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

Blunt-ended siRNA has been previously reported to be stronger inducers of IFN than the siRNAs with overhangs (47) . Indeed, a previously reported IFN-inducing shRNA, sh-PCAF (p300/CREB-binding protein-associated factor), contains a blunt end (38) and was more potent in activating IFN than sh-B971 ( Figure 5A ), which is predicted to form an overhang of 2-3 Ts at each end of the final siRNA. We therefore constructed a version of the sh-B971 that would be blunt at the end that is not processed by Dicer by adding two extra As to the 5 0 -end of the shRNA. This modification (Blunt sh-B971) did not increase the ability of sh-B971 to activate IFN-b promoter ( Figure 5A ). We confirmed, in two independent experiments, that IFN induction by sh-PCAF was also mediated by RIG-I. First, cotransfection of DN RIG-I resulted a 50-to 100-fold inhibition of IFN induction by sh-PCAF ( Figure 5B ), whereas wild-type RIG-I increased IFN induction by several fold in the same assay. Second, when HCV NS3-4A protease, which cleaves MAVS, thereby blocking the RIG-I pathway, was coexpressed with either sh-B971 or sh-PCAF, IFN induction by these shRNAs were severely compromised ( Figure 5C ), further substantiating a role of the RIG-I and MAVS pathway in mediating IFN induction by both the blunt-ended sh-PCAF and the sh-B971 with overhang. The proper expression of NS3-4A protease was confirmed by western blotting ( Figure 5D ).