Human Genome-Wide RNAi Screen Identifies an Essential Role for Inositol Pyrophosphates in Type-I Interferon Response


The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified 226 novel regulatory proteins of RIG-I mediated interferon-β production. Furthermore, the screen identified a metabolic pathway that synthesizes the inositol pyrophosphate 1-IP7 as a previously unrecognized positive regulator of interferon production. Detailed genetic and biochemical experiments demonstrated that the kinase activities of IPPK, PPIP5K1 and PPIP5K2 (which convert IP5 to1-IP7) were critical for both interferon induction, and the control of cellular infection by Sendai and influenza A viruses. Conversely, ectopically expressed inositol pyrophosphate-hydrolases DIPPs attenuated interferon transcription. Mechanistic experiments in intact cells revealed that the expression of IPPK, PPIP5K1 and PPIP5K2 was needed for the phosphorylation and activation of IRF3, a transcription factor for interferon. The addition of purified individual inositol pyrophosphates to a cell free reconstituted RIG-I signaling assay further identified 1-IP7 as an essential component required for IRF3 activation. The inositol pyrophosphate may act by β-phosphoryl transfer, since its action was not recapitulated by a synthetic phosphonoacetate analogue of 1-IP7. This study thus identified several novel regulators of RIG-I, and a new role for inositol pyrophosphates in augmenting innate immune responses to viral infection that may have therapeutic applications.


Vyšlo v časopise: Human Genome-Wide RNAi Screen Identifies an Essential Role for Inositol Pyrophosphates in Type-I Interferon Response. PLoS Pathog 10(2): e32767. doi:10.1371/journal.ppat.1003981
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1003981

Souhrn

The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified 226 novel regulatory proteins of RIG-I mediated interferon-β production. Furthermore, the screen identified a metabolic pathway that synthesizes the inositol pyrophosphate 1-IP7 as a previously unrecognized positive regulator of interferon production. Detailed genetic and biochemical experiments demonstrated that the kinase activities of IPPK, PPIP5K1 and PPIP5K2 (which convert IP5 to1-IP7) were critical for both interferon induction, and the control of cellular infection by Sendai and influenza A viruses. Conversely, ectopically expressed inositol pyrophosphate-hydrolases DIPPs attenuated interferon transcription. Mechanistic experiments in intact cells revealed that the expression of IPPK, PPIP5K1 and PPIP5K2 was needed for the phosphorylation and activation of IRF3, a transcription factor for interferon. The addition of purified individual inositol pyrophosphates to a cell free reconstituted RIG-I signaling assay further identified 1-IP7 as an essential component required for IRF3 activation. The inositol pyrophosphate may act by β-phosphoryl transfer, since its action was not recapitulated by a synthetic phosphonoacetate analogue of 1-IP7. This study thus identified several novel regulators of RIG-I, and a new role for inositol pyrophosphates in augmenting innate immune responses to viral infection that may have therapeutic applications.


Zdroje

1. KumarH, KawaiT, AkiraS (2009) Pathogen recognition in the innate immune response. Biochem J 420: 1–16.

2. LiuSY, SanchezDJ, ChengG (2011) New developments in the induction and antiviral effectors of type I interferon. Curr Opin Immunol 23: 57–64.

3. YoneyamaM, FujitaT (2010) Recognition of viral nucleic acids in innate immunity. Rev Med Virol 20: 4–22.

4. BelgnaouiSM, PazS, HiscottJ (2011) Orchestrating the interferon antiviral response through the mitochondrial antiviral signaling (MAVS) adapter. Curr Opin Immunol 23: 564–572.

5. RamosHJ, GaleMJr (2011) RIG-I like receptors and their signaling crosstalk in the regulation of antiviral immunity. Curr Opin Virol 1: 167–176.

6. SethRB, SunL, ChenZJ (2006) Antiviral innate immunity pathways. Cell Res 16: 141–147.

7. YoneyamaM, KikuchiM, NatsukawaT, ShinobuN, ImaizumiT, et al. (2004) The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol 5: 730–737.

8. HiscottJ, PithaP, GeninP, NguyenH, HeylbroeckC, et al. (1999) Triggering the interferon response: the role of IRF-3 transcription factor. J Interferon Cytokine Res 19: 1–13.

9. LinR, MamaneY, HiscottJ (1999) Structural and functional analysis of interferon regulatory factor 3: localization of the transactivation and autoinhibitory domains. Mol Cell Biol 19: 2465–2474.

10. SuharaW, YoneyamaM, IwamuraT, YoshimuraS, TamuraK, et al. (2000) Analyses of virus-induced homomeric and heteromeric protein associations between IRF-3 and coactivator CBP/p300. J Biochem 128: 301–307.

