The structural basis of 5' triphosphate double-stranded RNA recognition by RIG-I C-terminal domain

Abstract

RIG-I is a cytosolic sensor of viral RNA that plays crucial roles in the induction of type I interferons. The C-terminal domain (CTD) of RIG-I is responsible for the recognition of viral RNA with 5' triphosphate (ppp). However, the mechanism of viral RNA recognition by RIG-I is still not fully understood. Here, we show that RIG-I CTD binds 5' ppp dsRNA or ssRNA, as well as blunt-ended dsRNA, and exhibits the highest affinity for 5' ppp dsRNA. Crystal structures of RIG-I CTD bound to 5' ppp dsRNA with GC- and AU-rich sequences revealed that RIG-I recognizes the termini of the dsRNA and interacts with the 5' ppp through extensive electrostatic interactions. Mutagenesis and RNA-binding studies demonstrated that similar binding surfaces are involved in the recognition of different forms of RNA. Mutations of key residues at the RNA-binding surface affected RIG-I signaling in cells.

Figure 1

RIG-I CTD binds RNA with or without a 5′ triphosphate. (A) Binding study of RIG-I CTD with a 14-bp GC-rich 5′ ppp dsRNA by gel filtration chromatography. RIG-I CTD is shown by the black chromatogram. The RNA is shown by the red chromatogram. The mixture of the protein and the RNA is shown by the green chromatogram. Elution volumes of four protein standards are shown above the chromatograms. (B) Stoichiometry between RIG-I CTD and the 14-bp GC-rich 5′ ppp dsRNA in a purified protein RNA complex determined by sedimentation velocity (inset) and equilibrium. The experimental molecular weight values from velocity (~38 kDa) and equilibrium (37,339 Da) data were consistent only with the formation of a 2:1 complex. (C) Binding study of RIG-I CTD with a 13-nt 5′ ppp ssRNA by gel filtration chromatography. (D) Binding study of RIG-I CTD with a 14-bp blunt-ended dsRNA without a 5′ triphosphate.

Figure 2

RIG-I CTD binds 5′ triphosphate dsRNA with high affinity. (A) Equilibrium binding study of RIG-CTD with the 14-bp GC-rich 5′ ppp dsRNA by surface plasmon resonance (SPR). The dissociation constant (K_d) was derived by fitting of the equilibrium binding data to a one site binding model (lower panels). (B) Equilibrium binding study of RIG-I CTD with the 14-bp blunt-ended dsRNA. (C) Equilibrium binding study of RIG-I CTD with a 13-nt 5′ ppp ssRNA. (D) Kinetic binding studies of RIG-CTD with the 14-bp GC-rich 5′ ppp dsRNA. The association and dissociation rate constants (k_on and k_off) were derived from global fitting of the binding data to a 1:1 binding model (red curves). Half – life (t_1/2) for each of the RNA: RIG-I CTD complex was calculated from the k_off. The calculated dissociation constant (K_{D, calc}) was derived by division of the dissociation rate constant (k_off) by the association rate constant (k_on). (E) Kinetic binding studies of RIG-I CTD with the 14-bp blunt-ended dsRNA. (F) Kinetic binding studies of RIG-I CTD with the 13-nt 5′ ppp ssRNA.

Figure 3

Structures of RIG-I CTD bound to 5′ triphosphate double-stranded RNA. (A) Crystal structure of RIG-I CTD bound to the 14-bp GC-rich 5′ ppp dsRNA. The nucleotides at the 5′ ends of the dsRNA containing the triphosphate are shown as space filling models. The zinc ions bound to RIG-I CTD are shown as gray spheres. (B) Structure of the RIG-I CTD bound to the 12-bp AU-rich 5′ ppp dsRNA. The orientations of RIG-I CTD (Chain A) relative to the dsRNA are the same in the two structures.

Figure 4

Structural basis of 5′ triphosphate dsRNA recognition by RIG-I CTD. (A) Stereo close up of the interface between RIG-I CTD (Chain A) and the terminus of the 14-bp 5′ ppp dsRNA. RIG-I CTD is shown as cyan ribbons and the dsRNA is shown as stick models. Key residues of RIG-I CTD involved in RNA binding are shown as stick models. (B) Stereo close up of the interactions between RIG-I CTD and the 5′ triphosphate of the 14-bp GC-rich dsRNA. (C) Surface representation of RIG-I CTD bound to 5′ ppp dsRNA showing the shape and charge complementarity between RIG-I CTD and the terminus of the 5′ ppp dsRNA. Positively charged surfaces are colored blue and negatively charged surfaces are red. The orientation of RIG-I CTD relative to the dsRNA on the left panel is similar to the orientation of the protein in panel A.

Figure 5

Distinct binding by RIG-I and LGP2 CTDs to dsRNA with and without 5′ triphosphate. Stereo representation of the structure of RIG-I CTD bound to the 14-bp GC-rich 5′ ppp dsRNA superimposed on the structure of LGP2 CTD bound to an 8-bp dsRNA without 5′ triphosphate. The 14-bp 5′ ppp dsRNA bound to RIG-I CTD is shown as blue ribbons. The 8-bp dsRNA bound to LGP2 CTD is colored magenta. A 14-bp dsRNA (orange) is superimposed on the 8-bp dsRNA in the LGP2 CTD:dsRNA complex structure to show the different orientations of the dsRNA relative the proteins in the two complexes.

Figure 6

Similar binding surfaces of RIG-I CTD mediate the interactions with different forms of RNA. (A) Binding studies of wild type and mutants of RIG-I CTD for the 14-bp 5′ ppp dsRNA by electrophoretic mobility shift assay (EMSA). (B) Binding studies of RIG-I CTD mutants with a 14-bp blunt-ended dsRNA lacking 5′ triphosphate. (C) Binding studies of RIG-I CTD mutants with a 13-nt 5′ ppp ssRNA.

Figure 7

Mutations of key residues at the RNA binding surface affect RIG-I signaling. (A) Western blot showing the expression of wild type and mutants of full-length RIG-I in transfected cells. (B) IFN-β luciferase assay showing the signaling of wild type and mutants of RIG-I in HEK 293T cells stimulated with a 24-bp 5′ ppp dsRNA. (C) IFN-β luciferase assay showing the signaling of wild type and mutants of RIG-I in cells stimulated with a 27-nt 5′ ppp ssRNA. (D) IFN-β luciferase assay showing the signaling of wild type and mutants of RIG-I in cells stimulated with a 27-bp blunt-ended dsRNA without the 5′ triphosphate.