Structural and functional insights into 5′-ppp RNA pattern recognition by the innate immune receptor RIG-I

Journal name:
Nature Structural & Molecular Biology
Volume:
17,
Pages:
781–787
Year published:
DOI:
doi:10.1038/nsmb.1863
Received
Accepted
Published online

Abstract

RIG-I is a cytosolic helicase that senses 5′-ppp RNA contained in negative-strand RNA viruses and triggers innate antiviral immune responses. Calorimetric binding studies established that the RIG-I C-terminal regulatory domain (CTD) binds to blunt-end double-stranded 5′-ppp RNA a factor of 17 more tightly than to its single-stranded counterpart. Here we report on the crystal structure of RIG-I CTD bound to both blunt ends of a self-complementary 5′-ppp dsRNA 12-mer, with interactions involving 5′-pp clearly visible in the complex. The structure, supported by mutation studies, defines how a lysine-rich basic cleft within the RIG-I CTD sequesters the observable 5′-pp of the bound RNA, with a stacked phenylalanine capping the terminal base pair. Key intermolecular interactions observed in the crystalline state are retained in the complex of 5′-ppp dsRNA 24-mer and full-length RIG-I under in vivo conditions, as evaluated from the impact of binding pocket RIG-I mutations and 2′-OCH3 RNA modifications on the interferon response.

At a glance

Figures

  1. Sequence alignment of RIG-I family of pattern-recognition receptors and ITC studies of the binding of RIG-I CTD domain to RNA as a function of 5[prime]-end phosphorylation and duplex/strand status.
    Figure 1: Sequence alignment of RIG-I family of pattern-recognition receptors and ITC studies of the binding of RIG-I CTD domain to RNA as a function of 5′-end phosphorylation and duplex/strand status.

    (a) Sequence alignment of C-terminal regulatory domains (CTDs) of RIG-I, MDA5 and LGP2. The secondary structure of RIG-I is shown over the alignments. Filled blue circles, key residues contacting the α and β phosphates of the 5′-pp dsRNA; open blue circles, residues proposed to contact the modeled γ-phosphate. (b,c) ITC binding curves for RIG-I CTD binding to blunt-end 5′-ppp dsRNA 12-mer (b) and single-stranded 5′-ppp ssRNA (c) in 100 mM NaCl and 2 mM MgCl2 buffer. (d,e) ITC binding curves for RIG-I CTD domain binding to blunt-end 5′-ppp dsRNA 12-mer (d) and blunt-end 5′-OH dsRNA 12-mer (e) in 250 mM NaCl and 2 mM MgCl2 buffer.

  2. Details of the crystal structure of the RIG-I CTD bound to blunt-end 5[prime]-pp dsRNA 12-mer.
    Figure 2: Details of the crystal structure of the RIG-I CTD bound to blunt-end 5′-pp dsRNA 12-mer.

    (a) Crystal structure of the RIG-I CTD bound to 5′-pp dsRNA 12-mer (green). CTDs (salmon) are bound to both ends of the 5′-pp dsRNA 12-mer. (b) Schematic representation highlighting intermolecular hydrogen bonding and stacking contacts in the complex. (c) Details of the intermolecular contacts in the structure of the complex. The CTD is in a ribbon representation (salmon), and the 5′-pp dsRNA 12-mer is in a stick representation (green, with backbone phosphorus atoms in yellow). Red dotted lines, intermolecular hydrogen bonds between the α and β 5′-phosphates to residue side chains lining the CTD recognition pocket. The shaded aromatic ring of Phe853 is stacked on both bases of the terminal base pair. (d) A view similar to panel c except for an electrostatic representation of the 5′-phosphorylated RNA-binding surface of the CTD. The RNA is in a ribbon representation and the CTD-binding pocket in an electrostatic surface representation; blue and red patches, basic and acidic regions, respectively.

