Production of infectious hepatitis C virus by well-differentiated, growth-arrested human hepatoma-derived cells

Abstract

Dimethyl sulfoxide (DMSO) has been shown to induce the differentiation of primary hepatocytes in vitro. When actively dividing poorly differentiated human hepatoma-derived (Huh7) cells were cultured in the presence of 1% DMSO, cells became cytologically differentiated and transitioned into a nondividing state, characterized by the induction of hepatocyte-specific genes. Moreover, these cells were highly permissive for acute hepatitis C virus (HCV) infection, and persistent long term infection of these cultures could also be achieved. As HCV naturally replicates in highly differentiated nondividing human hepatocytes, this system may more accurately mimic the conditions under which HCV replicates in vivo than previous models using poorly differentiated rapidly dividing hepatoma cells.

FIG. 1.

DMSO-induced differentiation of Huh7 cells. (A) Phase contrast micrograph of Huh7 cells cultured on BioCoat collagen-coated plates (Becton Dickinson, Franklin Lakes, NJ) in the presence of 1% DMSO (vol/vol) (Sigma-Aldrich, St. Louis, MO) and photographed 20 days after plating (magnification, ×200). (B) Hematoxylin and eosin staining of DMSO-treated Huh7 cells cultured in the presence of 1% DMSO for 20 days (magnification, ×400; inset magnification, ×800). Arrows indicate binucleated cells containing multiple distinct nucleoli. (C) Flow cytometric analysis of Huh7 cells cultured in the presence of 1% DMSO for 20 days. For cell cycle analysis, 1 × 10⁵ cells were stained with 15 μg/ml propidium iodide (PI) (Boehringer Mannheim, Indianapolis, IN), in the presence of 0.25% NP-40 (Sigma-Aldrich) for 30 min, and analyzed using a FACSCalibur flow cytometer (Becton Dickinson), and data were analyzed using FloJo software (Tree Star, Inc., Ashland, OR).

FIG. 2.

Growth kinetics and induction of hepatocyte-specific cellular genes in DMSO-treated Huh7 cells. For gene analysis, 8 × 10⁴ Huh7 cells were plated in multiple wells of 12-well BioCoat collagen-coated plates. Medium was supplemented with 1% DMSO (vol/vol) at 24 h postseeding (when cells reached 95% confluence), and cells were cultured for an additional 20 days in the presence of DMSO. At indicated times post-DMSO treatment, triplicate wells were trypsinized and the average total cell number was calculated (line). Cells were additionally pelleted at 1,400 rpm for 5 min, and total cellular RNA was extracted (43) and analyzed by RT-QPCR (Bio-Rad, Hercules, CA) for human albumin, A1AT, and HNF4-α mRNA expression. GAPDH amplification was used as a normalization control, and the results are expressed as induction (n-fold) of gene expression in Huh7 cells post-DMSO treatment (days 1 to 20 and day 200) relative to that of control, non-DMSO-treated, Huh7 cells (day 0).

FIG. 3.

HCV infection kinetics in DMSO-treated Huh7 cells. (A and B) Huh7 cells cultured for 6 (open square), 14 (open circle), or 20 (open diamond) days in the presence of 1% DMSO or subconfluent (closed squares) and confluent (closed triangles) non-DMSO-treated Huh7 cells 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 postinfection. (A) Intracellular HCV RNA was analyzed by RT-QPCR and displayed as HCV RNA copies/μg total RNA. (B) Titers of supernatant infectivity, expressed as FFU/ml, were determined by indirect immunofluorescence analysis (Axiovert 200 fluorescence microscope; Zeiss, Germany) of 10-fold serially diluted culture supernatants on naïve Huh7 cells, using a 96-well plate format as described previously (43). A human monoclonal antibody with high avidity and specificity to HCV E2 (43) was used to detect positive foci (5 to 10 positive grouped cells constitute one focus). The anti-HCV E2 antibody was used in lieu of the previously reported (43) NS5A antibody, as it performs equally well in titer assays (data not shown).

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 . 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.