Hepatitis C virus subgenomic replicons in the human embryonic kidney 293 cell line

Abstract

Hepatitis C virus (HCV) infects liver cells and its replication in other cells is incompletely defined. Human hepatoma Huh-7 cells harboring subgenomic HCV replicons were used in somatic cell fusion experiments with human embryonic kidney 293 cells as a means of examining the permissiveness of 293 cells for HCV subgenomic RNA replication. 293 cells were generally not permissive for replication of Huh-7 cell-adapted replicons. However, upon coculturing of the two cell lines, we selected rare replicon-containing cells, termed 293Rep cells, that resembled parental 293 cells. Direct metabolic labeling of cells with (33)P in the presence of actinomycin D and Northern blotting to detect the negative strand of the replicon demonstrated functional RNA replicons in 293Rep cells. Furthermore, Western blots revealed that 293Rep cells expressed the HCV nonstructural proteins as well as markers of the naïve 293 cells but not Huh-7 cells. Propidium iodide staining and fluorescence-activated cell sorting analysis of 293Rep cells revealed that clone 293Rep17 closely resembled naïve 293 cells. Transfection of total RNA from 293Rep17 into naïve 293 cells produced replicon-containing 293 cell lines with characteristics distinct from those of Huh-7-derived replicon cell lines. Relative to Huh-7 replicons, the 293 cell replicons were less sensitive to inhibition by alpha interferon and substantially more sensitive to inhibition by poly(I)-poly(C) double-stranded RNA. This study established HCV subgenomic replicons in nonhepatic 293 cells and demonstrated their utility in expanding the study of cellular HCV RNA replication.

FIG. 1.

Neomycin- and zeocin-resistant colony formation with Huh-7- and 293-derived cell lines. (A) Schematic of somatic cell fusion between replicon-containing Neo^r S22.3 cells and Zeo^r 293 cells. The generation of heterokaryons from S22.3 and 293 cells would indicate that 293 cells are recessive for an activity present in Huh-7 cells that is essential for RNA replication; the lack of clones would indicate that the restriction of the 293 cells is dominant over HCV RNA replication. (B) Cellular fusions performed with Huh-7^Neo and Huh-7^Zeo, two distinct Huh-7-derived cell lines with chromosomally integrated markers (i); Huh-7^Neo and 293^Zeo cells, each with the respective chromosomally integrated marker (ii); S22.3 and Huh-7^Zeo, two homologous cell lines with distinct RNA and DNA selectable markers (iii); and S22.3 and 293^Zeo, two heterologous cell lines with RNA and DNA selectable markers (iv). Cellular fusions were performed by coculturing the cells for 20 h and then either leaving cells untreated (−PEG) or treating them with a 50% PEG solution for 5 min (+PEG). Cells were then transferred to 100-mm-diameter plates and selection was done with neomycin and zeocin for 21 days. Colonies obtained were stained with crystal violet and counted (numbers in lower right corners).

FIG. 2.

293Rep cells contain the HCV subgenomic replicon. (A) ³³P metabolic labeling of replicon RNA in the presence of actinomycin D. Total cellular RNA was extracted from cells treated with ³³P and actinomycin D for 16 h and was resolved in a 0.6% denaturing agarose gel. (B) Northern blots detecting the negative strand of HCV subgenomic replicons. Total cellular RNA was extracted and resolved in a glyoxal-dimethyl sulfoxide agarose gel. The RNAs were transferred and hybridized with an HCV negative-strand-specific ³²P-labeled RNA probe. The first three lanes are positive controls of in vitro-synthesized negative strand RNA, with 10⁸, 10⁷, and 10⁶ copies used, as indicated.

FIG. 3.

293Rep cells express 293- and HCV-specific proteins. (A) Western blot analysis of 293Rep cells. Equivalent concentrations of total cellular proteins, obtained from cell lysates and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were immunoblotted with either anti-E1A monoclonal antibody or anti-AFP polyclonal antibody. The anti-AFP blot was stripped and then reblotted with anti-albumin polyclonal antibody. (B) Western blot analysis of proteins from 293Rep cells. Total cellular proteins, prepared as for panel A, were immunoblotted with either anti-NS3 polyclonal antibody or anti-NS5A monoclonal antibody.

FIG. 4.

Detection of HCV proteins in 293Rep cells by immunofluorescence. Fixed and permeabilized cells were incubated with either a polyclonal antibody against the NS3 protein (A) or a monoclonal antibody against the NS5A protein (B) and appropriate secondary antibodies. IF, immunofluorescence with the indicated primary antibody; DAPI, 4′,6′-diamidino-2-phenylindole DNA staining; LM, phase-contrast light microscopy of the same cells. Arrows define the characteristic cytoplasmic lipid droplets of Huh-7 cells.

FIG. 5.

PI staining and FACS analysis of cell lines. Huh-7, 293, S22.3, 293Rep5, and 293Rep17 cells were digested with trypsin and incubated in staining buffer containing PI for 10 min. DNA content was determined with a FACS analyzer. (A) Overlay of the FACS profiles of parental Huh-7 (black) and 293 (blue) cells depicting canonical G₁ and G₂/M phase peaks. (B) Overlay of FACS profile of S22.3 cells (red) onto the profile of panel A. (C) Overlay of FACS profile of Huh-7^Neo/293^Zeo hybrid cells (red) onto the profile of panel A, revealing different G₁ and G₂/M phase peaks. (D) Overlay of FACS profile of 293Rep17 (red) cells onto the profile of panel A. (E) Overlay of FACS profile of 293Rep5 (red) cells onto the profile of panel A.

FIG. 6.

Analysis of 293 cell lines generated by transfection of total RNA from 293Rep17 cells. (A) ³³P labeling of total RNA in the presence of actinomycin D. Selected 293 cell clones (LR1, DM1, DM3, DM4, DM6, DM8, LIP3, LIP5, and LIP7) were metabolically labeled with ³³P and actinomycin D for 16 h and visualized as described for Fig. 2. (B) Western blot with anti-NS3 or anti-NS5A antibody as described for Fig. 3. (C) Immunofluorescence detection of viral proteins in replicon-containing LR1 and naïve 293 cells was performed as described for Fig. 4.

FIG. 7.

Inhibition of subgenomic RNA replicon in 293 cells. (A) An HCV NS3 protease inhibitor (4C) decreases intracellular replicon levels. S22.3 and 293Rep17 cells were cultured with the protease inhibitor and labeled with ³³P and actinomycin D; total cellular RNA was resolved as described for Fig. 2. Protease inhibitor was tested at 0, 0.1, 0.3, and 1 μM concentrations. (B) Replicon cells were cultured in the presence of different doses of IFN-α, ranging from 0.023 to 50 U/ml, for 72 h. Total cellular RNA was extracted and the replicon copy number was quantified by Taq-Man real-time RT-PCR to obtain the percent inhibition of treated samples with respect to untreated control levels. Circles, S22.3 cells; squares, 293Rep17 cells; triangles, LR1 cells. (C) Inhibition of replicon in cells as described for panel B, but cultured in the presence of different doses (0.9 to 2,000 μg/ml) of poly(I)-poly(C). The plotted percentages of inhibition represent average values (with error bars) from at least three determinants.