title: Flow cytometric assay using two fluorescent proteins for the function of the internal ribosome entry site of hepatitis C virus.
abstract: The initiation of translation in hepatitis C virus (HCV) occurs at the internal ribosome entry site (IRES) located at the 5'-end of its genomic RNA. To study the function of HCV IRES, we constructed a reporter plasmid that generates a bicistronic mRNA encoding two fluorescent proteins: cap-dependent DsRed2 and IRES-dependent Azami Green (AG). We introduced the plasmid into Huh7.5.1 and HEK293 cells and measured the relative IRES activity from the ratio of AG's signal to DsRed2's in individual cells using flow cytometry. To compare our method and a conventional biochemical method, we constructed a structurally similar reporter in which Renilla and Firefly luciferases replace DsRed2 and AG, respectively. With these systems, we found that the IRES A164G substitution decreased its activity, that interferon alpha affected the IRES activity in a cell type-specific manner, and that a synthetic micro-RNA targeting IRES was able to suppress the gene expression. In conclusion, the two methods were comparable in sensitivity in the studies of IRES mutations and host cell types. We discussed the significance of our findings and potential advantage of the cytometric assay: application to the molecular study of the HCV translation and to screening anti-IRES drugs.
authors.affiliation: China-Japan Joint Laboratory of Molecular Immunology and Molecular Microbiology, Institute of Microbiology, Chinese Academy of Sciences, Chaoyang District, Beijing 100101, China.
authors.forename: Guoli
authors.initials: G
authors.lastname: Shi
authors.forename: Fumihiro
authors.initials: F
authors.lastname: Yagyu
authors.forename: Yohko
authors.initials: Y
authors.lastname: Shimizu
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Shimizu
authors.forename: Masamichi
authors.initials: M
authors.lastname: Oshima
authors.forename: Aikichi
authors.initials: A
authors.lastname: Iwamoto
authors.forename: Bin
authors.initials: B
authors.lastname: Gao
authors.forename: Wenjun
authors.initials: W
authors.lastname: Liu
authors.forename: George Fu
authors.initials: GF
authors.lastname: Gao
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kitamura
mesh_terms: D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D057809:HEK293 Cells; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D022781:Hepatocytes; D006801:Humans; D016898:Interferon-alpha / Q000378:metabolism; D049409:Luciferases, Firefly / Q000378:metabolism*; D049410:Luciferases, Renilla / Q000378:metabolism*; D008164:Luminescent Proteins; D035683:MicroRNAs / Q000235:genetics; D014176:Protein Biosynthesis; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis*
chemical_list: D016898:Interferon-alpha; D008164:Luminescent Proteins; D035683:MicroRNAs; D014764:Viral Proteins; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly