FIG. 6.

Second-site mutations, Q1112R in the protease domain and S1369R in the helicase domain of NS3, enhance RNA replication of Con1-H77 NS4B chimeras. At 96 h posttransfection, NS5A protein expression was analyzed by immunoblotting and the number of HCV RNA molecules in 1 μg of total cellular RNA was quantified by real-time RT-PCR. Below the blot for NS5A, HCV RNA levels (% RNA) are shown as percentages of the C1/WT level, which has been set at 100%. (A) Replication abilities of the Con1 chimeras C1/HN₄₇Tm, C1/HN₄₇C₃₄, and C1/HTmC₃₄ (Fig. 1), containing two domains from H77 NS4B. (B) Replication abilities of the single domain Con1 chimeras C1/HN₄₇, C1/HTm, and C1/HC₃₄ (Fig. 1). NS5A protein expression and HCV RNA levels are representative of at least two independent experiments.