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. Author manuscript; available in PMC: 2015 Oct 1.
Published in final edited form as: Hepatology. 2015 May 12;62(4):1059–1069. doi: 10.1002/hep.27815

Fig. 2.

Fig. 2

Adaptation of HCV to infect CLDN1 KO Huh-7.5 cells. (A) Immunoblotting analysis for either GFP or actin was performed on lysates from naïve or CLDN1 KO clone 4 Huh-7.5 cells either not expressing a transgene (not transduced) or transduced with lentiviral particles to express GFP alone or the indicated GFP-CLDN fusion proteins. Approximate molecular mass (kDa) marker positions are indicated to the left of each blot. (B) Above cells were infected with GLuc expressing Jc1 in the presence of DMSO (vehicle, light gray columns) or 2′CMA (dark gray columns). Parallel VSVGpp infections were performed as a positive control (white columns). Luciferase values are expressed as RLU. Mean and SE of two independent experiments, each performed in triplicate, are shown. (C) HCV persistence in serially passaged Jc1 HCV-RNA-transfected CLDN1 KO clone 4 cells expressing either GFP alone or GFP-tagged CLDNs was monitored at the indicated days post-transfection and postmixing with fresh target cells. Shown are representative fields of 80%-100% confluent monolayers of cells stained for the HCV NS5A protein (dark cells). At Day 12 postmixing essentially all cells stained NSSA positive. (D) A stock of WT Jc1, as a reference point, and supernatants containing virus passed in the cell populations indicated in the figure key were titered by limiting dilution assay on naïve Huh-7.5 cells and CLDN1 KO clone 4 cells either not transduced or expressing the indicated transgene. Means and SEs of three independent TCID50 assays are shown. Limit of detection is indicated by a dashed line. (E) CLDN1 KO clone 4 cells expressing either GFP alone or GFP-CLDN1 were either not transfected (open symbols and dotted lines) or transfected with Jc1 HCV RNA (filled symbols and solid lines). Cells were passaged and analyzed for GFP expression by FACS at the indicated days post-transfection to monitor transgene expression. Abbreviations: DMSO, dimethyl sulfoxide; ND, not detected; NT, not tested; RLU, relative light units; TCID50/mL, 50% tissue culture infectious dose per milliliter.