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 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.