Immune Netw.  2016 Aug;16(4):249-255. 10.4110/in.2016.16.4.249.

Dependence of RIG-I Nucleic Acid-Binding and ATP Hydrolysis on Activation of Type I Interferon Response

• Baek YM
• Yoon S
• Hwang YE
• Kim DE

• KMID: 2349886
• DOI: http://doi.org/10.4110/in.2016.16.4.249

Abstract

Exogenous nucleic acids induce an innate immune response in mammalian host cells through activation of the retinoic acid-inducible gene I (RIG-I). We evaluated RIG-I protein for RNA binding and ATPase stimulation with RNA ligands to investigate the correlation with the extent of immune response through RIG-I activation in cells. RIG-I protein favored blunt-ended, double-stranded RNA (dsRNA) ligands over sticky-ended dsRNA. Moreover, the presence of the 5'-triphosphate (5'-ppp) moiety in dsRNA further enhanced binding affinity to RIG-I. Two structural motifs in RNA, blunt ends in dsRNA and 5'-ppp, stimulated the ATP hydrolysis activity of RIG-I. These structural motifs also strongly induced IFN expression as an innate immune response in cells. Therefore, we suggest that IFN induction through RIG-I activation is mainly determined by structural motifs in dsRNA that increase its affinity for RIG-I protein and stimulate ATPase activity in RIG-I.

Keyword

Retinoic acid inducible gene I (RIG-I); Interferon induction; 5'-terminal triphosphate; ATP hydrolysis; RNA binding

MeSH Terms

Adenosine Triphosphatases
Adenosine Triphosphate*
Hydrolysis*
Immunity, Innate
Interferon Type I*
Ligands
Nucleic Acids
RNA
RNA, Double-Stranded
Adenosine Triphosphatases
Adenosine Triphosphate
Interferon Type I
Ligands
Nucleic Acids
RNA
RNA, Double-Stranded

Figure

• 

  Figure 1 Binding affinities of various types of duplex nucleic acid ligands to RIG-I protein. (A) Increasing amounts of RIG-I protein were incubated with each duplex nucleic acid ligand; the dsRNA ligands included 3'-end overhang (47R/25RI), 5'-end overhang (47R/25RII), double overhang (47R/25RIII)' and RNA:DNA hybrids including 3'-end overhang (47R/25DI) and 5'-end overhang (47R/25DII). Nucleic acid ligands complexed with the RIG-I protein appeared as slowly migrating bands on 2% agarose gel after the electrophoretic mobility shift assay (EMSA). The nucleic acid band intensities were quantitated by ImageJ software, and the extent of nucleic acid ligands bound to the RIG-I protein were plotted as normalized values relative to the control (No RIG-I protein). Each curve was fitted to the Hill equation; Y=A×Lh/(Bh+Lh), where Y represents for relative fraction of RNA ligands bound to RIG-I at each RNA ligand concentration (L), A represents for the amount of RNA ligands complexed to RIG-I at equilibrium, and B and h represents apparent dissociation constant (i.e. Kd) and the Hill coefficient, respectively. Each apparent Kd (in µM) value was obtained from the fitting of the curve to the above equation using Sigma Plot, and represented as the bar graph (inset). (B) Increasing amounts of RIG-I protein were incubated with double-stranded nucleic acids with 3'-end overhang and 5'-end triphosphate (3P-47R/25RI and 3P-47R/25DI) or 5'-end-OH (47R/25RI and 47R/25DI). After the EMSA, the migration of the nucleic acid ligands was visualized on the gel. The upper band represents nucleic acids bound to RIG-I, whereas the bottom band shows unbound free nucleic acid ligands. The nucleic acid band intensities were quantitated by ImageJ software, and the extent of nucleic acid ligands bound to the RIG-I protein were plotted as normalized values relative to the control (No RIG-I protein). Each curve was fitted to the Hill equation; the amplitudes of each curve were different at equilibrium.

• 

  Figure 2 Stimulation of ATP hydrolysis by RIG-I with various types of nucleic acid ligands. ATP (200 M) was mixed with RIG-I (20 nM) in the presence of each nucleic acid ligand (0.5 nM). (A) ATP hydrolysis by RIG-I was monitored over time in the presence of dsRNAs including 3'-end overhang (47R/25RI), 5'-end overhang (47R/25RII), and double overhang (47R/25RIII). The thin-layer chromatography plates show the progress of the ATP hydrolysis reaction, in which ATP hydrolysis products (radioactive inorganic phosphate, Pi*) and radioactive substrates ATP* were resolved and quantified. (B) The ATPase activity of RIG-I over time was monitored in the presence of ssRNAs including 5'-ppp RNA (3P-47R) and ssRNA or ssDNA with a 5'-end hydroxyl group (47R, 25RI, and 25DI).

• 

  Figure 3 Interferon induction in lung epithelial cells with various types of nucleic acid ligands through RIG-I activation. A549 cells were transfected with the 5'-ppp or 5'-HO RNA substrates or the RNA:DNA hybrid substrate. A549 cells containing the pGL3-IFNβ reporter gene were transfected with each duplex RNA ligand at two different concentrations (10 nM or 50 nM). The luciferase reporter assay was conducted at 18 h post-transfection with each RNA ligand. Activation of the IFN-β promoter with the exogenous RNA ligand is shown in the graph as the fold-index relative to a mock transfection control (transfection reagent only). Transfections with yeast tRNA and poly I:C were performed for negative and positive controls, respectively. The unit fold was set to the IFN expression observed in the mock transfection. The values shown are the means±S.D. of triplicate experiments.

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