FIG. 4.

Establishment of a long-term persistent HCV infection in DMSO-treated Huh7 cells. Huh7 cells (8 × 10⁴) were plated in multiple wells of 12-well BioCoat collagen-coated plates. Medium was supplemented with 1% DMSO (vol/vol) at 24 h postseeding and replenished every 3 days thereafter. At 20 days post-DMSO treatment, multiple wells were infected with JFH-1 HCV at an MOI of 0.01 FFU/cell and the culture supernatant and intracellular RNA were collected at the indicated times p.i. for up to 63 days. (A) Intracellular HCV RNA was analyzed by RT-QPCR and displayed as HCV RNA copies/μg total RNA (line). Titers of supernatant infectivity were determined for naïve Huh7 cells and are expressed as FFU/ml (bars). The data presented are representative of three independent experiments. (B to D) NS5A immunostaining of DMSO-treated Huh7 cells at days (B) 12, (C) 36, and (E) 62 p.i. (magnification, ×100) was performed as described in reference 43. Image brightness and contrast were adjusted using Adobe Photoshop (San Jose, CA). (E) HCV RNA replication in DMSO-treated Huh7 cells is sensitive to the effects of interferons. At 30 days p.i., DMSO-treated Huh7 cultures were treated with 100 U/ml of IFN-α, IFN-β, or IFN-γ (PBL Biomedical Laboratories, New Brunswick, NJ). On the indicated days posttreatment, total RNA was extracted and intracellular HCV RNA copies/μg of cellular RNA was quantitated by RT-QPCR. Reductions (n-fold) in HCV copy numbers were calculated as follows: number of intracellular HCV RNA copies per μg of cellular RNA in IFN-treated cultures/number of intracellular HCV RNA copies per μg of cellular RNA in diluent-treated cultures. The data presented are representative of three independent experiments.