Limits...
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.

Show MeSH

Related in: MedlinePlus

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

License
getmorefigures.php?uid=PMC3105406&req=5

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).

Mentions: The tet-regulated system comprises a regulator vector that expresses tet-controlled trans-activators and a response vector consisting of TRE within the promoter that controls expression of the gene of interest. The tet-controlled trans-activators are classified into tTA and rtTA that binds to the TRE promoter and activates expression from the TRE promoter in the absence and presence of Dox, respectively (23,24). We speculated that a tet-regulated vector system would minimize the influence of the HCV replicon on the preparation of Ad vector particles. First, we optimized the chimeric promoter of TRE and the RNA pol I promoter. As shown in Figure 2A, the RNA pol I promoter is a 412-bp fragment containing an upstream control element (UCE) and the binding site of a transcription factor (Core). We constructed three chimeric promoter-driven plasmid vectors and checked the expression profiles using luciferase as a reporter gene. The chimeric vector was co-transfected into Huh7 cells with response vectors coding tTA or rtTA (23,24). As shown in Figure 2B and C, co-transfection with tTA exhibited a higher expression level than that of rtTA. The PI235 promoter had the lowest luciferase expression in the absence of response vectors (Supplementary Figure S2). We used tTA and the PI235 promoter in further studies. To investigate whether the chimeric RNA pol I promoter works in the Ad vector, we prepared Ad5 vector coding the chimeric RNA pol I-driven luciferase gene. AdPI235-EL (MOI of 5) was co-transduced with Ad-tTA at MOI of 10 and 50. As shown in Figure 2D, the luciferase expression was increased in an Ad-tTA concentration-dependent manner.Figure 2.


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)

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

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

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).
Mentions: The tet-regulated system comprises a regulator vector that expresses tet-controlled trans-activators and a response vector consisting of TRE within the promoter that controls expression of the gene of interest. The tet-controlled trans-activators are classified into tTA and rtTA that binds to the TRE promoter and activates expression from the TRE promoter in the absence and presence of Dox, respectively (23,24). We speculated that a tet-regulated vector system would minimize the influence of the HCV replicon on the preparation of Ad vector particles. First, we optimized the chimeric promoter of TRE and the RNA pol I promoter. As shown in Figure 2A, the RNA pol I promoter is a 412-bp fragment containing an upstream control element (UCE) and the binding site of a transcription factor (Core). We constructed three chimeric promoter-driven plasmid vectors and checked the expression profiles using luciferase as a reporter gene. The chimeric vector was co-transfected into Huh7 cells with response vectors coding tTA or rtTA (23,24). As shown in Figure 2B and C, co-transfection with tTA exhibited a higher expression level than that of rtTA. The PI235 promoter had the lowest luciferase expression in the absence of response vectors (Supplementary Figure S2). We used tTA and the PI235 promoter in further studies. To investigate whether the chimeric RNA pol I promoter works in the Ad vector, we prepared Ad5 vector coding the chimeric RNA pol I-driven luciferase gene. AdPI235-EL (MOI of 5) was co-transduced with Ad-tTA at MOI of 10 and 50. As shown in Figure 2D, the luciferase expression was increased in an Ad-tTA concentration-dependent manner.Figure 2.

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