11. BordenEC, SenGC, UzeG, SilvermanRH, RansohoffRM, et al. (2007) Interferons at age 50: past, current and future impact on biomedicine. Nat Rev Drug Discov 6: 975–990.

12. GackMU, ShinYC, JooCH, UranoT, LiangC, et al. (2007) TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature 446: 916–920.

13. JiangX, KinchLN, BrautigamCA, ChenX, DuF, et al. (2012) Ubiquitin-induced oligomerization of the RNA sensors RIG-I and MDA5 activates antiviral innate immune response. Immunity 36: 959–973.

14. LiuHM, LooYM, HornerSM, ZornetzerGA, KatzeMG, et al. (2012) The mitochondrial targeting chaperone 14-3-3epsilon regulates a RIG-I translocon that mediates membrane association and innate antiviral immunity. Cell Host Microbe 11: 528–537.

15. OshiumiH, MiyashitaM, InoueN, OkabeM, MatsumotoM, et al. (2010) The ubiquitin ligase Riplet is essential for RIG-I-dependent innate immune responses to RNA virus infection. Cell Host Microbe 8: 496–509.

16. SaitohT, Tun-KyiA, RyoA, YamamotoM, FinnG, et al. (2006) Negative regulation of interferon-regulatory factor 3-dependent innate antiviral response by the prolyl isomerase Pin1. Nat Immunol 7: 598–605.

17. VersteegGA, RajsbaumR, Sanchez-AparicioMT, MaestreAM, ValdiviezoJ, et al. (2013) The E3-ligase TRIM family of proteins regulates signaling pathways triggered by innate immune pattern-recognition receptors. Immunity 38: 384–398.

18. WiesE, WangMK, MaharajNP, ChenK, ZhouS, et al. (2013) Dephosphorylation of the RNA sensors RIG-I and MDA5 by the phosphatase PP1 is essential for innate immune signaling. Immunity 38: 437–449.

19. MunkC, SommerAF, KonigR (2011) Systems-biology approaches to discover anti-viral effectors of the human innate immune response. Viruses 3: 1112–1130.

20. GardyJL, LynnDJ, BrinkmanFS, HancockRE (2009) Enabling a systems biology approach to immunology: focus on innate immunity. Trends Immunol 30: 249–262.

21. ShapiraSD, Gat-ViksI, ShumBO, DricotA, de GraceMM, et al. (2009) A physical and regulatory map of host-influenza interactions reveals pathways in H1N1 infection. Cell 139: 1255–1267.

22. SutharMS, BrassilMM, BlahnikG, McMillanA, RamosHJ, et al. (2013) A systems biology approach reveals that tissue tropism to West Nile virus is regulated by antiviral genes and innate immune cellular processes. PLoS Pathog 9: e1003168.

23. MennitiFS, MillerRN, PutneyJWJr, ShearsSB (1993) Turnover of inositol polyphosphate pyrophosphates in pancreatoma cells. J Biol Chem 268: 3850–3856.

24. StephensL, RadenbergT, ThielU, VogelG, KhooKH, et al. (1993) The detection, purification, structural characterization, and metabolism of diphosphoinositol pentakisphosphate(s) and bisdiphosphoinositol tetrakisphosphate(s). J Biol Chem 268: 4009–4015.

25. TsuiMM, YorkJD (2010) Roles of inositol phosphates and inositol pyrophosphates in development, cell signaling and nuclear processes. Adv Enzyme Regul 50: 324–337.

26. JadavRS, ChanduriMV, SenguptaS, BhandariR (2013) Inositol pyrophosphate synthesis by inositol hexakisphosphate kinase 1 is required for homologous recombination repair. J Biol Chem 288: 3312–3321.

27. KoldobskiyMA, ChakrabortyA, WernerJKJr, SnowmanAM, JuluriKR, et al. (2010) p53-mediated apoptosis requires inositol hexakisphosphate kinase-2. Proc Natl Acad Sci U S A 107: 20947–20951.

28. MorrisonBH, BauerJA, KalvakolanuDV, LindnerDJ (2001) Inositol hexakisphosphate kinase 2 mediates growth suppressive and apoptotic effects of interferon-beta in ovarian carcinoma cells. J Biol Chem 276: 24965–24970.

29. IlliesC, GromadaJ, FiumeR, LeibigerB, YuJ, et al. (2007) Requirement of inositol pyrophosphates for full exocytotic capacity in pancreatic beta cells. Science 318: 1299–1302.

30. ChakrabortyA, KoldobskiyMA, BelloNT, MaxwellM, PotterJJ, et al. (2010) Inositol pyrophosphates inhibit Akt signaling, thereby regulating insulin sensitivity and weight gain. Cell 143: 897–910.

