A reporter-based assay for identifying hepatitis C virus inhibitors based on subgenomic replicon cells.
Hepatitis C virus (HCV) encodes a polyprotein that needs to be processed proteolytically by cellular and viral proteases into mature functional proteins. One of the viral proteins, NS3/4A, has serine protease activity that is critical for virus maturation. The generation and characterization of an engineered HCV replicon cell line (Ava5) is described which constitutively expresses EGdelta4AB)SEAP reporter protein and the cell line was designated as Ava5-EG(delta4AB)SEAP. EG(delta4AB)SEAP is a fusion protein in which Enhanced Green Fluorescent Protein (EGFP) was fused to SEcreted Alkaline Phosphatase (SEAP) through the NS3/4A protease decapeptide recognition sequence, delta4AB, which spans the NS4A and NS4B junction region. The secretion of SEAP into culture medium has been shown to depend on the cleavage of delta4AB by HCV NS3/4A protease. It is demonstrated that the amount of NS3/4A in Ava5-EG(delta4AB)SEAP cells correlated well with the copy numbers of HCV subgenomic RNA. It is also shown that replication of HCV subgenomic RNA inside cells is reflected by the alkaline phosphatase (SEAP) levels in culture medium. SEAP activity in the culture medium of Ava5-EG(delta4AB)SEAP was approximately 50-fold higher than the parental Ava5 cells. Ava5-EG(delta4AB)SEAP was validated as a drug screening system since several known HCV inhibitors were shown to reduce SEAP activities in culture media of Ava5-EG(delta4AB)SEAP cells. In conclusion, Ava5-EG(delta4AB)SEAP cells can be used to monitor HCV sub-genomic replication and the assay can be readily adapted to high throughput screening format to identify prospective anti-HCV drugs.
Lee
Jin-Ching
JC
Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Taipei 115, Taiwan, ROC.
Chang
Ching-Fung
CF
Chi
Ya-Hui
YH
Hwang
Der-Ren
DR
Hsu
John T A
JT
D000469:Alkaline Phosphatase / Q000235:genetics; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D003470:Culture Media / Q000737:chemistry; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D049452:Green Fluorescent Proteins; D016174:Hepacivirus / Q000187:drug effects* / Q000254:growth & development; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012093:Replicon; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000502:physiology; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology; D014779:Virus Replication / Q000187:drug effects*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000469:Alkaline Phosphatase; D000998:Antiviral Agents; D003470:Culture Media; D049452:Green Fluorescent Proteins; D016898:Interferon-alpha; D007371:Interferon-gamma; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors; D014108:Tosylphenylalanyl Chloromethyl Ketone; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases