FIG. 4.

Foreign H77 NS4B sequences do not affect polyprotein processing. Huh-7.5 cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase were transfected with the indicated plasmid DNA and labeled with [³⁵S]methionine-cysteine. Labeled NS3, NS4B, and NS5A was immunoprecipitated using patient serum before separation on an SDS-9% polyacrylamide gel. Proteins were visualized using autoradiography and quantified by phosphorimaging. The NS4B signal in each sample was normalized to the corresponding NS3 signal to account for differences in transfection efficiencies. Shown below the gel is the percentage of normalized NS4B expressed in each sample relative to the NS4B level expressed from C1/WT, which has been set at 100%. As controls, Huh-7.5 cells were transfected with the expression plasmid pcDNA (Invitrogen) or the H77 replicon plasmid (H/WT) (2) and treated as described above. The molecular mass standard in kilodaltons is labeled on the left and the mobilities of the various HCV proteins are indicated on the right. The difference in migration of Con1 NS4B compared to the migration of H77 NS4B may be attributed to differences in amino acid sequence.