FIG. 5.

NS2 complex formation is enhanced by cotranslation as a polyprotein. (A) Schematic of bicistronic HCV constructs containing insertions of a stop codon and ECMV IRES element (indicated by E·I). Protein cleavage sites are labeled as in Fig. 1A; the AP tag is indicated by a black box. (B) Affinity purification of NS2(AP)-containing complexes. Huh-7.5(BirA) cells were transfected with the indicated constructs and samples were prepared and analyzed as in Fig. 2A. (C) Bicistronic HCV constructs replicate efficiently. Huh-7.5(BirA) cells were split at 48 h posttransfection with the indicated viral RNAs, reseeded, and allowed to grow for an additional 48 h. Total cellular RNAs were extracted, and the level of HCV RNA was quantified by quantitative reverse transcription (qRT)-PCR as previously described (37). Jc1 GNN was a replication-defective Jc1 genome containing inactivating mutations in the NS5B RNA polymerase active site. The y axis represents copies of HCV genome per 10 ng of total RNA. (D) Bicistronic HCVcc constructs produced reduced viral titers. Media were collected from Huh-7.5(BirA) cells at 48 h posttransfection with the indicated viral RNAs, and titers were determined on Huh-7.5 cells. Values represent averages of results from three independent experiments; error bars represent the standard deviations from the means.