Short-hairpin RNAs delivered by lentiviral vector transduction trigger RIG-I-mediated IFN activation

Kenworthy, Rachael; Lambert, Diana; Yang, Feng; Wang, Nan; Chen, Zihong; Zhu, Haizhen; Zhu, Fanxiu; Liu, Chen; Li, Kui; Tang, Hengli

Typical IFN-inducing structure patterns include dsRNA of certain length, single-stranded RNA (ssRNA) containing 5 0 -triphosphates (5 0 -ppp), the dsRNA analogue polyinosinic-polycytidylic acid (poly I:C), and certain dsDNA molecules. These RNA patterns are generally believed to possess 'non-self' properties to allow the cell to recognize foreign (often viral) RNAs specifically. Various forms of siRNA duplexes have been reported to trigger IFN induction both in vitro and in vivo (5) (6) (7) (8) (9) , probably through the cell surface-and/or endosomeexpressed Toll-like receptors (TLRs), including TLR3 and TLR7 (6, 8, 9) . Short-hairpin RNAs (shRNAs) expressed from a DNA plasmid have also been shown to activate IFN (10) . The double-stranded form of these RNAs is below the size limit of the stem-loop RNAs that can be detected by the RNA-activated protein kinase (PKR) (11) and is probably detected by other cytoplasmic PRRs. Two cytoplasmic RNA helicases, retinoic-acid-inducible gene I (RIG-I) and melanoma differentiation associated gene 5 (MDA5), signal to the IFN-b promoter when activated by specific RNA structures (12) (13) (14) . Although both PRRs signal through the mitochondrial antiviral signaling protein MAVS/Cardif/VISA/IPS-1 (15) (16) (17) (18) , studies of ligand specificity suggest that RIG-I and MDA5 are parallel sensors with overlapping substrates. For example, although both PRRs are activated by poly I:C in cell culture systems (12, (19) (20) (21) (22) (23) , MDA5 appears to be more important in mediating the poly I:C response in vivo (13, 14) . In addition, RIG-I can bind and respond to ssRNAs bearing 5 0 -ppp, whereas MDA5 is not activated by 5 0 -ppp-containing RNA (24, 25) . Finally, several cytosolic sensors for dsDNA has been recently reported (26) (27) (28) (29) (30) (31) . Nevertheless, current data on what constitutes effective substrates for either PRR are incomplete and sometimes controversial. Here we report for the first time that shRNAs delivered by lentiviral transduction triggered IFN activation and that RIG-I and MAVS, but not MDA5 or TLR3, mediated the IFN activation triggered by intracellularly expressed shRNA, which could activate both IFN-a and IFN-b promoters. IFN activation depended on sequence, a 5 0 -ppp and correct processing of the RNA hairpin by Dicer; it was independent of promoter choice, presence of blunt ends, route of delivery and RNAi potency.