Hepatitis C virus non-structural 5A abrogates signal transducer and activator of transcription-1 nuclear translocation induced by IFN-alpha through dephosphorylation

Abstract

Aim: To study the effect of Hepatitis C virus non-structural 5A (HCV NS5A) on IFNalpha induced signal transducer and activator of transcription-1 (STAT1) phosphorylation and nuclear translocation.

Methods: Expression of STAT1 Tyr701 phosphorylation at different time points was confirmed by Western blot, and the time point when p-STAT1 expressed most, was taken as the IFN induction time for further studies. Immunocytochemistry was used to confirm the successful transient transfection of NS5A expression plasmid. Immunofluorescence was performed to observe if there was any difference in IFNalpha-induced STAT1 phosphorylation and nuclear translocation between HCV NS5A-expressed and non-HCV NS5A-expressed cells. Western blot was used to compare the phosphorylated STAT1 protein of the cells.

Results: Expression of HCV NS5A was found in the cytoplasm of P(CNS5A-transfected ) Huh7 cells, but not in the PRC/CMV transfected or non-transfected cells. STAT1 Tyr701 phosphorylation was found strongest in 30 min of IFN induction. STAT1 phosphorylation and nuclear import were much less in the presence of HCV NS5A protein in contrast to P( RC/CMV-transfected ) and non-transfected cells under fluorescent microscopy, which was further confirmed by Western blot.

Conclusion: HCV NS5A expression plasmid is successfully transfected into Huh7 cells and HCV NS5A protein is expressed in the cytoplasm of the cells. IFN-alpha is able to induce STAT1 phosphorylation and nuclear translocation, and this effect is inhibited by HCV NS5A protein, which might be another possible resistance mechanism to interferon alpha therapy.

Figure 1

HCV NS5A protein staining of Huh7 cells after 48 h transfection (DAB, × 400). A: P^CNS5A-transfected cells; B: P^RC/CMV-transfected cells; C: Non-transfected cells.

Figure 2

Tyr701 phosphorylated STAT1 in Huh7 cells induced by IFNα-2b (10 000 U/mL) (Western blot).

Figure 3

STAT1 or Phosphorylated STAT1 (Tyr701) in Huh7 cells stimulated by IFNα-2b (10 000 U/mL) for 30 min (FITC, × 400). A: STAT1; B: Tyr701.

Figure 4

STAT1 or phosphorylated STAT1 in cytoplasm and nuclei of Huh7 cells after 30 min IFN induced (FITC, × 400): A and D: P^CNS5A-transfected; B and E: P^RC/CMV-transfected; C and F: non-transfected.

Figure 5

Phosphorylated STAT1 from IFN induced Huh7 cells (FITC, × 400): A: P^CNS5A-transfected; B: P^RC/CMV-transfected; C: Non-transfected.