  3. Role of 5[prime]-phosphorylated ends in the crystal structure of the RIG-I CTD bound to blunt-end 5[prime]-pp dsRNA 12-mer and comparison of the electrostatics of the binding surfaces of RIG-I, MDA5 and LGP2 CTDs for 5[prime]-ppp dsRNA.
    Figure 3: Role of 5′-phosphorylated ends in the crystal structure of the RIG-I CTD bound to blunt-end 5′-pp dsRNA 12-mer and comparison of the electrostatics of the binding surfaces of RIG-I, MDA5 and LGP2 CTDs for 5′-ppp dsRNA.

    (a) Modeling of the γ 5′-phosphate onto the bound 5′-pp dsRNA 12-mer in the structure of the complex. The RNA is in a ribbon representation and the CTD-binding pocket is in an electrostatic surface representation. (b) Comparison in stereo of the superposed crystal structures of the RIG-I CTD bound to blunt-end 5′-pp dsRNA 12-mer (salmon; for clarity, only two terminal base pairs are shown) with RIG-I CTD in the free state22 (blue; PDB 2QFB). Conformational changes are seen for the loop spanning positions 847–856 and also in the alignment of Phe853, between the superposed structures; these are highlighted in darker colors. (c) The electrostatics of the binding surface of RIG-I CTD recognized by blunt-end 5′-ppp dsRNA 12-mer (shown by dashed red circle). (d) The electrostatics of the corresponding binding surface of MDA5 CTD. (e) The electrostatics of the corresponding binding surface of LGP2 CTD.

  4. ITC and electrophoretic mobility shift studies of the binding of blunt-end 5[prime]-ppp dsRNA 12-mer to wild-type and mutants of RIG-I CTD.
    Figure 4: ITC and electrophoretic mobility shift studies of the binding of blunt-end 5′-ppp dsRNA 12-mer to wild-type and mutants of RIG-I CTD.

    ITC binding curves for blunt-end 5′-ppp dsRNA 12-mer binding to wild-type (a), H847A single mutant (b), H847A K858A double mutant (c) and H847A K858A K851A triple mutant (d) RIG-I CTD in 100 mM NaCl and 2 mM MgCl2 buffer. (e) Palindromic radiolabeled 5′-ppp dsRNA 12-mer was incubated at increasing concentrations of recombinant protein under the same salt and buffer conditions as those of the ITC experiment. The complexes were resolved on a native polyacrylamide gel. Increasing numbers of mutation in the triphosphate-binding pocket of the protein weaken RNA binding and reduce the distinct gel shift seen for the wild-type protein to a smear. To ensure that the palindromic 5′-ppp RNA was exclusively present as dsRNA rather than a hairpin, we preannealed the RNA at 5 μM strand concentration and only diluted it to 20 nM before the incubation with protein.

  5. In vivo analysis of the impact of point mutants on the biological activity of RIG-I.
    Figure 5: In vivo analysis of the impact of point mutants on the biological activity of RIG-I.

    (a) Human RIG-I (WT) or RIG-I mutants as indicated were overexpressed in human HEK 293 cells and stimulated with 5 nM single-stranded (5′-ppp ssRNA, 5′-ppp GFP2) or double-stranded (5′-ppp dsRNA, 5′-ppp GFP2 + AS GFP2) synthetic triphosphorylated RNA or nonmodified double-stranded RNA (5′-OH dsRNA, 5′-OH GFP2 + As GFP2). IP-10 was analyzed in the supernatants of cells 24 h after stimulation. Data from three independent experiments are depicted as mean values ± s.e.m. (b) Positions of residues that were mutated in the crystal structure of the 5′-pp dsRNA 12-mer bound to RIG-I CTD.

  6. In vivo analysis of 5[prime]-ppp RNA strand 2[prime]-OCH3 substituent effects.
    Figure 6: In vivo analysis of 5′-ppp RNA strand 2′-OCH3 substituent effects.

