To this point, all the IFN induction experiments were done with transient transfection of DNA vectors and it was possible that certain features of the double-stranded plasmid DNA are responsible for IFN induction. We first tried to address this point by transfecting just the shRNAexpressing cassette, generated either by PCR or restriction enzyme digestion, into 293FT cells and confirming that these fragments of $200 bp were sufficient to trigger IFN induction (Supplementary Figure S1) . To definitively rule out any contribution by dsDNA, we used a lentiviral transduction system which has been suggested to express shRNAs that can escape detection by PRRs and IFN activation (48) . We produced lentiviral particles containing shRNAs from 293FT cells using standard . Sh-B971 expressed from an H1 promoter triggers IFN activation. Sh-B971 expressed from an H1 promoter was capable of (A) activating IFN-b promoter and (B) triggering IFN production to inhibit HCV replication in GS5 cells. (C) Intracellular levels of U6-and H1-driven sh-B971 products. RNA extraction and northern blotting were performed as described in Figure 4B . (D) Knockdown of CyPB expression by sh-B971 expressed from an H1 promoter. methods, centrifuged them to separate the vectors from the IFN-containing media, and then used them to infect naı¨ve 293FT cells ( Figure 7A ). Both sh-B971 and sh-PCAF vectors induced IFN production when delivered as concentrated lentiviral particles, measured both by HCV suppression ( Figure 7B ) and by OAS induction ( Figure 7C ) in Huh-7 cells. To rule out the possibility that residual IFN in the concentrated viral particles was responsible for these results, we added 100 U/ml IFN to the negative control vector sample before the concentration step. This preparation, designated sh-NTC*, was not able to trigger IFN production in naı¨ve 293FT cells, suggesting that the concentration step effectively removed the soluble IFN from the viral particle pellet. Proper knockdown of the siRNA target of sh-B971 was confirmed by this route of shRNA delivery ( Figure  7D ). To prove definitively that IFN induction by the shRNAs was mediated by the lentiviral infection route, we tested the effect of an inhibitor of HIV reverse transcriptase, Nevirapine, on IFN induction by sh-B971 and sh-PCAF. As shown in Figure 7E , inclusion of Nevirapine at the time of transduction effectively blocked the ability of both shRNAs to induce IFN in the transduced cells, suggesting the importance of the reverse transcription step in the expression of the shRNAs delivered by the lentiviruses. To determine whether lentiviral vector-delivered shRNA can trigger IFN induction in cells other than 293FT cells, we transduced a human hepatoma cell line, LH86, which has been reported to produce IFN upon viral infection (33) , and examined IFN induction in these cells. Culture medium from LH86 cells transduced with sh-PCAF contained biologically active IFN, which suppressed HCV replication in GS5 cells ( Figure 7F ), indicating that the ability of shRNAs delivered by lentivirus to induce IFN response was not limited to 293FT cells.