Nucleotide sequences and modifications that determine RIG-I/RNA binding and signaling activities.
Cytoplasmic viral RNAs with 5' triphosphates (5'ppp) are detected by the RNA helicase RIG-I, initiating downstream signaling and alpha/beta interferon (IFN-alpha/beta) 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-beta 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-beta 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.
Uzri
Dina
D
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Gehrke
Lee
L
D000225:Adenine / Q000378:metabolism; D001483:Base Sequence; D045744:Cell Line, Tumor; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016899:Interferon-beta / Q000494:pharmacology; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism*; D015398:Signal Transduction; D020506:Untranslated Regions / Q000235:genetics; D014529:Uridine / Q000378:metabolism
D012367:RNA, Viral; D020506:Untranslated Regions; D016899:Interferon-beta; D053487:DEAD-box RNA Helicases; D000225:Adenine; D014529:Uridine