    (a) Synthetic 5′-ppp GFP2 and the corresponding 2′-O-methyl derivatives (5′-ppp GFP2 OMe1–OMe6) were hybridized with the complementary antisense strand (GFP2 AS) and transfected into human chloroquine-treated PBMCs. GFP2 AS was used as a control. IFN-α production was analyzed 20 h after stimulation. Data from four independent donors are depicted as normalized mean values ± s.e.m. (b) Position of 2′-OH groups (red) of nucleosides 1, 2 and 3 adjacent to the 5′-phosphorylated end in the crystal structure of the 5′-pp dsRNA 12-mer bound to RIG-I CTD. Residues that either hydrogen bond to or are in close proximity of the 2′-OH groups are also labeled in the figure.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Takeuchi, O. & Akira, S. Pattern recognition receptors and inflammation. Cell 140, 805820 (2010).
  2. Yoneyama, M. & Fujita, T. Recognition of viral nucleic acids in innate immunity. Rev. Med. Virol. 20, 422 (2010).
  3. Wilkins, C. & Gale, M. Jr. Recognition of viruses by cytoplasmic sensors. Curr. Opin. Immunol. 22, 4147 (2010).
  4. Rehwinkel, J. & Reis e Sousa, C. RIGorous detection: exposing virus through RNA sensing. Science 327, 284286 (2010).
  5. Coch, C. et al. Higher activation of TLR9 in plasmacytoid dendritic cells by microbial DNA compared with self-DNA based on CpG-specific recognition of phosphodiester DNA. J. Leukoc. Biol. 86, 663670 (2009).
  6. Poeck, H. et al. 5′-triphosphate-siRNA: turning gene silencing and Rig-I activation against melanoma. Nat. Med. 14, 12561263 (2008).
  7. Barchet, W., Wimmenauer, V., Schlee, M. & Hartmann, G. Accessing the therapeutic potential of immunostimulatory nucleic acids. Curr. Opin. Immunol. 20, 389395 (2008).
  8. Barral, P.M. et al. Functions of the cytoplasmic RNA sensors RIG-I and MDA-5: key regulators of innate immunity. Pharmacol. Ther. 124, 219234 (2009).
  9. Takeuchi, O. & Akira, S. Innate immunity to virus infection. Immunol. Rev. 227, 7586 (2009).
  10. Yoneyama, M. & Fujita, T. RNA recognition and signal transduction by RIG-I-like receptors. Immunol. Rev. 227, 5465 (2009).
  11. Yoneyama, M. et al. The RNA helicase RIG-I has an essential function in dsRNA-induced innate antiviral response. Nat. Immunol. 5, 730737 (2004).
  12. Besch, R. et al. Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon-independent apoptosis in human melanoma cells. J. Clin. Invest. 119, 23992411 (2009).
  13. Poeck, H. et al. Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1 β production. Nat. Immunol. 11, 6369 (2010).
  14. Hornung, V. et al. 5′-triphosphate RNA is the ligand for RIG-I. Science 314, 994997 (2006).
  15. Pichlmair, A. et al. RIG-I mediated antiviral responds to single-stranded RNA bearing 5′-phosphates. Science 314, 9971001 (2006).
  16. Schmidt, A. et al. 5′-triphosphate RNA requires base-paired structures to activate antiviral signaling via RIG-I. Proc. Natl. Acad. Sci. USA 106, 1206712072 (2009).
  17. Schlee, M. et al. Recognition of the 5′-phosphate by RIG-I helicase requires short blunt dsRNA as contained in the panhandle of negative-strand virus. Immunity 31, 2534 (2009).
  18. Ablasser, A. et al. RIG-I dependent sensing of poly(dA:dT) through the induction of an RNA pol III-transcribed RNA intermediate. Nat. Immunol. 10, 10651072 (2009).
  19. Chiu, Y.H. et al. RNA pol III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell 138, 576591 (2009).
  20. Myong, S. et al. Cytosolic viral sensor RIG-I is a 5′-triphosphate-dependent translocase on dsRNA. Science 323, 10701074 (2009).
  21. Takahasi, K. et al. Non-self RNA-sensing mechanism of RNA-I helicase and activation of antiviral immune responses. Mol. Cell 29, 428440 (2008).
  22. Cui, S. et al. The C-terminal regulatory domain is the RNA 5′-triphosphate sensor of RIG-I. Mol. Cell 29, 169179 (2008).
  23. Ma, J.-B., Ye, K. & Patel, D.J. Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain. Nature 429, 318322 (2004).
  24. Ma, J.B. et al. Structural basis for 5′-end-specific recognition of the guide RNA strand by the A. fugidus PIWI protein. Nature 434, 666670 (2005).
  25. Teplova, M. et al. Structural basis for recognition and sequestration of UUUOH 3′-terminii of nascent mRNA polymerase III transcripts by La autoantigen. Mol. Cell 21, 7585 (2006).
  26. Li, X. et al. The RIG-I-like receptor LGP2 recognizes the termini of dsRNA. J. Biol. Chem. 284, 1388113891 (2009).
  27. Leung, D.W. et al. Structural basis for dsRNA recognition and interferon antagonism by Ebola VP35. Nat. Struct. Mol. Biol. 17, 165172 (2010).
  28. Liu, L. et al. Structural basis of Toll-like receptor 3 signaling with double-stranded RNA. Science 320, 379381 (2008).
  29. Utyanskaya, E.Z., Lidskii, B.V., Neihaus, M.G. & Shilov, A.E. Mathematical modeling of kinetics of adenosine 5′-triphosphate hydrolysis catalyzed by Zn2+ ion in the pH range 7.1 to 7.4. J. Inorg. Biochem. 81, 239258 (2000).
  30. Ludwig, J. & Eckstein, F. Rapid and efficient synthesis of nucleoside 5′-O-(1-thiotriphosphates), 5′-triphosphates and 2′,3′-cyclophosphorothioates using 2-chloro-4h-1,3,2-benzodioxaphosphorin-4-one. J. Org. Chem. 54, 631635 (1989).
  31. Anderson, A.C. et al. HPLC purification of RNA for crystallography and NMR. RNA 2, 110117 (1996).
  32. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307326 (1997).
  33. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658674 (2007).
  34. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 21262132 (2004).
  35. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213221 (2010).
  36. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283291 (1993).

