Abstract
BackgroundWe have developed multiple stable cell lines containing subgenomic HCV RNA that are resistant to treatment with interferon alpha (IFN-α. Characterization of these IFN-α resistant replicon cells showed defects in the phosphorylation and nuclear translocation of STAT1 and STAT2 proteins due to a defective Jak-STAT pathway.Methodology/Principal FindingsIn this study, we have developed an alternative strategy to overcome interferon resistance in a cell culture model by improving intracellular STAT1 signaling. An engineered STAT1-CC molecule with double cysteine substitutions in the Src-homology 2 (SH2) domains of STAT1 (at Ala-656 and Asn-658) efficiently phosphorylates and translocates to the nucleus of IFN-resistant cells in an IFN-γ dependent manner. Transfection of a plasmid clone containing STAT1-CC significantly activated the GAS promoter compared to wild type STAT1 and STAT3. The activity of the engineered STAT1-CC is dependent upon the phosphorylation of tyrosine residue 701, since the construct with a substituted phenylalanine residue at position 701 (STAT1-CC-Y701F) failed to activate GAS promoter in the replicon cells. Intracellular expression of STAT1-CC protein showed phosphorylation and nuclear translocation in the resistant cell line after IFN-γ treatment. Transient transfection of STAT1-CC plasmid clone into an interferon resistant cell line resulted in inhibition of viral replication and viral clearance in an IFN-γ dependent manner. Furthermore, the resistant replicon cells transfected with STAT1-CC constructs significantly up regulated surface HLA-1 expression when compared to the wild type and Y to F mutant controls.ConclusionsThese results suggest that modification of the SH2 domain of the STAT1 molecule allows for improved IFN-γ signaling through increased STAT1 phosphorylation, nuclear translocation, HLA-1 surface expression, and prolonged interferon antiviral gene activation.
Highlights
Hepatitis C virus (HCV) infection is a major public health concern with a prevalence of approximately 3% of the world population chronically infected by the virus [1]
These results suggest that modification of the Src-homology 2 (SH2) domain of the STAT1 molecule allows for improved IFN-c signaling through increased STAT1 phosphorylation, nuclear translocation, HLA-1 surface expression, and prolonged interferon antiviral gene activation
We showed that due to Jak-STAT signaling defects, the phosphorylation and nuclear translocation of STAT1 and STAT2 proteins are blocked in the IFN-a resistant cell line
Summary
Hepatitis C virus (HCV) infection is a major public health concern with a prevalence of approximately 3% of the world population chronically infected by the virus [1]. These chronically infected HCV patients experience a slow progressive disease of the liver that can result in end stage liver disease such as liver cirrhosis and hepatocellular carcinoma [4]. We have developed multiple stable cell lines containing subgenomic HCV RNA that are resistant to treatment with interferon alpha Characterization of these IFN-a resistant replicon cells showed defects in the phosphorylation and nuclear translocation of STAT1 and STAT2 proteins due to a defective Jak-STAT pathway
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