Nucleotide sequences and modifications that determine RIG-I/RNA binding and signaling activities

Abstract

Cytoplasmic viral RNAs with 5' triphosphates (5'ppp) are detected by the RNA helicase RIG-I, initiating downstream signaling and alpha/beta interferon (IFN-alpha/beta) expression that establish an antiviral state. We demonstrate here that the hepatitis C virus (HCV) 3' untranslated region (UTR) RNA has greater activity as an immune stimulator than several flavivirus UTR RNAs. We confirmed that the HCV 3'-UTR poly(U/UC) region is the determinant for robust activation of RIG-I-mediated innate immune signaling and that its antisense sequence, poly(AG/A), is an equivalent RIG-I activator. The poly(U/UC) region of the fulminant HCV JFH-1 strain was a relatively weak activator, while the antisense JFH-1 strain poly(AG/A) RNA was very potent. Poly(U/UC) activity does not require primary nucleotide sequence adjacency to the 5'ppp, suggesting that RIG-I recognizes two independent RNA domains. Whereas poly(U) 50-nt or poly(A) 50-nt sequences were minimally active, inserting a single C or G nucleotide, respectively, into these RNAs increased IFN-beta expression. Poly(U/UC) RNAs transcribed in vitro using modified uridine 2' fluoro or pseudouridine ribonucleotides lacked signaling activity while functioning as competitive inhibitors of RIG-I binding and IFN-beta expression. Nucleotide base and ribose modifications that convert activator RNAs into competitive inhibitors of RIG-I signaling may be useful as modulators of RIG-I-mediated innate immune responses and as tools to dissect the RNA binding and conformational events associated with signaling.

FIG. 1.

IFN-β induction potentials of HCV and flavivirus UTR RNAs. (A) Twenty-four hours after being plated, Huh7 cells were cotransfected with plasmids encoding firefly or Renilla luciferase under the control of the IFN-β promoter or constitutive cytomegalovirus promoter, respectively. Following a 24-hour further incubation period, the cells were mock transfected or transfected in triplicate with equal numbers of moles of renatured in vitro-transcribed viral 5′- or 3′-UTR or 3′-SL RNAs. Twenty-four hours later, the cells were lysed, and aliquots of the extracts were analyzed using a dual-luciferase assay. The firefly luciferase light unit values were divided by the Renilla light units (transfection efficiency control) to generate the relative luciferase (luc) value. The bars show average relative luciferase values plus standard deviations. (B) Twenty-eight hours after viral-RNA transfection, the cells were lysed and analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting for ISG56 and actin. (C) Huh7 cells were transfected or infected with increasing amounts of HCV 3′-UTR RNA (50 ng, 250 ng, 650 ng, and 1 μg) or Sendai virus (SenV) (50, 100, 250, or 500 hemagglutinin units), and IFN-β reporter activation was measured 24 h later as described in the legend to Fig. 1A.

FIG. 2.

Identification of the poly(U/UC) region of the HCV 3′ UTR as the determinant of RIG-I activation. (A) HCV poly(U/UC) and X RNAs were transcribed in vitro, and equal numbers of moles of the RNAs were transfected into Huh7 cells. Their potencies in activating the IFN-β reporter were determined as for Fig. 1A. The bars show average relative luciferase (luc) values plus standard deviations. (B) Wild-type (WT) or RIG-I knockout (KO) MEFs were mock transfected or transfected in triplicate with equal numbers of moles of in vitro-transcribed HCV 3′-UTR, poly(U/UC), or X RNA. After a 24-hour incubation period, an enzyme-linked immunosorbent assay was used to measure IFN-β protein levels from cell culture media. The bars show average amounts of mouse IFN-β protein levels plus standard deviations. (C) One microgram of biotinylated poly(U/UC) RNA was incubated with or without a 2.5-fold molar excess of nonbiotinylated competitor RNA and 30 μg of FLAG-RIG-I-containing Huh7 cell extract. RNA-protein complexes were recovered by pull-down assay using streptavidin magnetic particles. FLAG-tagged RIG-I protein within the pull-down fraction was analyzed by immunoblotting using M2 anti-FLAG antibody.

