prepared the figures.

Corresponding authors

Correspondence to Chaitan Khosla or Michael C Bassik.

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Stanford University has filed a patent application based on the findings in this report.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–7 and Supplementary Tables 1–4. (PDF 7399 kb)

Supplementary Data Set 1

Complete gene rankings from the genome-wide shRNA screen (XLSX 2220 kb)

Supplementary Data Set 2

Complete gene rankings from the genome-wide CRISPRCas9 screen (XLSX 1571 kb)

Supplementary Note

Synthetic Procedures (PDF 202 kb)

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Deans, R., Morgens, D., Ökesli, A. et al. Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification. Nat Chem Biol 12, 361–366 (2016).

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