title: HCV IRES-mediated core expression in zebrafish.
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.
authors.affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Zhao
authors.forename: Wei
authors.initials: W
authors.lastname: Qin
authors.forename: Jing-Pu
authors.initials: JP
authors.lastname: Zhang
authors.forename: Zhan-Ying
authors.initials: ZY
authors.lastname: Hu
authors.forename: Jun-Wei
authors.initials: JW
authors.lastname: Tong
authors.forename: Cun-Bao
authors.initials: CB
authors.lastname: Ding
authors.forename: Zong-Gen
authors.initials: ZG
authors.lastname: Peng
authors.forename: Li-Xun
authors.initials: LX
authors.lastname: Zhao
authors.forename: Dan-Qing
authors.initials: DQ
authors.lastname: Song
authors.forename: Jian-Dong
authors.initials: JD
authors.lastname: Jiang
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D006526:Hepatitis C / Q000188:drug therapy* / Q000821:virology; D054884:Host-Pathogen Interactions; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000494:pharmacology; D007814:Larva / Q000187:drug effects / Q000821:virology; D008099:Liver / Q000187:drug effects / Q000821:virology; D050506:Mutant Chimeric Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000037:antagonists & inhibitors* / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D012254:Ribavirin / Q000494:pharmacology; D014758:Viral Core Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D014805:Vitamin B 12 / Q000494:pharmacology; D015027:Zebrafish / Q000821:virology*
chemical_list: D000998:Antiviral Agents; D015415:Biomarkers; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D050506:Mutant Chimeric Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D049452:Green Fluorescent Proteins; D012254:Ribavirin; D014805:Vitamin B 12