Figure 3. TIP47 is involved in HCV RNA replication via interaction with NS5A.

A) Schematic representation of the HCV Replicon (genotype 1b) and Luc-JFH1 (genotype 2a), with mutations at position 9 and 12 in the NS5A N-terminus. B) Crystal violet-stained Huh7.5 cells transfected with wild-type or mutant HCV replicon RNA (genotype 1b). 24 h post-transfection, G418 was added to the medium (1 mg/ml). Cells were kept under selective pressure for 4 weeks before being stained with crystal violet. These results are representative of three independent experiments. C) Luciferase assay of Huh7.5 cells transfected with wild-type Luc-JFH1 RNA (genotype 2a) or Luc-JFH1 RNA containing indicated NS5A mutations. Values are expressed as fold increase of luciferase (RLU) per well, over a 4 h time point (mean ± standard deviation; n = 3, *P<0.05, **P<0.01, ***P<0.001). The decrease of A₉-I₁₂ and W₉-I₁₂ is significant in relation to WT (indicated in grey *), whereas the increase in A₉-V₁₂ is significant in relation to A₉-I₁₂ where indicated (indicated in black *). D) Amino acid sequences of the NS5A N-terminus of HCV replicon (genotype 1b) and HCV-JFH1 (genotype 2a). Positions 9 and 12 are highlighted in bold black and mutations are highlighted in color. Summary of respective replication phenotypes in the HCV replication assay (genotype 2a, Figure 3B, C). Wild-type replication phenotypes were set as equal to (++++) in their respective assays, while reduced replication levels were defined in the Replicon assay as (+++) for ∼90% of wild-type colonies, (++) for ∼20% colonies, or (−) for no colonies. In the Luciferase assay, luciferase values were described as (++) for values ∼19% of wild-type, (+) for ∼2%, and (−) for values below 1%. E) Coimmunoprecipitation assays in Huh7 cells transfected with expression vectors for Myc-TIP47 and NS5A-Flag or NS5A-Flag protein carrying either A₉-I₁₂ (W9A), W₉-I₁₂, or A₉-V₁₂ mutations. After immunoprecipitation with Flag antibodies, TIP47 was detected by western blotting with antibodies specific for Myc.