Abstract

RNA-specific adenosine-deaminase (ADAR)-mediated adenosine to inosine (A-to-I) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of non-self RNA by innate immune sensors, and accordingly investigated the impact of inosine-modified RNA on Toll-like receptor (TLR) 7/8 sensing. Inosine incorporation into synthetic single-stranded (ss) RNA potentiated tumor necrosis factor alpha (TNF-alpha) or interferon alpha (IFN-alpha) production in human peripheral blood mononuclear cells (PBMCs), in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs, and was not seen with inosine-containing short double-stranded RNAs, nor with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated IFN-alpha production in human PBMCs through TLR7 recruitment, as established through the use of a TLR7 antagonist and Tlr7-deficient cells. There was a correlation between hyper-editing of influenza A viral ssRNA and its ability to stimulate TNF-alpha, independent of 5' -triphosphate residues, and involving Adar-1. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate TLR7/8 sensing of phagocytosed viral RNA.