FIG. 1.

Effect of cyclosporine on the PK sensitivity of HCV and cellular proteins. (A) Cyclosporine (CsA) reduces the PK resistance of NS5B but not that of NS3 or NS5A. GS5 cells were treated with cyclosporine and PK where indicated. The cell lysate was then subjected to Western blotting for the detection of HCV proteins. PK treatment sometimes generates a doublet band of Con1 NS5B by SDS-PAGE; both were recognized by the antibody and were affected by cyclosporine treatment. (B) PK treatment or cyclosporine treatment did not compromise the integrity of ER membranes. The samples in A were subjected to the detection of two ER-associated chaperone proteins, Grp94 and calnexin. The antibody against calnexin recognizes the N terminus of the protein, which resides in the ER lumen. (C) IFN-α treatment did not alter the PK sensitivity of the NS5B protein. Results shown are representative of results from three to six independent experiments. (D) Cyclosporine increases the sensitivity of the NS5B protein to trypsin in replicon cells. GS5 cells were treated with cyclosporine for 22 h and lysed. The cell lysate was then digested with trypsin at the indicated concentrations before being subjected to Western blotting and detection with anti-NS5B antibodies. The protein loaded into each lane was obtained from 1 × 10⁵ cells.