Inhibitory effects of IRF-3ER dimerization on HCV JFH-1 virus replication

Huh7.5-IRF3ER cells were further examined for its inhibitory effects on HCV JFH-1 viral replication after 4-HT treatment. Huh7.5-IRF3ER cells were inoculated with 0.5 MOI of JFH-1 virus stock and cultured for 14 days to achieve full HCV JFH-1 infected Huh7.5-IRF3ER cell state. The infected Huh7.5-IRF3ER cells were treated with 4-HT (1 μM) at the indicated times and harvested at the last-sample collection point for analysis of HCV RNA by real-time PCR. The infected Huh7.5-IRF3ER cells were used as control without 4-HT treatment for 72 hours. HCV JFH-1 replication decreased to 50% of control after 24 hours of 4-HT treatment. This data indicates that IRF-3ER dimerization after 4-HT treatment has inhibitory effects on HCV JFH-1 replication and was correlated with the production of IFN-α and IFN-β. To further separate HCV JFH-1 viral RNA replication and viral translation, the plasmid pRL-HL, containing Cap-dependent Renilla luciferase translation and HCV IRES-mediated Firefly luciferase translation start sites, was used in this study. After transfection of pRL-HL, Huh7.5-IRF3ER cell lysates were harvested at various times after 4-HT treatment for analysis of luciferase activity. In Figure 4B, both Cap-dependent and HCV IRES-mediated translation was reduced in Huh7.5-IRF3ER cells after 4-HT treatment in a time-dependent fashion. This data shows strong evidence that activation of the IRF-3ER fusion protein not only inhibits JFH-1 viral RNA replication but also inhibits Cap-dependent and HCV IRES-mediated translation.

Expression of ISGs in Huh7.5-IRF3ER cells

All of the IFN types activate JAK/STAT pathways, regulating the expression of over 300 ISGs in order to achieve their anti-viral effects. In our previous studies, we demonstrated a novel pathway by which IFN inhibits HCV IRES-mediated translation through up-regulating 1-8U gene expression and down-regulating expression of the hnRNP M gene (unpublished data). In this study, we measured 1-8U and hnRNP M expression in Huh7.5-IRF3ER cells with and without 4-HT treatment. The 1-8U protein was detected by Western blotting and was up-regulated in Huh7.5-IRF3ER cells after 4-HT treatment. Due to auto-dimerization of IRF-3ER fusion protein in Huh7.5-IRF3ER cells, the fold-induction of 1-8U protein is not as robust as described in our previous report in which the STAT1 gene was activated. Real-time quantitative reverse-transcription PCR was used to detect and measure hnRNP M mRNA expression in Huh7.5-IRF3ER cells. After 4-HT treatment, hnRNP M mRNA levels were down-regulated in a time-dependent fashion. This data confirms that activation of the IRF-3ER fusion protein triggers a cellular anti-HCV state through inducing IFNs production and regulating ISG expression.