Expression of an IRF-3 fusion protein and mouse estrogen receptor, inhibits hepatitis C viral replication in RIG-I-deficient Huh 7.5 cells
Abstract
Interferon Regulatory Factor-3 (IRF-3) plays a central role in the induction of interferon (IFN) production and succeeding interferon-stimulated genes (ISG) expression en route for restraining hepatitis C virus (HCV) infection. Here, we established a stable Huh7.5-IRF3ER cell line expressing a fusion protein of IRF-3 and mouse estrogen receptor (ER) to examine IFN production and anti-HCV effects of IRF-3 in retinoic acid inducible-gene-I (RIG-I) deficient Huh 7.5 cells. Homodimerization of the IRF-3ER fusion protein was detected by Western blotting after treatment with the estrogen receptor agonist 4-hydrotamoxifen (4-HT) in Huh7.5-IRF3ER cells. Expression of IFN-α, IFN-β, and their inhibitory effects on HCV replication were demonstrated by real-time polymerase chain reaction (PCR). Peak expression of IFN-α and IFN-β was achieved 24-hours post 4-HT treatment, coinciding with the appearance of phosphorylated signal transducer and activator of transcription (STAT) proteins. Additionally, HCV viral replication declined in time-dependent fashion. In previous studies, a novel IFN-mediated pathway regulating expression of 1-8U and heterogeneous nuclear ribonucleoprotein M (hnRNP M) inhibited HCV internal ribosomal entry site (IRES)-dependent translation. When expression of ISGs such as 1-8U and hnRNP M were measured in 4-HT-treated Huh7.5-IRF3ER cells, both genes were positively regulated by activation of the IRF-3ER fusion protein. In conclusion, the anti-HCV effects of IRF-3ER homodimerization inhibited HCV RNA replication as well as HCV IRES-dependent translation in Huh7.5-IRF3ER cells. The results of this study indicate that IRF-3ER homodimerization is a key step to restore IFN expression in Huh7.5-IRF3ER cells and in achieving its anti-HCV effects.
Introduction
Hepatitis C virus infection causes chronic liver diseases, cirrhosis, and hepatic cellular carcinoma (HCC) with 170 million people worldwide and 4 million people in the United States reportedly infected (CDC, 1998). In addition to its global health problem, future projections suggest that HCV related mortality will increase 2-3-fold over the next decade with more than 180 billion US dollars estimated total social economic cost in the United States. The standard treatment of chronic HCV is anti-viral therapy with IFN and ribavirin (RBV) but no HCV vaccine available. Despite additional chemotherapeutics is on hand for treatment of genotype I HCV patients recently, the anti-viral mechanisms of IFN-based therapies are not well defined, but most likely involve the activation of host innate immunity to limit HCV replication.
During microbial infection, the recognition of microbial components is mediated by host-specific cellular pathogen-recognition receptors (PPRs). PPRs are members of the toll-like receptor (TLRs) family and are localized either to cellular plasma (TLR4 for lipopolysaccharide (LPS) and viral envelops) or endosomal membranes (TLR3 for dsRNA, TLR7/8 for ssRNA and TLR9 for DNA). Conversely, intracellular dsRNA is also recognized by the RIG-I cytosolic RNA helicase or melanoma differentiation associated gene (MDA)-5. RIG-I RNA helicase was found to be an essential mediator of anti-HCV effects due to its binding to un-capped 5′-end and 3′-end HCV dsRNA, triggering host innate immunity.
IFNs bind to the IFN-α/β receptor (IFNAR) in either an autocrine and/or paracrine manner to initiate a positive feedback loop that results in the production of more type I IFNs. IFNARs trigger the activation of the JAK/STAT pathway to phosphorylate the STAT proteins. The STAT transcription factors associated with IRF-9 to form a heterotrimeric complex, IFN-stimulated gene factor 3 (ISGF3), initiating the transcription of IFN-stimulated genes (ISGs) and inhibiting the different stages of virus replication and eliciting an anti-viral state in the host. During HCV infection, these anti-viral effects include the inhibitory effects on host and HCV translation, regulation of cellular proliferation and apoptosis, regulation of adaptive immunity, and recruitment of NK cells to the site of infection to clear HCV infection by inhibiting HCV gene expression and HCV replication. Patients with cleared HCV infection without IFN-based treatment provides strong evidence for the host innate immune response during acute HCV infection.