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HCV IRES-mediated core expression in zebrafish.

Zhao Y, Qin W, Zhang JP, Hu ZY, Tong JW, Ding CB, Peng ZG, Zhao LX, Song DQ, Jiang JD - PLoS ONE (2013)

Bottom Line: The results show that vitamin B12 inhibited core expression in mRNA and protein levels in dose-dependent manner, but failed to impact gfp expression.Also VB12 down-regulated some gene transcriptions involved in fat liver, liver fibrosis and HCV-associated pathological process in the larvae.IFNα-2b was not active, which maybe resulted from its degradation in vivo for the long time.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

ABSTRACT
The lack of small animal models for hepatitis C virus has impeded the discovery and development of anti-HCV drugs. HCV-IRES plays an important role in HCV gene expression, and is an attractive target for antiviral therapy. In this study, we report a zebrafish model with a biscistron expression construct that can co-transcribe GFP and HCV-core genes by human hepatic lipase promoter and zebrafish liver fatty acid binding protein enhancer. HCV core translation was designed mediated by HCV-IRES sequence and gfp was by a canonical cap-dependent mechanism. Results of fluorescence image and in situ hybridization indicate that expression of HCV core and GFP is liver-specific; RT-PCR and Western blotting show that both core and gfp expression are elevated in a time-dependent manner for both transcription and translation. It means that the HCV-IRES exerted its role in this zebrafish model. Furthermore, the liver-pathological impact associated with HCV-infection was detected by examination of gene markers and some of them were elevated, such as adiponectin receptor, heparanase, TGF-β, PDGF-α, etc. The model was used to evaluate three clinical drugs, ribavirin, IFNα-2b and vitamin B12. The results show that vitamin B12 inhibited core expression in mRNA and protein levels in dose-dependent manner, but failed to impact gfp expression. Also VB12 down-regulated some gene transcriptions involved in fat liver, liver fibrosis and HCV-associated pathological process in the larvae. It reveals that HCV-IRES responds to vitamin B12 sensitively in the zebrafish model. Ribavirin did not disturb core expression, hinting that HCV-IRES is not a target site of ribavirin. IFNα-2b was not active, which maybe resulted from its degradation in vivo for the long time. These findings demonstrate the feasibility of the zebrafish model for screening of anti-HCV drugs targeting to HCV-IRES. The zebrafish system provides a novel evidence of using zebrafish as a HCV model organism.

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Verification for the efficiency of the HCV IRES-mediating expression zebrafish model with anti-HCV drugs.RT-PCR (upper panel) and Western blotting (lower panel) were used for detecting expression of core and gfp in pFL-GIC-injected larvae that were exposed to ribavirin (A) and vitamin B12 (B) drugs at gradient concentrations from 5-dpf to 10 dpf. C. Result of IFNα-2b co-injected with plasmid pFL-GIC. All the larvae were collected at 10-dpf for RT-PCR and Western blotting assays. Untreated larvae were as a control. Both cDNA and protein bands were scanned against β-actin cDNA or β-ACTIN protein respectively for semiquantitative evaluation of core and gfp expression (right histograms).
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pone-0056985-g006: Verification for the efficiency of the HCV IRES-mediating expression zebrafish model with anti-HCV drugs.RT-PCR (upper panel) and Western blotting (lower panel) were used for detecting expression of core and gfp in pFL-GIC-injected larvae that were exposed to ribavirin (A) and vitamin B12 (B) drugs at gradient concentrations from 5-dpf to 10 dpf. C. Result of IFNα-2b co-injected with plasmid pFL-GIC. All the larvae were collected at 10-dpf for RT-PCR and Western blotting assays. Untreated larvae were as a control. Both cDNA and protein bands were scanned against β-actin cDNA or β-ACTIN protein respectively for semiquantitative evaluation of core and gfp expression (right histograms).

Mentions: To learn whether this HCV-zebrafish model was suitable for the efficacy evaluation of anti-HCV drugs that target HCV IRES, the pFL-GIC injected larvae at 5dpf were treated with ribavirin or vitamin B12, respectively, for another five days. In addition, another drug IFNα-2b was coinjected with pFL-GIC into embryos. Then, core and gfp were examined in the 10-dpf larvae at both transcription and translation levels. As shown in Figure 6B, the expression of HCV core gene was significantly reduced in a dose-dependent manner in the larvae incubated in the vitamin B12-containing water; however, the expression of GFP gene was not affected apparently, indicating that HCV IRES mediated core expression probably was inhibited by the drug. It is also observed that the transcription level of core was also decreased in a dose-dependent manner comparing with gfp group. The result hints that vitamin B12 might have certain degree of interaction with HCV IRES. The similar result was not observed in ribavirin and IFNα-2b treated groups (Fig. 6A, 6C), suggesting that ribavirin might have different mechanisms from vitamin B12 in its action against HCV; and IFNα-2b inefficiency may be resulted from its degradation in vivo for the long time since once injection at one-cell stage, which was proved by western blotting with IFNα-2b antibody (data not shown). Further, in order to confirm and evaluate vitamin B12 action in the HCV-IRES zebrafish model, mRNA levels of some gene markers involved in Fat Livers (adipor1b and acox3), Fibrosis (heparanase, pdgf-α, pdgf-β and tgf-β) and HCV infection (chemekine 1, erlin 1, etfa and lengpcr) were examined by RT-PCR in the larvae that were treated by pFL-GIC injection and vitamin B12 exposure. The results indicated that vitamin B12 exposure indeed down-regulated the gene mRNA levels which were elevated in pFL-GIC injected larvae (Fig. 7). Thus, we consider this zebrafish system a suitable small animal model to evaluate anti-HCV drugs that work through inhibition of HCV IRES.


HCV IRES-mediated core expression in zebrafish.

