Dimerization of IRF-3ER fusion protein induced by 4-HT in Huh7.5-IRF3ER cells

Activation of IRF-3 or IRF-7 is a critical step during virus infection, promoting the most potent type I IFN production. Previous studies showed the constitutively active forms (serines replaced by phosphomimetic aspartate amino acids) of human IRF-3 protein exerts the ability to modulate the apoptotic and anti-tumor properties after being delivered by recombinant adenovirus into macrophages. In our studies, a fusion protein of IRF-3 and C-terminal sequences (310 a.a.) of mouse estrogen receptor was used to establish the stable Huh7.5-IRF3ER cell line. In previous studies, mouse estrogen receptor was effective at inducing dimerization of STAT1 and STAT3 fusion proteins after 4-HT treatment. In these studies from us and others, 4-HT was titrated to a concentration of 1 μM that achieved the highest expression of STAT1ER and STAT3ER dimerization and the strongest inhibitory effects on HCV RNA replication. In our studies, 4-HT-treatment alone was also demonstrated to have no anti-HCV effects. In this study, similar sequences from the mouse ER C-terminal domain were fused to the C-terminus of the IRF-3 gene. In Figure 1, Western blotting with anti-IRF-3 antibody was used to detect the IRF-3 as well as the IRF-3ER monomer and dimer proteins. Lane 1 shows endogenous IRF3 protein (56.1 kd) but no IRF-3ER fusion protein in Huh 7.5 cells treated with 4-HT. Lane 2 shows both IRF-3 and IRF-3ER (monomer) (90 kd) in Huh7.5-IRF3ER cells without 4-HT treatment. Lane 3 shows that 4-HT treatment of Huh7.5-IRF3ER cells induces IRF-3ER fusion protein dimer formation (180 kd) in addition to IRF-3 protein and IRF-3ER monomers. The density of IRF-3ER dimers was less than the density of IRF-3ER monomers, which could be explained by the denaturing conditions used in the analysis as suggested in our previous report, including SDS-polyacrylamide gel electrophoresis, RIPA lysis buffer, and boiling during Western blotting. Interestingly, a small amount of IRF-3ER dimer formation was detected in Huh7.5-IRF3ER cells without 4-HT treatment. This may be due either to auto-dimerization of IRF-3ER or dimer formation induced by trace estrogen in the tissue culture medium. Multiple forms of the IRF-3ER fusion protein were also detected. Our data indicates the IRF-3ER fusion protein approach is an effective means to achieve IRF-3 homodimerization with 4-HT treatment.

Expression of IFNs after activation of the IRF-3ER fusion protein

Due to deficient RIG-I gene function in Huh 7.5 cells, virus infection will not lead to IRF-3 activation and IFN secretion. This phenomenon allows us to study IRF-3 gene function against HCV infection by establishing a stable Huh7.5-IRF3ER cell line. Fusion proteins of STAT1 and STAT3 with the mouse estrogen receptor provided a useful means to study dimerization of those proteins and resulting in anti-HCV status. In this study, the IRF-3 gene was fused with same C-terminal sequences of mouse estrogen receptor as reported for inducing IRF-3ER dimerization by 4-HT treatment. Expression of type I IFNs (α and β) was examined after 4-HT treatment by real-time PCR. IFN-α and IFN-β increased and peaked 24 hours after 4-HT induction. To further demonstrate the biological activities of IFN-α and IFN-β after IRF-3ER dimerization, Western blotting was used to detect phosphorylated STAT1 and STAT3. In Figure 3A, phosphorylated STAT1 was detected with an antibody against STAT1 (S727) in Huh 7.5 and Huh7.5-IRF3ER cells. Different amounts of phosphorylated STAT1 were observed in both Huh 7.5 cells and Huh7.5-IRF3ER cells. There were no appreciable time-dependent differences in phosphorylated STAT1 in Huh7.5-IRF3ER cells with or without 4-HT treatment. This observation is consistent with the auto-dimerization of IRF-3ER fusion protein to produce IFNs. In Figure 3B, phosphorylated STAT3 was examined; there was no difference between Huh 7.5, Huh7.5-IRF3ER cells with or without 4-HT treatment. This phenomenon could be explained by the constant activation of IRF-7 to induce expression of IFN-α which activates the type I IFN pathway through STAT3 phosphorylation. Total STAT1 and STAT3 proteins was used as internal controls and demonstrated no differences with or without 4-HT treatment on Huh7.5-IRF3ER cells or Huh 7.5 cells.