FIG. 4.
CyPA interacts with CRC-associated NS5B and is recruited to the MF and CRC. (A) CRC-associated NS5B interacts with CyPA in a cyclosporine (CsA)-sensitive manner. The PK-digested CRC was resuspended in lysis buffer and then incubated with GST proteins in a pulldown assay in the absence or in the presence of increasing amounts of cyclosporine. (B) CyPA is recruited to MFs in replicon cells. Huh-7.5 or GS5 cells were treated with 4 μg/ml of cyclosporine for 22 h before being lysed. MFs were then isolated and analyzed for the presence of CyPA by Western blotting. Proteins isolated from 4 × 105 cells were loaded into each lane. (C) The protein bands were quantified using Quantity One software. The total level of CyPA without cyclosporine treatment was set at 100%. Data represent results from three independent experiments. (D) Cyclosporine reduces the PK resistance of CyPA in replicon cells. GS5 cells were treated with cyclosporine and PK where indicated. The resulting cell lysate was then subjected to SDS-PAGE and Western blotting for the detection of CyPA. The PK concentration used was 0.5 μg/ml. (E) CyPA, but not CyPB, is incorporated into the CRC in a cyclosporine-sensitive manner. The fractions depicted in Fig. 3A were analyzed for the presence of both CyPA and CyPB. For total lysate and the MF, proteins isolated from 4 × 105 cells were loaded into each lane, and for the CRC-PK fraction, proteins isolated from 1 × 106 cells were loaded.