Zhao Y, Qin W, Zhang JP, Hu ZY, Tong JW, Ding CB, Peng ZG, Zhao LX, Song DQ, Jiang JD - PLoS ONE (2013)

Verification for the efficiency of the HCV IRES-mediating expression zebrafish model with anti-HCV drugs.RT-PCR (upper panel) and Western blotting (lower panel) were used for detecting expression of core and gfp in pFL-GIC-injected larvae that were exposed to ribavirin (A) and vitamin B12 (B) drugs at gradient concentrations from 5-dpf to 10 dpf. C. Result of IFNα-2b co-injected with plasmid pFL-GIC. All the larvae were collected at 10-dpf for RT-PCR and Western blotting assays. Untreated larvae were as a control. Both cDNA and protein bands were scanned against β-actin cDNA or β-ACTIN protein respectively for semiquantitative evaluation of core and gfp expression (right histograms).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585870&req=5

pone-0056985-g006: Verification for the efficiency of the HCV IRES-mediating expression zebrafish model with anti-HCV drugs.RT-PCR (upper panel) and Western blotting (lower panel) were used for detecting expression of core and gfp in pFL-GIC-injected larvae that were exposed to ribavirin (A) and vitamin B12 (B) drugs at gradient concentrations from 5-dpf to 10 dpf. C. Result of IFNα-2b co-injected with plasmid pFL-GIC. All the larvae were collected at 10-dpf for RT-PCR and Western blotting assays. Untreated larvae were as a control. Both cDNA and protein bands were scanned against β-actin cDNA or β-ACTIN protein respectively for semiquantitative evaluation of core and gfp expression (right histograms).
Mentions: To learn whether this HCV-zebrafish model was suitable for the efficacy evaluation of anti-HCV drugs that target HCV IRES, the pFL-GIC injected larvae at 5dpf were treated with ribavirin or vitamin B12, respectively, for another five days. In addition, another drug IFNα-2b was coinjected with pFL-GIC into embryos. Then, core and gfp were examined in the 10-dpf larvae at both transcription and translation levels. As shown in Figure 6B, the expression of HCV core gene was significantly reduced in a dose-dependent manner in the larvae incubated in the vitamin B12-containing water; however, the expression of GFP gene was not affected apparently, indicating that HCV IRES mediated core expression probably was inhibited by the drug. It is also observed that the transcription level of core was also decreased in a dose-dependent manner comparing with gfp group. The result hints that vitamin B12 might have certain degree of interaction with HCV IRES. The similar result was not observed in ribavirin and IFNα-2b treated groups (Fig. 6A, 6C), suggesting that ribavirin might have different mechanisms from vitamin B12 in its action against HCV; and IFNα-2b inefficiency may be resulted from its degradation in vivo for the long time since once injection at one-cell stage, which was proved by western blotting with IFNα-2b antibody (data not shown). Further, in order to confirm and evaluate vitamin B12 action in the HCV-IRES zebrafish model, mRNA levels of some gene markers involved in Fat Livers (adipor1b and acox3), Fibrosis (heparanase, pdgf-α, pdgf-β and tgf-β) and HCV infection (chemekine 1, erlin 1, etfa and lengpcr) were examined by RT-PCR in the larvae that were treated by pFL-GIC injection and vitamin B12 exposure. The results indicated that vitamin B12 exposure indeed down-regulated the gene mRNA levels which were elevated in pFL-GIC injected larvae (Fig. 7). Thus, we consider this zebrafish system a suitable small animal model to evaluate anti-HCV drugs that work through inhibition of HCV IRES.

Bottom Line: The results show that vitamin B12 inhibited core expression in mRNA and protein levels in dose-dependent manner, but failed to impact gfp expression.Also VB12 down-regulated some gene transcriptions involved in fat liver, liver fibrosis and HCV-associated pathological process in the larvae.IFNα-2b was not active, which maybe resulted from its degradation in vivo for the long time.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

ABSTRACT
The lack of small animal models for hepatitis C virus has impeded the discovery and development of anti-HCV drugs. HCV-IRES plays an important role in HCV gene expression, and is an attractive target for antiviral therapy. In this study, we report a zebrafish model with a biscistron expression construct that can co-transcribe GFP and HCV-core genes by human hepatic lipase promoter and zebrafish liver fatty acid binding protein enhancer. HCV core translation was designed mediated by HCV-IRES sequence and gfp was by a canonical cap-dependent mechanism. Results of fluorescence image and in situ hybridization indicate that expression of HCV core and GFP is liver-specific; RT-PCR and Western blotting show that both core and gfp expression are elevated in a time-dependent manner for both transcription and translation. It means that the HCV-IRES exerted its role in this zebrafish model. Furthermore, the liver-pathological impact associated with HCV-infection was detected by examination of gene markers and some of them were elevated, such as adiponectin receptor, heparanase, TGF-β, PDGF-α, etc. The model was used to evaluate three clinical drugs, ribavirin, IFNα-2b and vitamin B12. The results show that vitamin B12 inhibited core expression in mRNA and protein levels in dose-dependent manner, but failed to impact gfp expression. Also VB12 down-regulated some gene transcriptions involved in fat liver, liver fibrosis and HCV-associated pathological process in the larvae. It reveals that HCV-IRES responds to vitamin B12 sensitively in the zebrafish model. Ribavirin did not disturb core expression, hinting that HCV-IRES is not a target site of ribavirin. IFNα-2b was not active, which maybe resulted from its degradation in vivo for the long time. These findings demonstrate the feasibility of the zebrafish model for screening of anti-HCV drugs targeting to HCV-IRES. The zebrafish system provides a novel evidence of using zebrafish as a HCV model organism.

Show MeSH
Related in: MedlinePlus