Limits...
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.

Show MeSH

Related in: MedlinePlus

Biological impact on development in zebrafish larvae after pFL-GIC injection.A. The body length of pFL-GIC injected zebrafish larvae, compared with pFL-G injected zebrafish and WT larvae at 3-, 6- and 9-dpf (p>0.05). B. The mortality curve of zebrafish larvae after pFL-GIC injection, compared to that of pFL-G injected zebrafish and that of WT in the first 9 days during the embryonic development (p>0.05).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585870&req=5

pone-0056985-g004: Biological impact on development in zebrafish larvae after pFL-GIC injection.A. The body length of pFL-GIC injected zebrafish larvae, compared with pFL-G injected zebrafish and WT larvae at 3-, 6- and 9-dpf (p>0.05). B. The mortality curve of zebrafish larvae after pFL-GIC injection, compared to that of pFL-G injected zebrafish and that of WT in the first 9 days during the embryonic development (p>0.05).

Mentions: The toxic effect in pFL-GIC injection larvae was assessed by measuring body length and mortality in comparison with that of wild type larvae as well as pFL-G control injection. As shown in Figure 4A, expression of HCV core protein did not slow down the growth of the larvae (p>0.05) at least in the first 9 days of embryonic development; deformity phenotypes were not observed as well (data not shown). Difference in mortality was not detected among the three groups, pFL-GIC, pFL-G and wildtype larvae during the development from zygote to larva (10 dpf) (Fig. 4B), suggesting that the HCV construct injection as well as the subsequent HCV-core gene expression had no toxicity to embryonic development in zebrafish.


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)

Biological impact on development in zebrafish larvae after pFL-GIC injection.A. The body length of pFL-GIC injected zebrafish larvae, compared with pFL-G injected zebrafish and WT larvae at 3-, 6- and 9-dpf (p>0.05). B. The mortality curve of zebrafish larvae after pFL-GIC injection, compared to that of pFL-G injected zebrafish and that of WT in the first 9 days during the embryonic development (p>0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056985-g004: Biological impact on development in zebrafish larvae after pFL-GIC injection.A. The body length of pFL-GIC injected zebrafish larvae, compared with pFL-G injected zebrafish and WT larvae at 3-, 6- and 9-dpf (p>0.05). B. The mortality curve of zebrafish larvae after pFL-GIC injection, compared to that of pFL-G injected zebrafish and that of WT in the first 9 days during the embryonic development (p>0.05).
Mentions: The toxic effect in pFL-GIC injection larvae was assessed by measuring body length and mortality in comparison with that of wild type larvae as well as pFL-G control injection. As shown in Figure 4A, expression of HCV core protein did not slow down the growth of the larvae (p>0.05) at least in the first 9 days of embryonic development; deformity phenotypes were not observed as well (data not shown). Difference in mortality was not detected among the three groups, pFL-GIC, pFL-G and wildtype larvae during the development from zygote to larva (10 dpf) (Fig. 4B), suggesting that the HCV construct injection as well as the subsequent HCV-core gene expression had no toxicity to embryonic development in zebrafish.

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