5′-triphosphate RNA requires base-paired structures to activate antiviral signaling via RIG-I

Abstract

The ATPase retinoid acid-inducible gene (RIG)-I senses viral RNA in the cytoplasm of infected cells and subsequently activates cellular antiviral defense mechanisms. RIG-I recognizes molecular structures that discriminate viral from host RNA. Here, we show that RIG-I ligands require base-paired structures in conjunction with a free 5-triphosphate to trigger antiviral signaling. Hitherto unavailable chemically synthesized 5-triphosphate RNA ligands do not trigger RIG-I-dependent IFN production in cells, and they are unable to trigger the ATPase activity of RIG-I without a base-paired stretch. Consistently, immunostimulatory RNA from cells infected with a virus recognized by RIG-I is sensitive to double-strand, but not single-strand, specific RNases. In vitro, base-paired stretches and the 5-triphosphate bind to distinct sites of RIG-I and synergize to trigger the induction of signaling competent RIG-I multimers. Strengthening our model of a bipartite molecular pattern for RIG-I activation, we show that the activity of supposedly ''single-stranded'' 5-triphosphate RNAs generated by in vitro transcription depends on extended and base-paired by-products inadvertently, but commonly, produced by this method. Together, our findings accurately define a minimal molecular pattern sufficient to activate RIG-I that can be found in viral genomes or transcripts.immunostimulatory RNA ͉ melanoma differentiation-associated protein 5 ͉ retinoid acid-inducible gene-I-like helicases ͉ virus infection ͉ interferon production

Preference of RIG-I for short viral RNA molecules in infected cells revealed by next-generation sequencing

“…The preference of RIG-I for shorter substrates might explain why influenza virus has been shown to be exclusively recognized by RIG-I unlike most other RNA viruses that normally rely on both RIG-I and MDA5 recognition. All of the molecules we have identified as specifically interacting with RIG-I also contain some dsRNA regions directly adjacent to the triphosphate, supporting conclusions from previous work that this panhandle-type architecture of the RNA molecule may be preferentially recognized by RIG-I (12,13). The identification of the influenza virus NS segment and SeV DI RNA as preferred substrates of RIG-I also agrees with their predicted roles as RIG-I PAMPs on the basis of the U-rich composition of this RNA (22).…”

“…Some viruses, such as picornaviruses and influenza virus, appear to be recognized by only one of the sensors, with picornaviruses being sensed by MDA5 and influenza viruses by RIG-I (1,8,9). The substrate specificities of RIG-I and MDA5 have not been clearly established, although from RNA transfection experiments in knockout cells it appears that RIG-I recognizes RNA of various lengths with 5′-triphosphates and some partial double-stranded characteristics, whereas MDA5 senses only very long dsRNA molecules (>2,000 nt) in a phosphate-independent manner (10)(11)(12)(13)(14). All RLRs are members of the DExD/H family of RNA helicases and contain an ATP-dependent helicase domain and a C-terminal regulatory domain (RD).…”

“…Additionally, 5′ppp containing RNAs rich in U residues have been found to act as more potent inducers of RIG-I, indicating that sequence composition might play a role in activation (22). It is yet unclear whether ssRNA, even in the presence of a 5′-triphosphate group, is capable of inducing RIG-I activity, and at least in the case of shorter RNA molecules it appears that some double-stranded characteristics are required for its activation (12,13).…”

Influenza A virus-generated small RNAs regulate the switch from transcription to replication

“…S3B) (40). These results are consistent with the model that cellular antiviral helicases require an exposed 5′ triphosphate, with additional secondary structure and length requirements (41)(42)(43).…”

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].…”