FIG. 3.

Cyclosporine blocks the incorporation of NS5B into the CRC of HCV. (A) Schematic representation of the different subcellular fractions analyzed for cyclosporine sensitivity. (B) CRC incorporation, but not membrane association, of NS5B is reduced by cyclosporine (CsA) treatment. The amounts of NS3 and NS5A in CRC-PK were not affected by cyclosporine treatment. The loading amounts of the different fractions were obtained from 1 × 10⁵ cells for total lysate, from 4 × 10⁶ cells for the MF, and from 1 × 10⁷ cells for CRC-PK. The detection of the different proteins was achieved by the reprobing of either the same membrane or membranes that resulted from gels loaded with identical samples. (C) A fraction of the HCV RNA exhibits resistance to S7 nuclease treatment. RNA samples were extracted from replicon lysates incubated with or without S7 nuclease and subjected to real-time RT-PCR with HCV IRES- or GAPDH-specific primers; the untreated samples were normalized to 100% for HCV IRES and GAPDH, respectively. (D) Cyclosporine treatment did not influence the level of CRC-associated, nuclease-resistant HCV RNA. GS5 cells were treated with cyclosporine for 22 h before being lysed for RNA extraction (total) or CRC isolation and nuclease treatment (CRC + S7). The HCV RNA level was determined by quantitative RT-PCR with HCV IRES primers, and the value for the untreated total sample was normalized to 100%.