We next investigated the role of the different viral/ exogenous RNA sensors, RIG-I, MDA5 and TLR3, in sh-B971-triggered IFN production. Mammalian expression plasmids encoding each of these proteins, as well as the dominant negative (DN) mutants of RIG-I and MDA5, were transfected into 293FT cells with shRNAs and an IFN-b promoter reporter construct. The signaling to IFN-b promoter and the expression of the PRR proteins were then examined 48 h after transfection. In the absence of sensor proteins, the sh-B971 increased activation of the IFN-b promoter by 2.6-fold ( Figure 2A ). Coexpression of MDA5 or TLR3 did not increase or decrease sh-B971's ability to activate IFN-b promoter relatively to the negative control shRNA (sh-NTC), but in the presence of RIG-I coexpression, the induction of IFN-b promoter by sh-B971 was increased to $30-fold. Moreover, ectopic expression of a DN mutant of RIG-I (RIG-I C), but not that of MDA5 (MDA5-C), completely abrogated IFN promoter activation by sh-B971. With the exception of TLR3, which required prolonged exposure of the western blot to be detected, the cytoplasmic sensors and their mutants were expressed at comparable levels ( Figure 2B ). Moreover, activation of IRF-3 ( Figure 1E ) and IFN promoters ( Figure 1F ) in 293FT cells, which do not contain a functional TLR3 signaling pathway (42) , indicates that TLR3 plays a negligible role, if any, in IFN induction by sh-B971. The combination of sh-B971 and RIG-I produced the highest level of IFN-b promoter activity, which were confirmed by western blotting showing that endogenous ISG15 induction was only detectable in cells cotransfected with sh-B971 and wild-type RIG-I ( Figure 2B ). To confirm further that biologically active IFN was released from these cells, we applied the culture medium of the transfected 293FT cells to an HCV replicon cell line (GS5) in which NS5A-GFP expression is used for monitoring viral RNA replication (43) . HCV replication in this cell line is extremely sensitive to IFN, and the effect of the cytokine can be readily measured as the change in the mean GFP intensity of the treated cells. As shown in Figure 2C , culture medium from sh-B971 efficiently suppressed HCV replication, resulting in a decrease in Figure 1 . A small-hairpin RNA directed at CyPB induces IFN production in human embryonic kidney cells. (A) Sequence of sh-B971, which was expressed from a self-inactivating human immunodeficiency virus (HIV) vector with a murine U6 promoter (59) . (B) Inhibition of HCV expression by culture media of sh-B971-transfected 293FT cells. GS5 cells were treated with culture supernatant taken from 293FT cells transfected with various shRNA plasmids with (left) or without (right) the packaging plasmids overnight. Cells were then cultured in fresh media for an additional 6 days before being lysed for western blotting. (C) OAS1 induction by culture supernatant from 293FT cells transfected with sh-B971. Huh 7 and GS5 cells were treated with culture supernatant from 293FT cells transfected with either sh-Luc or sh-B971 for 24 h before RNA extraction and real-time RT-PCR analysis. OAS1 RNA level was normalized to that of GAPDH RNA. (D) Transfected culture media failed to suppress HCV replication in an IFN-resistant cell line. HCV replicon cells were cultured as described earlier (34) and then treated with the indicated culture medium from transfected 293FT cells. HCV RNA was analyzed with real-time RT-PCR. (E) IRF-3 dimerization in response to sh-B971 expression. Flag-IRF-3 was cotransfected with a shRNA into 293FT cells. Cells were lysed 24 h after transfection, and total cell lysate was separated on a polyacrylamide gel under non-denaturing conditions, transferred and stained with an anti-flag antibody. (F) IFN-a and IFN-b promoter activation by sh-B971 expression. Sh-NTC, sh-C454 (an shRNA directed at CyPC), or sh-B971 was cotransfected along with luciferase reporter plasmids with or without IRF-7. The ratios of firefly luciferase readings to Renilla luciferase readings were plotted. the NS5A-GFP intensity within 48 h of treatment. Cotransfecting wild-type RIG-I produced a medium with stronger inhibition, whereas the RIG-C drastically suppressed the antiviral effect of the medium. Finally, real-time RT-PCR analysis revealed that sh-B971, but not the negative control shRNA, strongly activated expression of endogenous RIG-I, a well-characterized ISG whose induction requires paracrine/autocrine action of IFN (44, 45) . As expected, poly I : C activated RIG-I expression in the same assay ( Figure 2D ). These results, taken together, show that RIG-I is the cellular sensor that mediates the IFN induction by sh-B971.