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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 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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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).

Mentions: The packaging limit of a foreign gene in the conventional Ad5 vector has been estimated to be 8.1–8.2-kb (25). The HCV replicon is ∼8.9-kb and contains a 1.7-kb firefly luciferase gene and sequence derived from the HCV genome. Thus, another reason for no previous reports regarding the preparation of Ad5 vector coding the HCV replicon appears to be the packaging limit of the Ad5 vector. Mizuguchi and Hayakawa found that Ad5/35 vector containing chimeric fibers of Ad5 and Ad35 increased the size limit of foreign genes to 8.8-kb (26). We were successful in preparing Ad5/35 vector particles (9.53 × 108 IFU/ml) coding the TRE/RNA pol I chimeric promoter-driven HCV replicon containing the 1.0-kb renilla luciferase gene and sequence derived from the HCV genome (Figure 3A). To investigate the expression of the HCV replicon, Huh7 cells were transfected with the Ad vector coding the HCV replicon and Ad-tTA at MOI of 10 and 50, respectively. As shown in Figure 3B, western blot analysis showed that NS5A was expressed in Huh7 cells transfected with the vectors in the absence of Dox. Luciferase was also expressed (Figure 3C). Dox dose-dependently attenuated expression of luciferase (Supplementary Figure S3). To discriminate between translation of the RNA pol I-transribed HCV RNA derived from the vector DNA and translation of HCV RNA derived from autonomous HCV replication in the transcribed cells, we prepared replication-incompetent HCV replicon deleting GDD motif in NS5B. Luciferase expression was attenuated in the cells transfected with the GDD-deleted Ad vector (AdPI235-ΔGDD) (Figure 3D). A fragment of the HCV negative strand RNA, an essential replication intermediate, amplified by RT-PCR has been detected in the cells transfected with AdPI235-HCV but not AdPI235-ΔGDD (Figure 3E). Autonomous replication of the HCV RNA may occur in this system. To evaluate whether the Ad vector systems could be used to evaluate inhibitors of HCV replication, we investigated the effect of IFN on luciferase expression from HCV replicon in the Ad vector. As shown in Figure 3F, treatment of cells with 5 pg/ml of IFN reduced luciferase expression (33.3% of vehicle-treated cells). Cell viability was not affected by IFN treatment (Figure 3G). These findings indicate that the tet-controllable RNA pol I Ad vector may be useful for evaluation of anti-HCV activity.Figure 3.


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 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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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).
Mentions: The packaging limit of a foreign gene in the conventional Ad5 vector has been estimated to be 8.1–8.2-kb (25). The HCV replicon is ∼8.9-kb and contains a 1.7-kb firefly luciferase gene and sequence derived from the HCV genome. Thus, another reason for no previous reports regarding the preparation of Ad5 vector coding the HCV replicon appears to be the packaging limit of the Ad5 vector. Mizuguchi and Hayakawa found that Ad5/35 vector containing chimeric fibers of Ad5 and Ad35 increased the size limit of foreign genes to 8.8-kb (26). We were successful in preparing Ad5/35 vector particles (9.53 × 108 IFU/ml) coding the TRE/RNA pol I chimeric promoter-driven HCV replicon containing the 1.0-kb renilla luciferase gene and sequence derived from the HCV genome (Figure 3A). To investigate the expression of the HCV replicon, Huh7 cells were transfected with the Ad vector coding the HCV replicon and Ad-tTA at MOI of 10 and 50, respectively. As shown in Figure 3B, western blot analysis showed that NS5A was expressed in Huh7 cells transfected with the vectors in the absence of Dox. Luciferase was also expressed (Figure 3C). Dox dose-dependently attenuated expression of luciferase (Supplementary Figure S3). To discriminate between translation of the RNA pol I-transribed HCV RNA derived from the vector DNA and translation of HCV RNA derived from autonomous HCV replication in the transcribed cells, we prepared replication-incompetent HCV replicon deleting GDD motif in NS5B. Luciferase expression was attenuated in the cells transfected with the GDD-deleted Ad vector (AdPI235-ΔGDD) (Figure 3D). A fragment of the HCV negative strand RNA, an essential replication intermediate, amplified by RT-PCR has been detected in the cells transfected with AdPI235-HCV but not AdPI235-ΔGDD (Figure 3E). Autonomous replication of the HCV RNA may occur in this system. To evaluate whether the Ad vector systems could be used to evaluate inhibitors of HCV replication, we investigated the effect of IFN on luciferase expression from HCV replicon in the Ad vector. As shown in Figure 3F, treatment of cells with 5 pg/ml of IFN reduced luciferase expression (33.3% of vehicle-treated cells). Cell viability was not affected by IFN treatment (Figure 3G). These findings indicate that the tet-controllable RNA pol I Ad vector may be useful for evaluation of anti-HCV activity.Figure 3.

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