FIGURE 2.

ORF45 facilitates translation. A, ORF45 promotes assembly of the translation initiation complex. HEK293 cells were transfected with pCR3.1-ORF45, pCR3.1-ORF45-F66A, or empty vector. Forty-eight hours after transfection, cell lysates were prepared and incubated with m⁷GTP-Sepharose beads at 4 °C. After washes, the bead-bound proteins and the input cell lysates were analyzed by Western blots with antibodies as indicated. B, ORF45 promotes association of mRNAs with polysomes. Lysates from pCR3.1-ORF45 or empty vector-transfected HEK293 cells were loaded on 15–50% (w/v) sucrose gradients. After centrifugation, 1-ml fractions were collected (fraction 1, 15% sucrose; fraction 12, 50% sucrose) and used to purify RNA. The concentration of RNA was measured by absorbance at 260 nm (Ab260). The experiments were performed in duplicate (*, p < 0.05). C, schematic presentation of the bicistronic reporter plasmid (pGEM-REN-HCV IRES-LUC). T7, T7 promoter; REN, Renilla luciferase; IRES, internal ribosomal entry site of HCV; LUC, firefly luciferase. D, ORF45 promotes protein translation. HEK293 cells stably transduced with control siRNA or siRNAs against RSK1 and RSK2 were infected with a recombinant vaccinia virus, vTF7-3 (multiplicity of infection = 10), that expresses bacteriophage T7 RNA polymerase. The infected cells were then transfected with the reporter plasmid pGEM-REN-HCV IRES-LUC plus pCR3.1-ORF45, pCR3.1-ORF45-F66A, or empty vector pCR3.1. Luciferase activities were measured 36 h after transfection with the Dual-Luciferase assay kit (Promega). The experiments were performed three times, each in duplicate (**, p < 0.01). Error bar represents standard deviation.