FIG. 6.

Effect of CyPA on the PK sensitivity of NS5B in the absence of RC formation. (A) Recombinant NS5B Δ21 was readily digested by 250 ng/ml of PK in the presence of the GST-CyPA protein. Approximately 1,800 ng of NS5B and 600 ng of the GST proteins were used. Proteins were detected by Coomassie staining. (B) GST-CyPA does not protect NS5B from PK digestion under limiting PK concentrations. NS5B Δ21 was mixed with both GST fusion proteins (lane 1) or GST-EC2 (lanes 2 to 4) or GST-CyPA (lanes 5 to 7) individually prior to incubation with increasing concentrations of PK (lanes 2 to 4 and 5 to 7). Approximately 3,000 ng of NS5B and 1,800 ng of the GST proteins were used. Proteins were detected with Western blotting. (C) The PK sensitivity of NS5B expressed in the absence of RC formation is independent of CyPA short hairpin (sh) RNA-mediated knockdown. An NS5B-expressing plasmid was transfected into the indicated Huh-7.5 derivative cell lines. Forty-eight hours posttransfection, the cells were treated with digitonin and increasing concentrations of PK as described in Materials and Methods. The CyPA knockdown in the sh-A161 cell line was shown previously (71).