FIG 4.

Optimization of a luciferase-based reporter system for quantification of HCV cell-to-cell spread. Our previously described dual-reporter system for monitoring HCV infection (34) was modified by replacing the mCherry gene with a firefly luciferase gene. A stable Huh-7.5 cell line, designated NIrD-fluc-sc4, was constructed by using the newly modified reporter plasmid. This novel reporter cell line was used to optimize the conditions for the quantification of HCV cell-to-cell spread. The parameters for optimization included the coculture time of HCV donor and recipient cells, the MOI used for HCV infection of donor cells, the ratio between HCV donor and recipient cells, and restriction of HCV cell-free dissemination. NIrD-fluc-sc4 cells, serving as HCV recipient cells, were transfected with a DNA vector expressing Renilla luciferase as a control for normalization, whereas Huh-7.5 cells were infected with HCV as donor cells. At 24 h p.t. and at 24 h p.i., respectively, HCV donor and recipient cells were mixed and cocultured in the presence or absence of 2 μg/ml of Dox and a 2% methylcellulose overlay. (A) HCV cell-to-cell spread at different time points (24 h, 48 h, and 72 h) of coculture of HCV donor and recipient cells. Cell lysates collected at 24 h, 48 h, and 72 h p.i. were used for measurement of both Renilla and firefly luciferase activities with the Dual-Glo luciferase assay system. Uninfected cells (Dox only) and coculture of HCV donor and recipient cells without Dox (HCV only) served as negative controls. (B) Dependence of HCV cell-to-cell spread on HCV infection of donor cells. Huh-7.5 cells were infected with HCV at different MOIs (0 to 10) (x axis) and then cocultured with pcDNA6/TR-Rluc-transfected NIrD-fluc-sc4 recipient cells for 48 h in the presence of 2 μg/ml of Dox. The levels of Renilla luciferase and firefly luciferase expression were determined as described above for panel A. (C) Determination of the optimal ratio between HCV donor and recipient cells. Huh-7.5 cells were infected with HCV at an MOI of 10 for 24 h. HCV-infected Huh-7.5 cells were then cocultured with pcDNA6/TR-Rluc-transfected NIrD-fluc-sc4 cells at different ratios (8:1, 4:1, 2:1, 1:1, 1:2, 1:4, and 1:8) of donor to recipient cells in the presence of 2 μg/ml of Dox and a 2% methylcellulose overlay. After 48 h of coculture, cells were harvested for the measurement of Renilla and firefly luciferase activities. (D) Blockade of HCV cell-free dissemination by E2-specific monoclonal antibody CBH5 (60) or 2% methylcellulose. HCV-infected Huh-7.5 cells (MOI of 10) were cocultured with pcDNA6/TR-Rluc-transfected NIrD-fluc-sc4 cells in the presence of Dox (2 μg/ml) and CBH5 (10 μg/ml) or a 2% methylcellulose overlay. After 48 h of coculture, cells were lysed for the quantification of Renilla and firefly luciferase expression. The relative levels of firefly luciferase (average values derived from three experiments) were normalized to the levels of Renilla luciferase (average values derived from three experiments) and converted to a percentage of the control, considering the conditions with the highest level of firefly luciferase expression as 100%. *, P < 0.05; **, P < 0.01.