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

Yoshida T, Kondoh M, Ojima M, Mizuguchi H, Yamagishi Y, Sakamoto N, Yagi K - Nucleic Acids Res. (2011)

Bottom Line: 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.This is the first report of the development of an Ad vector-mediated HCV replicon system.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bio-Functional Molecular Chemistry, Department of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.

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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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).
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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).

Mentions: First, we constructed an RNA pol I-driven plasmid coding an HCV replicon in which structural coding genes were replaced by the luciferase gene (Figure 1A). To investigate the expression of the HCV replicon from the RNA pol I plasmid vector, we transfected the plasmid vector into Huh7 cells. As shown in Figure 1B, the luciferase activity was observed in the RNA pol I vector-transfected cells. IFN is the most popular agent used to inhibit HCV replication. To examine whether the RNA pol I plasmid vector functions as an assay system for anti-HCV activity, we investigated the effect of IFN on the expression of the HCV replicon in the RNA pol I plasmid-transfected Huh7 cells. IFN dose-dependently reduced the replication of the HCV genome (Figure 1C), reaching 29.2% of the control at 5 pg/ml. IFN treatment did not cause any cytotoxicity (Figure 1D). These data suggest that the RNA pol I plasmid coding the HCV replicon works as an assay system for HCV replication.Figure 1.


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

Yoshida T, Kondoh M, Ojima M, Mizuguchi H, Yamagishi Y, Sakamoto N, Yagi K - Nucleic Acids Res. (2011)

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).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105406&req=5

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).
Mentions: First, we constructed an RNA pol I-driven plasmid coding an HCV replicon in which structural coding genes were replaced by the luciferase gene (Figure 1A). To investigate the expression of the HCV replicon from the RNA pol I plasmid vector, we transfected the plasmid vector into Huh7 cells. As shown in Figure 1B, the luciferase activity was observed in the RNA pol I vector-transfected cells. IFN is the most popular agent used to inhibit HCV replication. To examine whether the RNA pol I plasmid vector functions as an assay system for anti-HCV activity, we investigated the effect of IFN on the expression of the HCV replicon in the RNA pol I plasmid-transfected Huh7 cells. IFN dose-dependently reduced the replication of the HCV genome (Figure 1C), reaching 29.2% of the control at 5 pg/ml. IFN treatment did not cause any cytotoxicity (Figure 1D). These data suggest that the RNA pol I plasmid coding the HCV replicon works as an assay system for HCV replication.Figure 1.

Bottom Line: 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.This is the first report of the development of an Ad vector-mediated HCV replicon system.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bio-Functional Molecular Chemistry, Department of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.

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.

Show MeSH
Related in: MedlinePlus