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- Title
SH2 Modified STAT1 Induces HLA-I Expression and Improves IFN-γ Signaling in IFN-α Resistant HCV Replicon Cells.
- Authors
Poat, Bret; Hazari, Sidhartha; Chandra, Partha K.; Gunduz, Feyza; Balart, Luis A.; Alvarez, Xavier; Dash, Srikanta
- Abstract
Background: We 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 Findings: 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-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. Conclusions: These 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.
- Subjects
RNA; NUCLEIC acids; RIBOSE; INTERFERONS; ANTINEOPLASTIC agents; ANTIVIRAL agents; GLYCOPROTEINS; CELL culture; CELL lines
- Publication
PLoS ONE, 2010, Vol 5, Issue 9, p1
- ISSN
1932-6203
- Publication type
Article
- DOI
10.1371/journal.pone.0013117