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

J Virol. 2009 May;83(9):4174-84. doi: 10.1128/JVI.02449-08. Epub 2009 Feb 18.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenine / metabolism
  • Base Sequence
  • Cell Line, Tumor
  • DEAD-box RNA Helicases / genetics
  • DEAD-box RNA Helicases / metabolism*
  • Hepacivirus / genetics
  • Hepacivirus / metabolism*
  • Humans
  • Interferon-beta / pharmacology
  • Protein Binding
  • RNA, Viral / metabolism*
  • Signal Transduction*
  • Untranslated Regions / genetics
  • Uridine / metabolism

Substances

  • RNA, Viral
  • Untranslated Regions
  • Interferon-beta
  • DEAD-box RNA Helicases
  • Adenine
  • Uridine