Abstract
The mechanisms underlying the Hepatitis C virus (HCV) resistance to interferon alpha (IFN-α) are not fully understood. We used IFN-α resistant HCV replicon cell lines and an infectious HCV cell culture system to elucidate the mechanisms of IFN-α resistance in cell culture. The IFN-α resistance mechanism of the replicon cells were addressed by a complementation study that utilized the full-length plasmid clones of IFN-α receptor 1 (IFNAR1), IFN-α receptor 2 (IFNAR2), Jak1, Tyk2, Stat1, Stat2 and the ISRE- luciferase reporter plasmid. We demonstrated that the expression of the full-length IFNAR1 clone alone restored the defective Jak-Stat signaling as well as Stat1, Stat2 and Stat3 phosphorylation, nuclear translocation and antiviral response against HCV in all IFN-α resistant cell lines (R-15, R-17 and R-24) used in this study. Moreover RT-PCR, Southern blotting and DNA sequence analysis revealed that the cells from both R-15 and R-24 series of IFN-α resistant cells have 58 amino acid deletions in the extracellular sub domain 1 (SD1) of IFNAR1. In addition, cells from the R-17 series have 50 amino acids deletion in the sub domain 4 (SD4) of IFNAR1 protein leading to impaired activation of Tyk2 kinase. Using an infectious HCV cell culture model we show here that viral replication in the infected Huh-7 cells is relatively resistant to exogenous IFN-α. HCV infection itself induces defective Jak-Stat signaling and impairs Stat1 and Stat2 phosphorylation by down regulation of the cell surface expression of IFNAR1 through the endoplasmic reticulum (ER) stress mechanisms. The results of this study suggest that expression of cell surface IFNAR1 is critical for the response of HCV to exogenous IFN-α.
Highlights
Hepatitis C virus (HCV) is a positive-stranded RNA virus that infects the liver
To establish the mechanisms of the defective Jak-Stat signaling, the expression levels of Jak-Stat signaling molecules in resistant replicon cell lines were examined in a representative interferon alpha (IFN-a) sensitive (S-5/15) and an IFN-a resistant cell line (R-17/3) by Western blot analysis using antibodies targeted to the phosphorylated and non-phosphorylated form of Jak1, tyrosine kinase 2 (Tyk2), Stat1 and Stat2 (Figure 2A)
Since the expression level of the cell surface receptors (IFNAR1 and IFN-a receptor 2 (IFNAR2)) is critical for the IFN-a induced signaling events leading to the phosphorylation of the Jak-Stat proteins, the expression levels of IFN-a receptor 1 (IFNAR1) and IFNAR2 proteins in cured sensitive and resistant Huh-7 cells were measured by Western blot analysis and found to be not significantly different (Figure 2A)
Summary
Hepatitis C virus (HCV) is a positive-stranded RNA virus that infects the liver. The majority of patients after initial exposure to the virus develop a chronic infection. The standard treatment option of chronic HCV infection is the combination of IFN-a and ribavirin [4]. Each of nine IFN-a resistant Huh-7 replicon cells showed reduced activation of pISRE-firefly luciferase promoter and impaired phosphorylation of Stat proteins [5,6,7]. All of the cured Huh-7 cell clones showed significant reduction in the ISRE promoter activation and a defect in the Jak-Stat signaling. Stable expression of either Jak or Tyk or both in resistant Huh-7 cells did not complement the defective Jak-Stat signaling and antiviral response of IFN-a
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