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

The majority of the shRNAs that we use in the lab do not activate RIG-I expression and IFN signaling despite having essentially the same structure as sh-B971, so we wanted to determine whether the sequence of sh-B971 is distinctive enough to trigger the production of IFN. We first tested a synthetic siRNA duplex with the same target sequence as sh-B971. This siRNA (si-B971-syn) should resemble the final Dicer product of sh-B971 except for the 5 0 -ends. The synthetic siRNA contains 5 0 -OH groups, whereas the Dicer products probably Figure 3A ) while failing to activate IFN production, as measured by the GFP-HCV assay ( Figure 3B ). To determine whether the sequence of the intact hairpin RNA before Dicer cleavage is sufficient to trigger IFN, we tested a synthetic shRNA (sh-B971-syn) that had exactly the same sequence as the predicted intracellular sh-B971 transcript generated by the U6 promoter. Again, the 5 0 -end of the synthetic sh-B971 had a 5 0 -OH group instead of any phosphate. Sh-B971-syn behaved similarly to si-B971-syn in that it knocked down CyPB expression without activating IFN response (Figure 3) . These results suggest that the 5 0 -end status of sh-B971 is important for IFN activation, consistent with the previously finding that a 5 0 -triphosphate is required for RIG-I activation (24, 25) .