Nonhepatic cell lines HeLa and 293 support efficient replication of the hepatitis C virus genotype 2a subgenomic replicon

Abstract

The hepatitis C virus (HCV) genotype 2a subgenomic replicon can replicate in two human non-hepatocyte-derived cell lines, HeLa and 293, with in vitro-transcribed replicon RNA. Sequencing analysis revealed that mutations in HCV-derived regions were not essential for replication in these cells, as some clones displayed no mutations.

FIG. 1.

Colony formation of JFH-1 HCV subgenomic RNA replicon in Huh7, HeLa, and 293 cell lines. Transcribed RNAs from pSGR-JFH1 were transfected into each cell line, and cells were cultured with G418 for 3 weeks before staining with crystal violet, as described in Materials and Methods. Representative staining examples are shown for 0.1 μg of synthetic RNA transfected onto Huh7 cells and 3 μg transfected for both HeLa and 293 cells.

FIG. 2.

Detection of replicon RNA in cloned HeLa (A) and 293 (B) cells. Total RNA from cloned cells in each cell line was analyzed by Northern blotting with DNA probes of the neo^r EMCV IRES and β-actin genes. In vitro synthesis of 10⁸ and 10⁷ copies of transcribed positive-strand RNAs was performed, and RNA was loaded (lanes 10⁸ and 10⁷) as positive controls, as indicated. Arrowheads indicate target positions of replicon RNA and β-actin. N, cellular RNA of HeLa or 293 cells as negative controls.

FIG. 3.

Detection of HCV NS5A antigens in cloned cells of HeLa (A) and 293 (B) cells by Western blot analysis. Cell lysates were prepared from pSGR-JFH1 RNA-transfected Huh7 cell clones (lanes 4-1 and C6) as positive controls or untransfected parental HeLa and 293 (lane N) as negative controls. Anti-NS5A polyclonal antibodies were used to detect HCV antigens. Target sizes of NS5A proteins are indicated by arrowheads.

FIG. 4.

Subcellular localization of HCV antigens determined by immunofluorescence. Replicon RNA-untransfected [Replicon(−)] or cloned HeLa, 293, and Huh7 [Replicon(+)] cells were cultured on coverslips, fixed in acetone-methanol, and incubated with patient serum, as described in the Materials and Methods. Representative clones of HeLa, 293, and Huh7 cells are indicated.

FIG. 5.

Effect of replicon RNA replication on cell growth rate in HeLa (A) and 293 (B) cell lines. Cell growth rates were compared between parental cells (closed circles) and replicon-containing cell lines (open triangles and diamonds) with a resazurin reduction assay. OD570/600, optical density at 570/600 nm. Names of replicon clones (in parentheses) were consistent with Tables 1 and 2. Experiments were performed with triplicate wells and repeated twice. Mean data and standard deviation bars are shown.

FIG. 6.

Dose-dependent inhibition of replicon RNA replication on interferon administration in the HeLa (A) and 293 (B) cell lines. Amounts of replicating replicon RNA were measured by RT-PCR for serial doses of interferon (IFN) administration and are represented as a percentage of the amount of replicon RNA in cells without interferon administration. Names of replicon clones (in parentheses) are consistent with Tables 1 and 2. Experiments were performed with duplicate wells for each interferon dose and repeated twice. Mean data and standard deviation bars are shown.