FIG 5.

Colony-forming ability of HCV subgenomic replicons expressing phosphorylation-defective NS5B proteins. (A) Schematic of the HCV subgenomic replicon. The substituted amino acid sequences are shown below the corresponding expanded NS5B region; ΔC and Neo represent the N-terminal part of the Core protein and neomycin phosphotransferase, respectively. Nonstructural (NS) proteins essential for HCV replication are shown. (B and C) Huh7 cells were electroporated with the indicated in vitro transcripts of subgenomic HCV replicons and seeded in 10-cm dishes. Colonies were selected by culturing with 0.5 mg/ml G418 for 3 weeks, fixed, and stained with crystal violet solution. The results represent the means (±SD) of three independent experiments (B), from which one representative set of stained cells is shown (C). ND, not detected. (D) Steady-state intracellular HCV subgenomic RNA levels of R-1 and the Huh7-derived stable cell line established with the NS5B_S42A subgenomic replicon were measured by real-time qRT-PCR. The results represent the means (±SD) of three independent experiments. Comparisons among the groups were made using the Student t test. *, P < 0.01.