Persistent replication of hepatitis C virus replicons expressing the beta-lactamase reporter in subpopulations of highly permissive Huh7 cells

Abstract

Progress toward development of better therapies for the treatment of hepatitis C virus (HCV) infection has been hampered by poor understanding of HCV biology and the lack of biological assays suitable for drug screening. Here we describe a powerful HCV replication system that employs HCV replicons expressing the beta-lactamase reporter (bla replicons) and subpopulations of Huh7 cells that are more permissive (or "enhanced") to HCV replication than naïve Huh7 cells. Enhanced cells represent a small fraction of permissive cells present among naïve Huh7 cells that is enriched during selection with replicons expressing the neomycin phosphotransferase gene (neo replicons). The level of permissiveness of cell lines harboring neo replicons can vary greatly, and the enhanced phenotype is usually revealed upon removal of the neo replicon with inhibitors of HCV replication. Replicon removal is responsible for increased permissiveness, since this effect could be reproduced either with alpha interferon or with an HCV NS5B inhibitor. Moreover, adaptive mutations present in the replicon genome used during selection do not influence the permissiveness of the resulting enhanced-cell population, suggesting that the mechanisms governing the permissiveness of enhanced cells are independent from viral adaptation. Because the beta-lactamase reporter allows simultaneous quantitation of replicon-harboring cells and reporter activity, it was possible to investigate the relationship between genome replication activity and the frequency with which transfected genomes can establish persistent replication. Our study demonstrates that differences in the replication potential of the viral genome are manifested primarily in the frequency with which persistent replication is established but modestly affect the number of replicons observed per replicon-harboring cell. Replicon copy number was found to vary over a narrow range that may be defined by a minimal number required for persistent maintenance and a maximum that is limited by the availability of essential host factors.

FIG. 1.

The %BC readout correlates with the number of Neo^r CFU. Left panel, Huh7 cells were lipotransfected with 5 μg of the indicated bla-NS3-3′ replicon and stained for bla expression at day 6. The fraction of cells harboring replicon was determined by DIP of stained cells (>1,000 cells/data point). Right panel, 2 × 10⁵ Huh7 cells were lipotransfected with 100 ng of the indicated neo-NS3-3′ replicon in T75 flasks. Cells were cultured in complete medium supplemented with 250 μg of G418/ml for 4 weeks, at which time colonies were counted.

FIG. 2.

Persistent replication of bla-NS3-3′/S2204I in MR1 cells. Naïve Huh7 cells and MR1 cells (G418-selected population of Huh7 cells harboring neo-NS2-3′/S2204A) were transfected with bla-NS2-3′/S2204I replicon. The fraction of cells harboring replicon was determined for each population by DIP at the indicated times. •, Huh7 cells; ○, Huh7 cells harboring neo-NS2-3′/S2204A.

FIG. 3.

Replicon-enhanced cells. (A) Total RNA was isolated from Huh7 cells harboring either neo-NS3-3′/S2204I (MR4) or neo-NS2-3′/S2204A (MR1) and from MR1 cells treated with IFN-α (MR2 cells) or with a specific NS5B inhibitor (MR3 cells). Replicon RNA levels were estimated by RNase protection assays by comparison with HBI10A cells that have 800 to 1,000 copies/cell (8). (B) MR1 (•), MR2 (○), and MR3 (▾) cells were transfected with bla-NS3-3′/S2204I replicon RNA. The fraction of cells harboring replicon was determined by DIP at the indicated times following transfection.

FIG. 4.

Replicon-cured MR4 cells are enhanced to replication of bla replicons. MR5 and MR6 cells were derived by treating MR4 cells with IFN-α and an NS5B specific inhibitor, respectively. Persistent replication was assayed in these cells as described in the text. •, Huh7 cells; ○, MR4 cells; ▾, MR5 cells; ▿, MR6 cells.

FIG. 5.

Level of cellular permissiveness is not dependent on the con1 adaptive mutation present in the neo replicon used to isolate Neo^r cells. (A) Replicon RNA per cell in MR1, MR4, MR7, and MR9 cells was measured by RNase protection assays. Data were normalized with respect to GAPDH. (B) Huh7 and replicon-cured MR2, MR5, MR8, and MR10 cells were transfected with bla-NS3-3′ replicons containing the indicated adaptive mutations. The fraction of cells harboring replicon 6 days after transfection was determined by DIP.

FIG. 6.

Persistent replication is RNA dose dependent. MR2 cells were transfected with various doses of bla-NS3-3′ replicons containing the indicated adaptive mutation. The fraction of cells harboring replicon was determined by DIP at days 1 (A) and 6 (B). The bla-NS3-3′ replicons used had the following mutations: S2204I (•), A2199T (○), R2884G (▾), and S2204I and the NS5B GAA inactivating mutation (▿).

FIG. 7.

Relative amounts of replicon per replicon-harboring cell. Cells were lipotransfected with the indicated bla-NS3-3′ replicon and then assayed at day 3. (A) The relative amounts of bla in each sample are proportional to the rate at which nitrocefin is cleaved to generate a product that absorbs at 490 nm. Cells were lysed by three cycles of freezing and thawing. Reactions were initiated by the addition of nitrocefin, and absorbance (Abs) at 490 nm was measured at 5-min intervals. •, S2204I; ○, A2199T; ▾, R2884G; ▿, wild type; ▪, GAA. (B) The %BC in the same population was determined by DIP. (C) Reporter activity per replicon-harboring cell (or intrinsic genome replication activity) was calculated by normalizing the reporter activity presented in panel A by the number of cells expressing bla at the time of the assay (total number of cells assayed multiplied by the fraction of cells that contain replicon [panel B]).