Expression of an IRF-3 fusion protein and mouse estrogen receptor, inhibits hepatitis C viral replication in RIG-I-deficient Huh 7.5 cells. Part 2
In order to study the direct anti-HCV response of IRF-3 activation, an inducible Huh7.5-IRF3ER cell line was established in RIG-I deficient Huh 7.5 cells that allow IRF-3 protein homodimer formation in a cytokine/receptor-independent fashion. Huh 7.5 cells are a highly adapted and poorly differentiated hepatoma cell line that lacks the ability to produce detectable interferon-α/β when infected with HCV JFH-1 virus. Therefore, Huh7.5-IRF3ER cells is an adequate system to study the downstream molecular events of IRF-3 activation and establishment of a subsequent anti-HCV state without RIG-I activation in Huh 7.5 cells.
Plasmids
A mammalian expression vector, pTIRF3ER, was constructed as a fusion protein of the IRF3 gene (51.6 Kd) [22] and C-terminal sequences of the mouse estrogen receptor (310 a.a.) in the pEF6/V5-His TOPO® TA vector (Invitrogen, Carlsbad, CA). The plasmid pJFH-1 contains a full-length HCV genomic cDNA. The plasmid pRL-HL is a dicistronic construct that mediates Cap-dependent and HCV IRES-dependent translation. Synthetic 4-hydroxytamoxifen (4-HT) was purchased from Sigma (Saint Louis, MO) and dissolved in ethanol as a 5 mM stock solution.
Cell lines
Human hepatoma Huh 7.5 cells were grown in Dulbecco’s modified Eagle’s medium (Invitrogen). To establish the Huh7.5-IRF3ER cell line, Huh 7.5 cells were transfected with the plasmid pTIRF3ER and Lipofectin (Invitrogen). Blasticidin (Invitrogen) (10 μg/ml) was used for the clone selection 24-hours after transfection. Medium was changed every 3 days with fresh Blasticidin until day 14, at which time, positive clones were propagated. The clones were amplified and IRF-3ER dimer formation was measured by Western blotting after 4-HT treatment.
Detection of IRF-3ER dimers, p-STAT1 (S727), p-STAT3 (Y705), and 1-8U protein by Western blotting
Huh7.5-IRF3ER cell monolayers were washed in phosphate buffered saline (PBS) post 4-HT treatment with protease inhibitor cocktail (Sigma). Preparation of Huh7.5-IRF3ER cell lysates was performed as reported previously. Cellular lysates were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) (6% gel for IRF-3ER dimers; 8% gel for p-STAT1 (S727) and p-STAT3 (Y705)). Western blotting was carried out as previously reported with antibodies for actin (Santz Cruz Biotechnology, Inc., Santa Cruz, CA), p-STAT3 (Y705) (Cell Signaling, Boston, MA), p-STAT3 (Y705) (Cell Signaling), and STAT1 (Santz Cruz). Western blotting of STAT1 and STAT3 proteins were performed with the same PVDF membrane used for detection of p-STAT1 (S727) and p-STAT3 (Y705) after stripping the blot (Bio-rad stripping buffer).