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 investigate the potential role of the cyclophilins (CyPs) in HCV replication (41), we delivered several shRNAs directed at mRNAs of three CyPs into HCV replicon cells by means of a lentiviral vector, using a murine U6 promoter to drive the expression of the shRNA ( Figure 1A ) (37) . We observed a discrepancy between two anti-CyPB shRNAs (B971 and B710) in their relative efficiency in knocking down CyPB expression and in suppressing HCV. Lentiviral vector sh-B971 was less efficient in knocking down CyPB expression but potently inhibited HCV NS5A expression in a human hepatoma cell line containing replicating HCV RNA ( Figure 1B , left). Viral inhibition was independent of CyPB knockdown, as control medium from transfected 293FT cells that did not contain any lentiviral vector particles, generated by omission of the packaging plasmids during transfection, also inhibited HCV replication ( Figure 1B , right) without affecting CyPB expression. The fast kinetics of viral inhibition (complete inhibition with 48 h, data not shown) was also more consistent with IFN than with RNAi-based inhibition. The presence of IFN in the lentiviral vector preparation of sh-B971 was confirmed by strong induction of 2 0 -5 0 -oligoadenylate synthetase 1 (OAS1), a classic IFN-induced gene, in both naı¨ve Huh-7 and the HCV replicon cell line (GS5) treated with the medium ( Figure 1C ). In addition, HCV replication in an IFN-resistant HCV replicon cell line (H801), in contrast to that in a wildtype replicon cell line (GSB1) (34), was not inhibited by the sh-B971 medium ( Figure 1D ), suggesting the lack of additional viral inhibiting agents in the sh-B971 medium. Expression of sh-B971 in 293FT cells also induced dimerization of IRF-3, confirming the activation of the IFN production pathway in these transfected cells ( Figure 1E ). Finally, sh-B971 was able to activate both IFN-a and IFN-b promoters, although the activation of the IFN-a promoter required coexpression of IRF-7, which is normally expressed at very low levels in 293-based cells ( Figure 1F ). These results demonstrate that sh-B971 is a potent activator of IRF-3 and IRF-7, master regulators of IFN expression in human cells.