Download references

Author information

  1. These authors contributed equally to this work.

    • Yanli Wang &
    • Janos Ludwig

Affiliations

  1. Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Yanli Wang,
    • Haitao Li,
    • Gang Sheng &
    • Dinshaw J Patel
  2. Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, University of Bonn, Bonn, Germany.

    • Janos Ludwig,
    • Christine Schuberth,
    • Marion Goldeck,
    • Martin Schlee &
    • Gunther Hartmann
  3. Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, New York, USA.

    • Stefan Juranek &
    • Thomas Tuschl
  4. Institute for Organic Chemistry, Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria.

    • Ronald Micura

Contributions

Y.W. expressed the RIG-I CTD domain, with G.S. assisting with protein expression and purification; Y.W. crystallized and solved the structure of the complex; J.L. was responsible for chemical synthesis of palindromic and non-palindromic 5′-ppp RNAs and 2′-O-methyl-modified 5′-ppp RNAs; C.S., M.G. and M.S. were responsible for the in vivo functional assays, including expression of full-length RIG-I mutants; H.L. and Y.W. were responsible for ITC titration assays; S.J. was responsible for gel-shift assays and prepared the plasmids for mutant RIG-I CTD expression; R.M. provided earlier batches of short 5′-ppp RNAs for crystallization trials; the paper was written by D.J.P., G.H. and T.T. with the assistance of the other authors.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (596K)

    Supplementary Tables 1–3 and Supplementary Figures 1–4

Additional data