FIG. 3.

Separating the 5′ppp and the poly(U/UC) region does not disrupt signaling. (A) Schematic representations of the chimeric RNAs, showing the activating poly(U/UC) region positioned upstream of ss1 and immediately adjacent to the 5′ppp or downstream of ss1 and distant from the 5′ppp. (B) The chimeric RNAs were in vitro transcribed, and their abilities to activate the IFN-β reporter were tested as for Fig. 1A. The bars show average relative luciferase (luc) values plus standard deviations.

FIG. 4.

Examining the roles of RNA sequence composition and length in RIG-I activation. (A) Schematic representations of the different HCV strain J4L6 poly(U/UC) 3′ deletion RNAs tested. The black bars at the top indicate (left to right) 28 contiguous U residues followed by 11 contiguous U residues followed by a downstream U/C region. The gaps between the bars correspond to single cytosine residues. (B to E) Activation of the IFN-β reporter by in vitro-transcribed RNAs. The bars show average relative luciferase (luc) values plus standard deviations. (B) Activation by poly(U/UC) 3′ deletion RNAs. (C) Activation by poly(AG/A) 100-nt (full-length), poly(AG/A) 60-nt, and poly(G/GC) 60-nt RNAs. (D) Activation by 50-nt and 35-nt homopolymeric uridine RNAs and 50-nt homopolymeric adenine RNA. (E) Activation by 50-nt homopolymeric uridine and adenine RNAs interrupted with a single C or G nucleotide, respectively. (F) One microgram of biotinylated poly(U/UC) RNA was incubated with or without threefold molar excess of nonbiotinylated competitor RNA and 30 μg of FLAG-RIG-I cell extract. The competitive-binding assays were carried out as described in the legend to Fig. 2C.

FIG. 5.

Ribose and base modifications affect RNA innate immune stimulation potential. (A and B) Activation of the IFN-β reporter by in vitro-transcribed RNAs. The bars show average relative luciferase (luc) values plus standard deviations. (A) Activation by HCV 3′-UTR and poly(U/UC) RNAs transcribed with 2′F-dUTP in place of UTP or 2′F-dCTP in place of CTP. (B) Activation by poly(U/UC) RNA transcribed with pseudouridine-5′ppp in place of UTP. (C) One microgram of biotinylated poly(U/UC) RNA was incubated with or without a threefold molar excess of nonbiotinylated competitor RNA and 30 μg of FLAG-RIG-I cell extract. The competitive-binding assays were carried out as described in the legend to Fig. 2C. (D) Unmodified poly(U/UC) RNA was transfected alone or with 2′F-dUTP- or pseudouridine-modified poly(U/UC) RNA at 2:1 or 4:1 (modified/unmodified) molar excesses. IFN-β reporter activity was measured 24 h posttransfection as described in the legend to Fig. 1A. The data are presented as percentages of the control unmodified poly(U/UC) RNA activity (100%). The bars show average relative luciferase values plus standard deviations.

FIG. 6.

Comparison of the HCV type 1b J4L6 and HCV type 2a JFH-1 strain poly(U/UC) and poly(AG/A) RNAs as activators of innate immune stimulation. (A) HCV J4L6 and JFH-1 RNAs were transcribed in vitro and transfected into Huh7 cells as described in the legend to Fig. 1A. The bars show average relative luciferase (luc) values plus standard deviations. (B) Twenty-four hours after Huh7 cells were transfected with equal numbers of moles of J4L6 or JFH-1 poly(U/UC), poly(AG/A), or X RNA, the cells were infected with VSV-luc at MOIs of 0.05, 0.1, and 0.15. Four hours postinfection, the cells were lysed and luciferase activity was assayed as a measure of VSV replication. The bars show average luciferase values plus standard deviations.

FIG. 7.

Model defining steps of RIG-I activation and where modified RNAs could block signaling. The actual order of steps may be different than what is shown here. Ribose 2′ hydroxyl and base modifications do not affect binding to RIG-I but may inhibit RIG-I activation at one of the downstream steps. Additional details can be found in the text.