FIG. 1.

An siRNA directed at CyPA inhibits replication of wild-type but not CsA-resistant replicons. (A) RS2 replicon is less sensitive than GS5 replicon to CsA. The two replicon cell lines were treated with CsA at the indicated concentrations for 4 days before being analyzed for NS5A expression by Western blotting. (B) Various shRNAs directed at three CyPs or firefly luciferase were introduced into the replicon cells and expressed for 7 days. The cells were then lysed and probed for the indicated proteins. (C) Suppression of CyPC RNA expression by sh-C454. Total RNA was then extracted 7 days posttransduction and subjected to real-time RT-PCR analysis to determine the level of CyPC mRNA. (D) After introduction of the shRNAs as in panel B, the cells were subjected to double selection with puromycin and G418. At the end of the selection, the cell colonies were stained with violet blue. (E) In vitro replication reaction of GS5 or RS2 lysate was allowed to progress in the presence of either IgG negative control or an anti-CyPA antibody for 4 h. The ³²P-labeled products of replication were detected as a single band of expected size on an RNA gel.