FIG. 1.

The host response and cellular permissiveness to HCV RNA replication. (A) Diagram of the HCV genome (upper) and related replicon (lower). The polyprotein coding region and positions of individual protein products are shown. Arrows indicate positions of the RNA motifs described in the present study, including the IRES within the 5′NTR. (B) Huh7 cells were mock transfected or transfected with the indicated HCV replicon RNA and harvested 3, 8, 12, or 24 h later. ISG, replicon (HCV), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript abundance was monitored by Northern blot analysis. (C) Huh7 cells were transfected with an IFN-β promoter-luc construct and 24 h later were mock treated (mock), infected with SenV, or transfected with the indicated RNA. Cells were harvested 20 h after virus infection or 8 h after RNA transfection and then assayed for firefly luciferase activity. Bars show the average values and standard deviation (SD) of firefly luciferase activity relative to a Renilla luciferase control from three experiments (relative luc activity). (D) In the left panel is shown RNA abundance in Huh7 cells that were mock transfected (mock) or transfected with luc-poly(A), HCV 5′NTR-luc, or encephalomyocarditis virus IRES-luc RNA were assessed by Northern blot with probes specific to the indicated ISGs or GAPDH. Cells were harvested at the time shown in hours above each lane, and transfected RNA was detected with a luciferase-specific probe (Luc). For the right panel, cells were mock transfected or transfected with purified L2198S HCV replicon RNA, luc-poly(A), or HCV 5′NTR-luc transcript and then harvested 12 h later. mRNA levels were quantified by real-time PCR. Bars show the average level ± the SD of IFN-β mRNA relative to GAPDH control from three experiments. (E) HCV RNA replication and cellular G418 resistance transduction efficiency were evaluated after transfection of Δ5B or HP replicon RNAs into Huh7 or Huh7.5 cells. On the left is shown the average fold increase ± the SD of HP replicon RNA relative to Δ5B replicon RNA at the indicated times. The replicon abundance was estimated from the luciferase activity expressed by a firefly luciferase sequence that was inserted in lieu of the Neo sequence in the replicon shown in Fig. 1A. This provides an accurate assessment of HCV RNA abundance (28). On the right, G418-resistant colony-forming efficiency of the indicated HCV replicon RNAs was determined by cell staining 3 weeks after transfection of Huh7 (panels 1 to 3) or Huh7.5 cells (panels 4 to 6) followed by G418 selection. The numbers at the bottom show the fold increase in numbers of Huh7.5 cell colonies compared to Huh7 colonies. The results shown are representative of three independent experiments.