Recognition of 5′ Triphosphate by RIG-I Helicase Requires Short Blunt Double-Stranded RNA as Contained in Panhandle of Negative-Strand Virus

Abstract

Antiviral immunity is triggered by immunorecognition of viral nucleic acids. The cytosolic helicase RIG-I is a key sensor of viral infections and is activated by RNA containing a triphosphate at the 5′end. The exact structure of RNA activating RIG-I remains controversial. Here we established a chemical approach for 5′triphosphate oligoribonucleotide synthesis and found that synthetic single-stranded 5′triphosphate oligoribonucleotides were unable to bind and activate RIG-I. Conversely, the addition of the synthetic complementary strand resulted in optimal binding and activation of RIG-I. Short double strand conformation with base pairing of the nucleoside carrying the 5′triphosphate was required. RIG-I activation was impaired by a 3′overhang at the 5′triphosphate end. These results define the structure of RNA for full RIG-I activation and explain how RIG-I detects negative strand RNA viruses which lack long double-stranded RNA but do contain panhandle blunt short double-stranded 5′triphosphate RNA in their single-stranded genome.

RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA

“…Binding of biotinylated ODNs to purified, His-tagged, human RAGE domains was assayed in a buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% ultrapure BSA, 0.01% Tween 20) using an amplified luminescent proximity assay with streptavidin-conjugated donor beads and nickel chelate acceptor beads (AlphaScreen; Perkin Elmer; Schlee et al, 2009). For cell-binding studies, HeLa cells coexpressing RAGE-CFP and Rab4a-YFP or expressing only Rab4a-YFP were incubated with 1 µM Alexa Fluor 647-labeled ODN 2336 at 37°C for 5 min, washed with medium, and imaged by confocal microscopy.…”

Cooperative assembly and dynamic disassembly of MDA5 filaments for viral dsRNA recognition

“…RIG-I detects a variety of positive and negative strand viruses through recognition of a 5′ triphosphate group and blunt ends of genomic RNAs (3,5,6). By contrast, MDA5 detects several positive strand and dsRNA viruses such as picornaviruses and reoviruses through recognition of dsRNA replication intermediates and genomic dsRNAs (3,4,7).…”

“…The proposed role of ATP hydrolysis as a conformational switch for signaling is supported by the requirement of ATP in the reconstituted signaling system of RIG-I (9, 10) and the observation that mutations in the active site for ATP hydrolysis either constitutively activate or inactivate interferon signaling by RIG-I and MDA5 (11,12). In addition, induction of ATP hydrolysis by dsRNA has been shown to correlate with stimulation of interferon signaling (6,13).…”

Evading innate immunity in nonviral mRNA delivery: don’t shoot the messenger

“…Recent studies however, challenged this hypothesis and demonstrated that activation of RIG-I requires base pairing of the nucleoside carrying the 5'ppp. Evidence was provided that RIG-I is triggered by double-stranded, but not single-stranded, RNA containing 5'ppp [35,36]. In addition, Goubau et al showed that also 5'-diphosphate (5'pp) dsRNA serves as an RIG-I ligand, thereby concluding that a minimal feature for RIG-I activation is a base-paired RNA with a free 5'pp [37].…”