Recombinant Dicer binds and cleaves HCV IRES in vitro. (A-B) Electrophoretic mobility shift assays (EMSA) ³²P-labeled HCV RNA nt 1-341 (A) or nt 1-515 (B) was incubated in the absence or presence of recombinant human Dicer (200 ng) and/or BSA (2 μg), and complex formation visualized by non-denaturing PAGE and autoradiography. (C-D) Dicer RNase activity assays. (C) ³²P-labeled HCV RNA nt 1-341 (left panel) or nt 1-515 (right panel) was incubated in the absence (-) or presence (+) of recombinant human Dicer (200 ng), and HCV RNA processing monitored by denaturing PAGE and autoradiography. Lanes 4, 5, 6 and 7 represent higher numerical exposition of lanes 2, 3, 8 and 9 respectively. M, indicates a 10-nt RNA size marker.

Dicer does not bind HCV IRES in vivo. HCV IRES nt 1-341 was amplified by RT-PCR from RNA extracted from Dicer immunoprecipitates (IPs) prepared from Huh-7 or 9–13 cells by ribonucleoprotein immunoprecipitation (RIP) assay. The amplified DNA products were analyzed by 1.5% agarose gel electrophoresis and stained with ethidium bromide (lower panel). Proteins (100 μg) were analyzed by 10% SDS-PAGE to visualize Dicer protein expression or immunoprecipitation in Huh-7 and 9–13 cells (upper panel).

miRNA-guided RNA silencing is not perturbed in cells harboring a subgenomic HCV replicon. (A) Schematic representation of the experimental strategy and reporter gene constructs. (B) HCV RNA expression in Huh-7 or 9–13 cells harbouring a subgenomic HCV replicon, treated or not with 100 IU/ml of interferon α-2B (IFNα-2B), was documented by Northern blot using a DNA probe complementary to HCV Internal ribosome entry site (nt 1 to 341). GAPDH was used as a loading control. (C) HCV NS3 and NS5B protein expression Huh-7 or 9–13 cells, treated or not with 100 IU/ml of IFNα-2B, was documented by Western blot using anti-NS3 1B6 (first panel) and anti-NS5B 5B-3B1 (third panel) antibodies, respectively. Actin was used as a loading control (second and fourth panels). (D) Huh-7 or 9–13 cells, treated or not with 100 IU/ml of IFNα-2B, were cotransfected using Lipofectamine 2000 with a Rluc:miRNA binding site construct, in which the Rluc reporter gene is coupled with 1 or 3 copies of perfectly complementary (PC) or natural wild-type (WT) binding sites (BS) for miR-328 (250 ng DNA), and a psiSTRIKE-based, pre-miR-328 expression construct (250 ng DNA). psiSTRIKE-Neg, which encodes a shRNA directed against a sequence deleted in the Rluc reporter mRNA, was used as a control. Results of Rluc activity were normalized with Fluc activity and expressed as a percentage of Rluc activity obtained with psiSTRIKE-Neg. Results are expressed as mean ± s.e.m. (n = 3 experiments, in duplicate).

HCV domains II, III and VI are processed into ~21 to 23-nt RNA species by recombinant human Dicer in vitro. (A) Predicted secondary structure of nt 1 to 515 of the HCV RNA genome. (B) Dicer RNase activity assays. ³²P-labeled HCV RNA domain II (left panel), domain VI (center panel) or domain III (right panel) was incubated in the absence (-) or presence (+) of recombinant human Dicer (65 ng) with MgCl₂. The samples were analyzed by denaturing PAGE and autoradiography. M, indicates a 10-nt RNA size marker.

Overexpression of Dicer has no effect on HCV IRES-mediated translation. (A) Schematic representation of the reporter gene construct with pRL-CMV-1-515. (B) Reporter gene activity assays. pRL-CMV-1-515 was co-transfected in HEK 293 cells with increasing amounts (0–300 ng DNA) of pcDNA3.1-5'Flag-Dicer. Cells were harvested seventy-two (72) hours later, lysates were prepared, and Rluc and Fluc activities were measured successively. The results were normalized with those obtained from cells cotransfected with pRL-CMV-1-515 with empty vector pcDNA3.1-5'Flag. Results are expressed as mean ± s.e.m. (n = 6 experiments, in duplicate).