31. PrasadA, JiaY, ChakrabortyA, LiY, JainSK, et al. (2011) Inositol hexakisphosphate kinase 1 regulates neutrophil function in innate immunity by inhibiting phosphatidylinositol-(3,4,5)-trisphosphate signaling. Nat Immunol 12: 752–760.

32. BozymRA, Delorme-AxfordE, HarrisK, MoroskyS, IkizlerM, et al. (2012) Focal adhesion kinase is a component of antiviral RIG-I-like receptor signaling. Cell Host Microbe 11: 153–166.

33. KokKH, LuiPY, NgMH, SiuKL, AuSW, et al. (2011) The double-stranded RNA-binding protein PACT functions as a cellular activator of RIG-I to facilitate innate antiviral response. Cell Host Microbe 9: 299–309.

34. ZhuJ, SmithK, HsiehPN, MburuYK, ChattopadhyayS, et al. (2010) High-throughput screening for TLR3-IFN regulatory factor 3 signaling pathway modulators identifies several antipsychotic drugs as TLR inhibitors. J Immunol 184: 5768–5776.

35. LiY, XieJ, WuS, XiaJ, ZhangP, et al. (2013) Protein kinase regulated by dsRNA downregulates the interferon production in dengue virus- and dsRNA-stimulated human lung epithelial cells. PLoS One 8: e55108.

36. YangK, ShiHX, LiuXY, ShanYF, WeiB, et al. (2009) TRIM21 is essential to sustain IFN regulatory factor 3 activation during antiviral response. J Immunol 182: 3782–3792.

37. BolenCR, RobekMD, BrodskyL, SchulzV, LimJK, et al. (2013) The blood transcriptional signature of chronic hepatitis C virus is consistent with an ongoing interferon-mediated antiviral response. J Interferon Cytokine Res 33: 15–23.

38. CaskeyM, LefebvreF, Filali-MouhimA, CameronMJ, GouletJP, et al. (2011) Synthetic double-stranded RNA induces innate immune responses similar to a live viral vaccine in humans. J Exp Med 208: 2357–2366.

39. IndraccoloS, PfefferU, MinuzzoS, EspositoG, RoniV, et al. (2007) Identification of genes selectively regulated by IFNs in endothelial cells. J Immunol 178: 1122–1135.

40. LeeSM, ChanRW, GardyJL, LoCK, SihoeAD, et al. (2010) Systems-level comparison of host responses induced by pandemic and seasonal influenza A H1N1 viruses in primary human type I-like alveolar epithelial cells in vitro. Respir Res 11: 147.

41. FennerBJ, ScannellM, PrehnJH (2010) Expanding the substantial interactome of NEMO using protein microarrays. PLoS One 5: e8799.

42. BelgnaouiSM, PazS, SamuelS, GouletML, SunQ, et al. (2012) Linear ubiquitination of NEMO negatively regulates the interferon antiviral response through disruption of the MAVS-TRAF3 complex. Cell Host Microbe 12: 211–222.

43. ShearsSB, GokhaleNA, WangH, ZarembaA (2011) Diphosphoinositol polyphosphates: what are the mechanisms? Adv Enzyme Regul 51: 13–25.

44. BarkerCJ, IlliesC, GaboardiGC, BerggrenPO (2009) Inositol pyrophosphates: structure, enzymology and function. Cell Mol Life Sci 66: 3851–3871.

45. BennettM, OnneboSM, AzevedoC, SaiardiA (2006) Inositol pyrophosphates: metabolism and signaling. Cell Mol Life Sci 63: 552–564.

46. ChoiJH, WilliamsJ, ChoJ, FalckJR, ShearsSB (2007) Purification, sequencing, and molecular identification of a mammalian PP-InsP5 kinase that is activated when cells are exposed to hyperosmotic stress. J Biol Chem 282: 30763–30775.

47. FridyPC, OttoJC, DollinsDE, YorkJD (2007) Cloning and characterization of two human VIP1-like inositol hexakisphosphate and diphosphoinositol pentakisphosphate kinases. J Biol Chem 282: 30754–30762.

48. MuluguS, BaiW, FridyPC, BastidasRJ, OttoJC, et al. (2007) A conserved family of enzymes that phosphorylate inositol hexakisphosphate. Science 316: 106–109.

49. WundenbergT, MayrGW (2012) Synthesis and biological actions of diphosphoinositol phosphates (inositol pyrophosphates), regulators of cell homeostasis. Biol Chem 393: 979–998.

50. BhandariR, JuluriKR, ResnickAC, SnyderSH (2008) Gene deletion of inositol hexakisphosphate kinase 1 reveals inositol pyrophosphate regulation of insulin secretion, growth, and spermiogenesis. Proc Natl Acad Sci U S A 105: 2349–2353.

