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

Plasmids pGL3-IFNA1, pGL3-IFNB, pRL-TK, pCMV-Flag-IRF-3 and pCR3.1-IRF-7A have been described earlier (36) . shRNAs were expressed from a human immunodeEciency virus (HIV)-based lentiviral vector (32, 37) , and sh-PCAF was constructed on the basis of a previously reported sequence (38) . Plasmid sh-B971/H1 was constructed by cloning of the DNA fragment encoding the sh-B971 RNA into pSilencer 3.0-H1 (Ambion, Austin, TX, USA) according to the manufacturer's instructions. The RIG-I and TLR3 constructs have been described (39, 40) . The RIG-I C construct encodes Flag-tagged, C-terminal 707 aa of human RIG-I cloned into a bicistronic expression vector modified from pBICEP-CMV-1 (Sigma-Aldrich, St Louis, MO, USA), in which the CMV promoter was replaced with the elongation-factor-1 promoter. The MDA5, MDA5-C constructs were kindly provided by Fujita (12) . HCV genotype 2a NS3-4A protease was expressed from the pCMV-3Tag-1a plasmid (Stratagene, La Jolla, CA, USA).