FIG. 8.

Effect of NS4B on HCV IRES-driven translation and polyprotein processing. (A) Eight micrograms of the polymerase-defective replicons (pol⁻) expressing H77 NS4B (H/pol⁻), Con1 NS4B (H/C4B/pol⁻), or a hybrid NS4B containing the first 47 amino acids from Con1 NS4B (H/CN₄₇/pol⁻) was electroporated into Huh-7.5 cells. Lysates collected at 10 h postelectroporation were subjected to immunoblot analysis with a polyclonal antiserum to NS5A (top). Relative NS5A levels measured by phosphorimaging were normalized to GAPDH expression (bottom) and to HCV RNA quantified at the same time point (data not shown). Samples collected 96 h after transfection of the replication-competent chimeric replicons, H/C4B and H/CN₄₇, were separated in parallel (lanes 1 and 2). The negative control (Huh) (lane 6) represents Huh-7.5 cells transfected with 8 μg of cellular RNA isolated from naïve Huh-7.5 cells. The migration of NS5A and GAPDH is shown on the left. These results are representative of two independent experiments. (B) Huh-7.5 cells infected with a vaccinia virus expressing T7 RNA polymerase were transfected with 1 μg of the indicated plasmid DNA and incubated for 1 h in the presence of [³⁵S]methionine-cysteine, as described in Materials and Methods. The labeled cells were lysed, and NS3, NS4B, and NS5A were analyzed by immunoprecipitation using a patient serum, followed by SDS-9% polyacrylamide gel electrophoresis, autoradiography, and phosphorimaging. The ³⁵S-labeled NS4B signal was corrected for DNA transfection efficiency by normalizing to NS3 expressed from the corresponding replicon DNA. Values below the gel represent the percentage of normalized NS4B expressed relative to the level of NS4B encoded by the H/WT replicon, which has been set at 100%. The negative control (Huh) represents vaccinia virus-infected Huh-7.5 cells transfected with an unrelated plasmid DNA that was immunoprecipitated with the same antiserum as described above. The mobilities of the molecular mass standards (in kilodaltons) are given on the left, and the migration of the HCV-specific proteins is indicated on the right. Con1 NS4B migrated more slowly than H77 NS4B, presumably reflecting differences in amino acid composition between the proteins.