51. SzijgyartoZ, GaredewA, AzevedoC, SaiardiA (2011) Influence of inositol pyrophosphates on cellular energy dynamics. Science 334: 802–805.

52. RehwinkelJ, TanCP, GoubauD, SchulzO, PichlmairA, et al. (2010) RIG-I detects viral genomic RNA during negative-strand RNA virus infection. Cell 140: 397–408.

53. PadmanabhanU, DollinsDE, FridyPC, YorkJD, DownesCP (2009) Characterization of a selective inhibitor of inositol hexakisphosphate kinases: use in defining biological roles and metabolic relationships of inositol pyrophosphates. J Biol Chem 284: 10571–10582.

54. KilariRS, WeaverJD, ShearsSB, SafranyST (2013) Understanding inositol pyrophosphate metabolism and function: kinetic characterization of the DIPPs. FEBS Lett 587: 3464–3470.

55. LonettiA, SzijgyartoZ, BoschD, LossO, AzevedoC, et al. (2011) Identification of an evolutionarily conserved family of inorganic polyphosphate endopolyphosphatases. J Biol Chem 286: 31966–31974.

56. ZengW, XuM, LiuS, SunL, ChenZJ (2009) Key role of Ubc5 and lysine-63 polyubiquitination in viral activation of IRF3. Mol Cell 36: 315–325.

57. WeaverJD, WangH, ShearsSB (2012) The kinetic properties of a human PPIP5K reveal that its kinase activities are protected against the consequences of a deteriorating cellular bioenergetic environment. Biosci Rep 33 (2) pii: e00022.

58. AzevedoC, BurtonA, Ruiz-MateosE, MarshM, SaiardiA (2009) Inositol pyrophosphate mediated pyrophosphorylation of AP3B1 regulates HIV-1 Gag release. Proc Natl Acad Sci U S A 106: 21161–21166.

59. BhandariR, SaiardiA, AhmadibeniY, SnowmanAM, ResnickAC, et al. (2007) Protein pyrophosphorylation by inositol pyrophosphates is a posttranslational event. Proc Natl Acad Sci U S A 104: 15305–15310.

60. RileyAM, WangH, WeaverJD, ShearsSB, PotterBV (2012) First synthetic analogues of diphosphoinositol polyphosphates: interaction with PP-InsP5 kinase. Chem Commun (Camb) 48: 11292–11294.

61. JinC, FlavellRA (2013) Innate sensors of pathogen and stress: linking inflammation to obesity. J Allergy Clin Immunol 132: 287–294.

62. WangH, FalckJR, HallTM, ShearsSB (2012) Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding. Nat Chem Biol 8: 111–116.

63. ChakrabortyA, KimS, SnyderSH (2011) Inositol pyrophosphates as mammalian cell signals. Sci Signal 4: re1.

64. LeeYS, MuluguS, YorkJD, O'SheaEK (2007) Regulation of a cyclin-CDK-CDK inhibitor complex by inositol pyrophosphates. Science 316: 109–112.

65. SaiardiA, BhandariR, ResnickAC, SnowmanAM, SnyderSH (2004) Phosphorylation of proteins by inositol pyrophosphates. Science 306: 2101–2105.

66. KawasakiT, TakemuraN, StandleyDM, AkiraS, KawaiT (2013) The second messenger phosphatidylinositol-5-phosphate facilitates antiviral innate immune signaling. Cell Host Microbe 14: 148–158.

67. RieberN, KnappB, EilsR, KaderaliL (2009) RNAither, an automated pipeline for the statistical analysis of high-throughput RNAi screens. Bioinformatics 25: 678–679.

68. ManicassamyB, ManicassamyS, Belicha-VillanuevaA, PisanelliG, PulendranB, et al. (2010) Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus. Proc Natl Acad Sci U S A 107: 11531–11536.

69. IwamuraT, YoneyamaM, YamaguchiK, SuharaW, MoriW, et al. (2001) Induction of IRF-3/-7 kinase and NF-kappaB in response to double-stranded RNA and virus infection: common and unique pathways. Genes Cells 6: 375–388.

70. LinR, HeylbroeckC, PithaPM, HiscottJ (1998) Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation. Mol Cell Biol 18: 2986–2996.

71. LossO, AzevedoC, SzijgyartoZ, BoschD, SaiardiA (2011) Preparation of quality inositol pyrophosphates. J Vis Exp e3027.

72. HoenigM, LeeRJ, FergusonDC (1989) A microtiter plate assay for inorganic phosphate. J Biochem Biophys Methods 19: 249–251.

Štítky
Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

Článok vyšiel v časopise

PLOS Pathogens


2014 Číslo 2
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa