FIG. 8.

The PPIase motif of CyPA is essential for HCV replication. (A) CyPA protein that bears mutations in the active site of PPIase retained binding to NS5B. GST or GST fusion proteins were incubated with GS5 lysate in a GST pulldown assay. Bound NS5B was detected by Western blotting (top), and the recombinant proteins were detected by Coomassie staining (bottom). (B) The PPIase mutants failed to rescue HCV replication in CyPA knockdown cells. In vitro-transcribed Rep1b RNA was coelectroporated into Huh-7.5/sh-A161 cells with myc-tagged CyPA expression plasmids (71). RNA was extracted at 7 h and 4 days after electroporation for analysis of HCV IRES and GAPDH RNA levels. After normalization to GAPDH levels, the ratio of the day 4 HCV RNA levels to the 7-h HCV RNA levels was calculated and plotted. The RNA replication level of the vector control sample was set at 1. wt, wild type.