Host immunity, including innate immunity and adaptive immunity, is an important and complicated system dedicated to the task of defending the host from microbial infection and cancer development. Innate immunity provides an immediate (first line) reply to a microbial infection, specifically for viral infections, while also controlling the later antigen-specific adaptive response. A key aspect of the antiviral innate immune response is the synthesis and secretion of type I INFs (α and β), which exhibit antiviral, anti-proliferative, and immunomodulatory functions. Two key steps are required to elicit an effective antiviral innate immune response: a. detection of the invading virus by immune system receptors; b. initiation of protein signaling cascades that regulate the synthesis of IFNs. Viruses are highly infectious pathogens that depend on host cellular machinery for survival and replication. Most viral infections, like the common cold caused by Rhinoviruses, are efficiently resolved by the host innate and adaptive immune system. For other viral infections, such as chronic hepatitis B or C viral infection, the host innate and adaptive immunity response is unable to clear them effectively and they become persistent infections. Several families of PPRs have been demonstrated to inspect the cellular micro-environment for microbial infection to target the pathogen-associated molecular patterns (PAMPs), a conserved structural moiety essential for microbial survival. Toll-like receptors (TLRs 3, 4, 7, 8, and 9) in addition to RIG-I are major PPRs that recognize different types of virally-derived nucleic acids or intracellular dsRNA to initiate signaling cascades leading to production of type I IFNs (details in reviews [11, 31, 32]). The mechanisms by which different viruses induce a unique IFN-mediated antiviral response appear to require selective activation of members of the IRF family of proteins (IRF-1 to IRF-9). Thus far, IRF-3 and IRF-7 have been shown to be major regulators of IFN gene expression [33, 10].
The type I IFNs, represented by multiple subtypes of IFN-α in addition to one subtype IFN-β, are key cytokines in this process, mounting an immediate antiviral response as well as adaptive immunity. IFN-mediated anti-viral effects are carried out using different mechanisms that are dependent on the type of viral infection, but these anti-viral effects are all dependent on IRF-3 activation [35, 34, 33, 21, 7]. Activation of IRF-3 proteins appears to recruit the Tank Binding Kinase 1 (TBK1) and inhibitor of IκB-related kinase epsilon (IKKε)  through their interaction with the RIG-I RNA helicase , resulting in phosphorylation of IRF-3, its dimerization, nuclear translocation, and transcriptional activation through binding to IFN-stimulated response elements (ISREs) . Activated IRF-3 interacts with nuclear factor-κB (NF-κB) and transcriptional factor-2/c-Jun to form a transcriptionally active enhanceosome complex on IFNA1 and IFNB gene promoters.
In our studies, we utilized an IRF-3/mouse ER fusion protein expressing plasmid in order to achieve IRF-3ER activation in a cytokine/receptor-independent fashion. Our results demonstrated that IRF-3ER homodimers (Figure 1, lane 3) triggered the downstream pathways to produce IFN-α and IFN-β (Figure 2A and 2B). The anti-HCV effects, induced by 4-HT in Huh7.5-IRF3ER cells, were achieved by decreasing HCV RNA replication and HCV IRES-mediated translation. This is consistent with our previous studies which achieved activation of STAT1/and STAT3/mouse ER fusion proteins. Activation of the IRF-3ER fusion protein by 4-HT treatment provides strong evidence that this is necessary and sufficient to increase IFN-α and IFN-β expression in Huh7.5-IRF3ER cells (Figure 2A and 2B). Our data showing that IRF-3ER activation triggers the downstream pathway, activating the JAK/STAT pathway and regulating ISG expression. Detection of p-STAT1 (S727) and p-STAT3 (Y705) in Huh7.5-IRF3ER cells provides a strong evidence for activation of Jak/STATs pathway by IFNs (Figure 3A and 3B). Although the mechanism of IFN action against HCV replication has not been well defined, recent studies suggest that IFNs have a great impact on HCV replication by interrupting HCV IRES-mediated translation [38, 12]. Clinical data confirmed these findings in a study of HCV IRES-mediated translation in chronic HCV patients receiving IFN treatment, in which the efficiency of HCV IRES-mediated translation was reduced in IFN-treated HCV patients [39, 40]. In our study, the inhibitory effects of HCV RNA replication and HCV IRES-mediated translation were confirmed in Huh7.5-IRF3ER cells after treatment with 4-HT (Figure 4A and 4B).
Here, we present data demonstrating that activation of the IRF-3 gene restores IFN production in RIG-I deficient Huh 7.5 cells. The anti-HCV effects were achieved in Huh7.5-IRF3ER cells by decreasing both HCV RNA replication and HCV IRES-mediated translation. Recently, two new genes, 1-8U and hnRNP M, were isolated in our studies due to their ability to modulate cellular Cap-dependent and HCV IRES-mediated translation and regulated by STAT1 pathway activation.