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Adenovirus vector-mediated assay system for hepatitis C virus replication - PubMed

Adenovirus vector-mediated assay system for hepatitis C virus replication

Takeshi Yoshida et al. Nucleic Acids Res. 2011 May.

Abstract

The efficient delivery of the hepatitis C virus (HCV) RNA subgenomic replicon into cells is useful for basic and pharmaceutical studies. The adenovirus (Ad) vector is a convenient and efficient tool for the transduction of foreign genes into cells in vitro and in vivo. However, an Ad vector expressing the HCV replicon has never been developed. In the present study, we developed Ad vector containing an RNA polymerase (pol) I-dependent expression cassette and a tetracycline-controllable RNA pol I-dependent expression system. We prepared a hybrid promoter from the tetracycline-responsive element and the RNA pol I promoter. Ad vector particles coding the hybrid promoter-driven HCV replicon could be amplified, and interferon, an inhibitor of HCV replication, reduced HCV replication in cells transduced with the Ad vector coding HCV replicon. This is the first report of the development of an Ad vector-mediated HCV replicon system.

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Figures

Figure 1.
Figure 1.

Preparation of plasmid expressing HCV replicon driven by RNA pol I promoter. (A) Schematic construct of HCV replicon-expression cassette. The HCV replicon gene was driven by the RNA pol I promoter (PI) and terminator (TI). (B) Transgene expression in Huh7 cells. Cells were transfected with pPol I-HCV. After 24 h of transfection, the luciferase activities were measured. Data are mean ± SD (n = 3). (C and D) Effect of IFN on HCV replication in RNA pol I vector-transfected cells. Huh7 cells were transfected with pPol I-HCV. After 2.5 h of transfection, the cells were treated with IFN at the indicated concentration. After an additional 72 h of incubation, the luciferase activity (C) and the cell viability (D) were measured. The luciferase activity (%) was calculated as a percentage of that in the vehicle-treated cells. Data are mean ± SD (n = 3).

Figure 2.
Figure 2.

Development of tet-controllable RNA pol I promoter. (A) Construct of the chimeric RNA pol I promoter. The 412-bp human RNA pol I promoter contains Core (from −40 to −1), the binding site of the transcription factor, and UCE (from −235 to −130). TRE is connected to the full or partial RNA pol I promoter at the indicated sites, resulting in PI235, PI311 and PI412. (B and C) Promoter activities of the chimeric promoter in Huh7 cells. Huh7 cells were co-transfected with the chimeric RNA pol I plasmid coding EGFPLuc, pCMVβ and trans-activator plasmid [rtTA (B) or tTA (C)]. After 2.5 h of transfection, the cells were treated with Dox at the indicated dose. After an additional 48 h of incubation, the luciferase and β-galactosidase activities were measured. The luciferase activity was normalized by the β-galactosidase activity and expressed relative to that of pPIWT-EL-transfected cells. Data are mean ± SD (n = 3). (D) Transgene activity of Ad vector coding the chimeric promoter construct. Huh7 cells were co-infected with AdPI235-EL and Ad-tTA. The MOI ratio of AdPI235-EL to Ad-tTA was 5:10 or 5:50. After an additional 48 h of incubation, the luciferase activity was measured. Data are the mean ± SD (n = 3).

Figure 3.
Figure 3.

Preparation of Ad vector to monitor HCV replication. (A) Construct of Ad vector. The Ad vector contained the chimeric RNA pol I promoter (PI235) and the HCV replicon to monitor HCV replication as the luciferase expression. (B) Expression of HCV NS5A protein in Huh7 cells transfected with AdPI235-HCV. The cells were transfected with AdPI235-HCV (10 MOI) and Ad-tTA (50 MOI). After 72 h of incubation, the cells were harvested, and the lysates (30 µg) were subjected to SDS–PAGE, followed by immunoblotting with antibody against NS5A. Huh7 cells and Huh7.5.1 1bFeo cells were used as the negative and positive controls, respectively. Lane 1, Huh7 cells; lane 2, Huh7 cells infected with AdPI235-HCV; lane 3, Huh7.5.1 1bFeo cells. (C) Expression of luciferase in the Ad vector-transfected cells. Huh7 cells were co-infected with AdPI235-HCV (10 MOI) and 0 or 50 MOI of Ad-tTA. After an additional 48 h of incubation, the luciferase activity was measured. Data represent the mean ± SD (n = 3). (D) Involvement of NS5B in expression of luciferase in the Ad vector-transfected cells. Huh7 cells were infected with AdPI235-HCV or AdPI235-ΔGDD (3 MOI) and Ad-tTA (15 MOI). After 24 h, the cells were treated with 10 µg/ml of Dox for 48 h. Then, the luciferase activity was measured. Data represent the mean ± SD (n = 3). (E) Expression of minus-stranded HCV RNA in the Ad vector-transfected cells. Huh7 cells were co-infected with AdPI235-HCV or AdPI235-ΔGDD at 3 MOI and Ad-tTA at 15 MOI. After 24 h, the cells were treated with 10 µg/ml of Dox for 48 h. Then RT-PCR analysis was performed for detection of minus-stranded HCV NS3 and GAPDH. The PCR products were separated on 2% agarose gel. Huh7 cells and Huh7.5.1 1bFeo cells were used as the negative and positive controls, respectively. Lane 1, Huh7 cells; lane 2, Huh7.5.1 1bFeo cells; lane 3, Huh7 cells infected with AdPI235-ΔGDD; lane 4, Huh7 cells infected with AdPI235-HCV. (F and G) Effect of IFN on the replication of HCV replicon. Huh7 cells were infected with AdPI235-HCV (10 MOI) and Ad-rtTA (50 MOI). After 1.5 h of infection, the cells were treated with IFN at the indicated concentration for 72 h. Then, the luciferase activity (F) and the cell viability (G) were measured. Data represent the percentage of vehicle-treated cells. Data are the mean ± SD (n = 3).

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