SH2 Modified STAT1 Induces HLA-I Expression and Improves IFN-[gamma] Signaling in IFN-[alpha] Resistant HCV Replicon Cells
We have developed multiple stable cell lines containing subgenomic HCV RNA that are resistant to treatment with interferon alpha (IFN-[alpha]. Characterization of these IFN-[alpha] resistant replicon cells showed defects in the phosphorylation and nuclear translocation of STAT1 and STAT2 proteins du...
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Veröffentlicht in: | PloS one 2010-09, Vol.5 (9), p.e13117 |
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Sprache: | eng |
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Zusammenfassung: | We have developed multiple stable cell lines containing subgenomic HCV RNA that are resistant to treatment with interferon alpha (IFN-[alpha]. Characterization of these IFN-[alpha] resistant replicon cells showed defects in the phosphorylation and nuclear translocation of STAT1 and STAT2 proteins due to a defective Jak-STAT pathway. In 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-[gamma] 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-[gamma] 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-[gamma] 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. These results suggest that modification of the SH2 domain of the STAT1 molecule allows for improved IFN-[gamma] signaling through increased STAT1 phosphorylation, nuclear translocation, HLA-1 surface expression, and prolonged interferon antiviral gene activation. |
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ISSN: | 1932-6203 1932-6203 |
DOI: | 10.1371/journal.pone.0013117 |