Nucleotide sequences and modifications that determine RIG-I/RNA binding and signaling activities

• Main Authors: Urzi, Dina (Contributor), Gehrke, Lee (Contributor)
• Other Authors: Harvard University- (Contributor)
• Format: Article
• Language: English
• Published: American Society for Microbiology, 2012-03-28T19:20:47Z.
• Subjects: Article
• Online Access: Get fulltext

Cytoplasmic viral RNAs with 5' triphosphates (5'ppp) are detected by the RNA helicase RIG-I, initiating downstream signaling and alpha/beta interferon (IFN-α/β) expression that establish an antiviral state. We demonstrate here that the hepatitis C virus (HCV) 3' untranslated region (UTR) RNA has greater activity as an immune stimulator than several flavivirus UTR RNAs. We confirmed that the HCV 3'-UTR poly(U/UC) region is the determinant for robust activation of RIG-I-mediated innate immune signaling and that its antisense sequence, poly(AG/A), is an equivalent RIG-I activator. The poly(U/UC) region of the fulminant HCV JFH-1 strain was a relatively weak activator, while the antisense JFH-1 strain poly(AG/A) RNA was very potent. Poly(U/UC) activity does not require primary nucleotide sequence adjacency to the 5'ppp, suggesting that RIG-I recognizes two independent RNA domains. Whereas poly(U) 50-nt or poly(A) 50-nt sequences were minimally active, inserting a single C or G nucleotide, respectively, into these RNAs increased IFN-β expression. Poly(U/UC) RNAs transcribed in vitro using modified uridine 2' fluoro or pseudouridine ribonucleotides lacked signaling activity while functioning as competitive inhibitors of RIG-I binding and IFN-β expression. Nucleotide base and ribose modifications that convert activator RNAs into competitive inhibitors of RIG-I signaling may be useful as modulators of RIG-I-mediated innate immune responses and as tools to dissect the RNA binding and conformational events associated with signaling.
United States. Public Health Service (award GM42504)
United States. Public Health Service (award P30 DK034854)