Nucleotide Modifications of RNA Suppress RIG-I Antiviral Signaling by Unique Mechanisms

Abstract

In order to counter pathogen infection while preventing autoimmune responses, the human innate immune system must be precisely regulated to distinguish “self” from “non-self”. Pattern recognition receptors detect “non-self” pathogen RNAs and initiate antiviral signaling. Accumulated evidence suggests that host “self” RNAs contain modified nucleotides that evade or suppress immune signaling; however, the precise mechanisms are not understood. Defining these mechanisms is relevant toward understanding the biology of immunity as well as the applied use of RNAs as therapeutic molecules, where reducing ligand immunogenicity is essential. Evidence from our lab and others’ suggests that the cytosolic RNA helicase RIG-I (retinoic acid inducible gene-I) detects not only the 5’ terminus and double-stranded nature of RNA, but also the presence/absence of modified nucleotides. In the present study, we use a model RNA ligand (polyU/UC), derived from the 3’ untranslated region of the hepatitis C virus RNA, to dissect the mechanisms by which RNAs containing nucleotide modifications suppress or evade RIG-I signaling. Five assays were developed to test our hypothesis that eight different nucleotide modifications, both natural and synthetic, share a common mechanism of innate immune evasion. In vitro transcribed 5’-triphosphate polyU/UC RNA containing canonical nucleotides potently activates the RIG-I signaling pathway in transfected cells, culminating in an antiviral state. When transcribed with any of eight modified nucleotides, the polyU/UC RNA suppressed the RIG-I antiviral response. Unexpectedly, the modified nucleotides had different effects on RIG-I:RNA binding affinity, as well as RIG-I conformational change. The data suggest that multiple RIG-I evasion/suppression mechanisms associated with different modified nucleotides may have evolved to effect a common result of interrupting innate immune signaling responses to “self” RNA. Our findings hold implications for understanding the co-evolution of the innate immune response and RNA modification pathways across domains of life, as well as for defining approaches for testing the multitude of naturally occurring and synthetic nucleotides that may have utility in the design of therapeutic RNAs.