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.

Importance: The innate immune response to pathogens is based on the discrimination between self-RNA and non-self-RNA. The main determinants of this detection for RNA viruses are specific pathogen-associated molecular patterns (PAMPs) of RNA, which are detected by dedicated cytoplasmic pattern recognition receptors (PRRs). RIG-I is a PRR that specifically detects short viral dsRNAs amid a sea of cellular RNAs. Here we study the determinants of this discrimination and how RIG-I ATPase activity, the only enzymatic activity of this sensor, contributes to its activation in a manner restricted to its interaction with non-self-RNAs. We also show how the innate immune response evolves during infection via IFN expression, from a state in which discrimination of self-RNA from non-self-RNA is most important to one in which this discrimination is sacrificed for the effectiveness of the antiviral response.