Abstract
Interferon alpha (IFN-α) binds to a cell surface receptor that activates the Jak-Stat signaling pathway. A critical component of this pathway is the translocation of interferon stimulated gene factor 3 (a complex of three proteins Stat1, Stat2 and IRF9) to the nucleus to activate antiviral genes. A stable sub-genomic replicon cell line resistant to IFN-α was developed in which the nuclear translocation of Stat1 and Stat2 proteins was prevented due to the lack of phosphorylation; whereas the nuclear translocation of IRF9 protein was not affected. In this study, we sought to overcome defective Jak-Stat signaling and to induce an antiviral state in the IFN-α resistant replicon cell line by developing a chimera IRF9 protein fused with the trans activating domain (TAD) of either a Stat1 (IRF9-S1C) or Stat2 (IRF9-S2C) protein. We show here that intracellular expression of fusion proteins using the plasmid constructs of either IRF9-S1C or IRF9-S2C, in the IFN-α resistant cells, resulted in an increase in Interferon Stimulated Response Element (ISRE) luciferase promoter activity and significantly induced HLA-1 surface expression. Moreover, we show that transient transfection of IRF9-S1C or IRF9-S2C plasmid constructs into IFN-α resistant replicon cells containing sub-genomic HCV1b and HCV2a viruses resulted in an inhibition of viral replication and viral protein expression independent of IFN-α treatment. The results of this study indicate that the recombinant fusion proteins of IRF9-S1C, IRF9-S2C alone, or in combination, have potent antiviral properties against the HCV in an IFN-α resistant cell line with a defective Jak-Stat signaling.
Highlights
Hepatitis C virus (HCV) infection is a major public health problem with 170 million infected individuals worldwide [1,2]
Interferon alpha (IFN-a) independent nuclear localization of IRF9 in the R153 and S9-13 cells The green fluorescence protein (GFP) fused Stat1, Stat2 and IRF9 plasmid clones illustrated in Fig. 1A were used to establish the dynamics of nuclear translocation in the S9-13 and R153 cells in the presence and absence of IFN-a
Using a transient transfection experiment, we demonstrate that both the Stat1-GFP and Stat2-GFP fusion proteins were expressed at high levels in the cytoplasm of both cell lines
Summary
Hepatitis C virus (HCV) infection is a major public health problem with 170 million infected individuals worldwide [1,2]. HCV infection establishes a chronic inflammatory liver disease in over 70 percent of patients. In chronically infected individuals the disease slowly progresses over decades resulting in liver cirrhosis, hepatocellular carcinoma (HCC), and liver failure [3]. The World Health Organization estimates that 3% of the world’s population is infected with HCV and approximately three to four million new cases of HCV infection occur globally per year [4]. Pegylated IFN-a plus ribavirin is the standard of care for the treatment of chronic HCV infection [5,6].
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