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Journal of Virology, May 2009, p. 4174-4184, Vol. 83, No. 9
0022-538X/09/$08.00+0     doi:10.1128/JVI.02449-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Nucleotide Sequences and Modifications That Determine RIG-I/RNA Binding and Signaling Activities {triangledown}

Dina Uzri and Lee Gehrke*

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, and Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received 27 November 2008/ Accepted 10 February 2009

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}/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.

* Corresponding author. Mailing address: HST Division, MIT E25-406, 77 Massachusetts Ave., Cambridge, MA 02139. Phone: (617) 253-7608. Fax: (509) 357-7835. E-mail: Lee_Gehrke{at}hms.harvard.edu

{triangledown} Published ahead of print on 18 February 2009.

Journal of Virology, May 2009, p. 4174-4184, Vol. 83, No. 9
0022-538X/09/$08.00+0     doi:10.1128/JVI.02449-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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