SH2 modified STAT1 induces HLA-I expression and improves IFN-γ signaling in IFN-α resistant HCV replicon cells.
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.


RESULTS
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.
Poat
Bret
B
Department of Pathology and Laboratory Medicine, New Orleans, Louisiana, United States of America.
Hazari
Sidhartha
S
Chandra
Partha K
PK
Gunduz
Feyza
F
Balart
Luis A
LA
Alvarez
Xavier
X
Dash
Srikanta
S
D000998:Antiviral Agents / Q000494:pharmacology; D002460:Cell Line; D024882:Drug Resistance, Viral; D015870:Gene Expression; D006680:HLA Antigens / Q000235:genetics* / Q000378:metabolism; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D006526:Hepatitis C / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012093:Replicon; D050794:STAT1 Transcription Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014779:Virus Replication / Q000187:drug effects; D018909:src Homology Domains
D000998:Antiviral Agents; D006680:HLA Antigens; D016898:Interferon-alpha; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D007371:Interferon-gamma