1. Volume 128, Issue 4, Pages 1034-1041 (April 2005)
Hepatitis C virus expression suppresses interferon signaling by degrading STAT1 
Wenyu Lin, Won Hyeok Choe, Yoichi Hiasa, Yoshitaka Kamegaya, Jason T. Blackard, Emmett V. Schmidt, Raymond T. Chung 
Gastroenterology 
Volume 128, Issue 4, Pages (April 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
STAT1 is a critical component in controlling HCV expression. (A) STAT1 is indispensable for control of HCV expression in cell lines defective for Jak-STAT kinases. The U3 (STAT1 knockout) cell line enhanced HCV core Ag production (48 pg/mL) by more than 2-fold compared with the parental 2fTGH (23 pg/mL) and other mutant cell lines (P = .002). The increased production of HCV in this line was successfully rescued by overexpression of STAT1 using a pcDNA3.mu-STAT1 expression vector. (B) Western blotting for STAT1 and STAT2 protein expression in 2fTGH and other mutant cell lines. Lane 1, 2fTGH/pH77; lane 2, U1A/pH77; lane 3, U2A/pH77; lane 4, U3A/pH77; lane 5, U3A/pcDNA3.STAT1/pH77; lane 6, U4A/pH77; lane 7, U6A/pH77; lane 8, 2fTGH cell. (C) HCV core Ag production in Huh-T7 cells. Overexpression of STAT1 decreased HCV core Ag production from pg/mL (pH77) to 65.8 pg/mL (pH77 + pcDNA3.mu-STAT1) (P = .001). siRNA knockdown of STAT1 increased HCV core Ag production to pg/mL (pH77 + STAT1 siRNA) (P = .002). The negative control siRNA had no effect in HCV core Ag production (215.8 pg/mL). (D) IFN-induced ISRE-mediated signaling. IFN treatment (12 hours) markedly induced ISRE-directed luciferase activity. However, in the presence of HCV, this IFN-stimulated activity was blocked by 2–4–fold. Overexpression of STAT1 (pH77 + pcDNA3.mu-STAT1) enhanced ISRE-mediated signaling in HCV transfected cells (pH77) by 2.1-fold (P = .005). In contrast, STAT1 siRNA knockdown (pH77 + STAT1 siRNA) inhibited ISRE-mediated signaling by 3.0-fold (P = .007). Relative luciferase activity in HCV cotransfected with negative control STAT1 siRNA (pH77 + Neg control STAT1 siRNA) was not altered compared to pH77-treated cells alone. (E) Western blotting for STAT1 expression in Huh-T7 cells. Lane 1, pGEM transfected; lane 2, pH77 transfected; lane 3, pcDNA3.mu-STAT1 transfected; lane 4, STAT1 siRNA-depleted STAT1 expression; lane 5, negative control siRNA.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
Western blotting of HCV transfection reduces STAT1 and P-STAT1. (A) Whole cell lysates. HCV transfection reduced STAT1 expression, and impaired IFN-α (1000 IU/mL for 12 hours) induced P-STAT1 expression. Actin protein was used as a control. (B) Cytoplasmic fraction. STAT1 localized to the cytoplasmic fraction. In contrast, HCV transfection inhibited STAT1 protein expression in Huh-T7 cells. (C) Nuclear fraction. IFN-α treatment (1000 IU/mL for 12 hours) induced P-STAT1 accumulation in the nuclear fraction. In contrast, HCV reduced P-STAT1 accumulation in the nuclear fraction. (D) ISGF3 electrophoretic mobility shift assay. IFN-α treatment (1000 IU/mL for 12 hours) activated the formation of ISGF3. HCV transfection blocked ISGF3 in gel shift assay. The ISGF3 complex is indicated by the arrows.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
HCV-induced degradation of STAT1 and P-STAT1 is proteasome dependent. (A) STAT1- and IFN-α– (1000 IU/mL for 12 hours) induced P-STAT1 levels were assessed in Huh-T7 cells by Western blot. HCV transfection is associated with degradation of STAT1 and P-STAT1. Addition of the proteasome inhibitor MG132 (10 μmol/L for 12 hours) blocks the degradation of STAT1 and P-STAT1. (B) HCV core Ag production. IFN-α treatment (1000 IU/mL for 12 hours) significantly reduced the HCV core Ag production from pg/mL (pH77) to 41.3 pg/mL (pH77/IFN) (P = .003) in Huh-T7 cells. The introduction of MG132 and MG132/IFN further decreased the HCV core Ag to 85.9 pg/mL (pH77/MG132) (P = .004) and 27.1 pg/mL (pH77/IFN/MG132) (P = .002).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
Multiple HCV proteins inhibit IFN signaling. (A) Multiple HCV proteins subvert ISRE-mediated signaling in luciferase assay (12 hours of IFN treatment). Full-length HCV polyprotein (pH77), HCV structural proteins (HCVst), NS3-4A, and NS4B significantly suppressed ISRE-mediated signaling. In contrast, pGEM, NS5A, and eGFP transfection did not inhibit IFN signaling. (B) Western blotting of STAT1 after HCV viral protein transfection without IFN treatment. Full-length HCV, HCV structural protein, and HCV NS3-4A transfection reduced STAT1 and P-STAT1 protein expression in Huh-T7 cells. Lane 1, pGEM; lane 2, pH77; lane 3, pHCVst; lane 4, pNS3-4A; lane 5, pNS4B; lane 6, pNS5A; lane 7, pNS5B; lane 8, eGFP.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
STAT1 binds to HCV core and STAT2 in immunoprecipitation/Western blot. (A) STAT1-immunoprecipitated Western blot. STAT1-immunoprecipitated cell lysates were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Anti-HCV core, anti-HCV E1, anti-HCV E2, anti-HCV NS3, anti-HCV NS4, anti-HCV NS5a, anti-STAT1, and anti-STAT2 monoclonal antibodies were used for Western blotting. With the introduction of proteasome inhibitor MG132 (10 μmol/L for 12 hours), HCV core protein and STAT2 bound to STAT1. HCV E1, E2, NS3, NS4, and NS5a proteins did not bind to STAT1. STAT2 expression was not affected by HCV transfection. (B) HCV core-immunoprecipitated Western blot. HCV core-immunoprecipitated cell lysates were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Anti-STAT1 and anti-STAT2 monoclonal antibodies were used for Western blotting. STAT1 bound to HCV core, but STAT2 was not associated with HCV core.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions