FIG. 5.

Second-site mutations in NS3 suppress the replication defect of the Con1 chimera encoding the entire H77 NS4B protein. At 96 h postelectroporation, NS5A expression was analyzed by immunoblotting. The numbers of HCV RNA molecules in 1 μg of total cellular RNA were quantified at 96 h posttransfection by real-time RT-PCR and are shown below the NS5A blot (% RNA) as percentages of the C1/WT level, which has been set at 100%. PR+SG refers to the chimera containing both P1115R and S1560G in NS3. Similar results were obtained in three independent experiments and the same trends in RNA replication efficiency were also observed at 24 h, 48 h, and 72 h posttransfection, except that the difference in HCV RNA levels between experimental RNAs and the C1/pol⁻ control was greater at later time points.