RIG-I ATPase Activity and Discrimination of Self-RNA versus Non-Self-RNA

Abstract

Many RNA viruses are detected by retinoic acid-inducible gene i (RIG-I), a cytoplasmic sensor that triggers an antiviral response upon binding non-self-RNA that contains a stretch of double-stranded RNA (dsRNA) bearing a base-paired 5′ ppp nucleotide. To gain insight into how RIG-I discriminates between self-RNA and non-self-RNA, we used duplexes whose complementary bottom strand contained both ribo- and deoxynucleotides. These duplexes were examined for their binding to RIG-I and their relative abilities to stimulate ATPase activity, to induce RIG-I dimerization on the duplex, and to induce beta interferon (IFN-β) expression. We show that the chemical nature of the bottom strand is not critical for RIG-I binding. However, two key ribonucleotides, at positions 2 and 5 on the bottom strand, are minimally required for the RIG-I ATPase activity, which is necessary but not sufficient for IFN-β stimulation. We find that duplexes with shorter stretches of dsRNA, as model self-RNAs, bind less stably to RIG-I but nevertheless have an enhanced ability to stimulate the ATPase. Moreover, ATPase activity promotes RIG-I recycling on RIG-I/dsRNA complexes. Since pseudo-self-RNAs bind to RIG-I less stably, they are preferentially recycled by ATP hydrolysis that weakens the helicase domain binding of dsRNA. Our results suggest that one function of the ATPase is to restrict RIG-I signaling to its interaction with non-self-RNA. A model of how this discrimination occurs as a function of dsRNA length is presented.

Mismatches in the Influenza A Virus RNA Panhandle Prevent Retinoic Acid-Inducible Gene I (RIG-I) Sensing by Impairing RNA/RIG-I Complex Formation

“…versus ssRNA). In EMSA, addition of ATP and MgCl 2 , which allows RIG-I ATPase activity, had no effect on RIG-I dimerization, as previously observed (12). Interestingly, although these mismatches are positioned in key residues for RIG-I ATPase activity (12), their presence had no effect on RIG-I ATPase activity in vitro, which was higher than that of 20r because of their greater dsRNA length (30 versus 20 bp) (Fig.…”

“…RNA pulldown assays were performed with 13 pmol of biotinylated RNA ligands (as previously described [12]) and 13 pmol of His-RIG-I (16). Reaction products were analyzed by Western blotting with antihistidine antibody (1:2,000, H1029; Sigma).…”

“…Radiolabeled RNAs (25 pmol) were incubated with purified His-RIG-I (50 pmol of RIG-I). Reaction products were analyzed on native gradient acrylamide gel and revealed by phosphorimaging (Typhoon; GE Healthcare Life Sciences) (12). (D) Relative (Rel.)…”

“…Data are presented as the mean Ϯ the standard error of the mean (n ϭ 4). Statistical significance: NS, P Ͼ 0.05; *, 0.01 Յ P Ͻ 0.05; **, P Ͻ 0.001 (12). (E) IFN-␤ promoter activation.…”

“…However, a fraction of the influenza virus genome promoters is likely not bound to the polymerase during infection. As residues close to the 5= ppp blunt end of RNA duplexes are critical for RIG-I ATPase activity (12), the presence of mismatches in influenza virus panhandles raises the possibility that, in addition to their primary role in RNA synthesis, mismatches represent a viral strategy to minimize detection by RIG-I.…”