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

To assess the contribution of the promoter choice in IFN activation by intracellular expressed shRNA, we expressed sh-B971 from another commonly used pol III promoter, the human H1 promoter. Both the original, mU6-driven sh-B971 and the H1-driven sh-B971 activated IFN-b promoter ( Figure 6A ) and resulted in secretion of IFN into the transfected cell-culture media, which in turn suppressed HCV replication ( Figure 6B ). Proper expression of the siRNA ( Figure 6C ) and the subsequent knockdown of CyPB expression ( Figure 6D ) all appeared normal for sh-B971 expressed from the H1 promoter plasmid, which has a backbone different from that of our lentiviral vector carrying the mU6 promoter. These data suggest that IFN induction by sh-B971 is not restricted to a particular promoter or expression construct. Further supporting this conclusion was the observation that the expression cassette by itself, removed and isolated from the lentiviral plasmid by restriction digestion, could also activate IFN production in transfected 293FT cells (data not shown).