FIG.1.

HCV RNA and protein synthesis in HuH6 Con1 replicons cells. Naive HuH6 cells were transfected via electroporation with a mixture of subgenomic in vitro-transcribed Con1 replicons and subjected to G418 selection (for details, see the text). Six cell clones were pooled and further passaged. (A) HCV RNAs were detected by Northern hybridization to a neo gene-specific riboprobe (lane 5). Different amounts of in vitro transcripts were used as standards (lanes 1 to 3). RNA isolated from naive HuH6 cells served as a negative control (lane 4). The positions of replicon RNA and 28S rRNA are indicated. (B) Detection of HCV protein expression by Western blotting. Equal amounts of total cell lysates of HuH6 Con1 replicon cells (lane 1) and naive control cells (lane 2) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by using antibodies with given specificities. (C) Detection of NS4B protein expression by indirect immunofluorescence in HuH6 Con1 replicon cells (top) and naive control cells (bottom). Cells were seeded onto glass coverslips, cultured for 24 h, fixed, permeabilized, and stained for NS4B (red) and DNA (turquoise) by using a specific antibody and DAPI, respectively. Bars, 25 μm.