Translation from the HCV IRES is selectively resistant to inhibition by PKR in vivo. Embryonic fibroblasts from PKR−/− mice were transfected with bicistronic constructs containing an upstream Renilla luciferase gene (expressed in a 5′ cap-dependent manner) and a downstream firefly luciferase gene under the control of the HCV IRES (pRHCVF), the EMCV IRES (pREMCVF), or no IRES (pRF). The cells were cotransfected either with an expression vector for wild-type human PKR or with pcDNA3 as a control plasmid. At 21–24 h after transfection, cell extracts were prepared and luciferase activities were determined as described in Materials and Methods. (A) Diagrammatic representation of the bicistronic vectors used. (B, C) Cap-dependent Renilla luciferase activity. (D, E) IRES-dependent firefly luciferase activity. The percent inhibitions by PKR are indicated in parentheses over the appropriate bars. (F) Ratios of IRES-dependent to cap-dependent activity.
