title: Real-time monitoring of human enterovirus (HEV)-infected cells and anti-HEV 3C protease potency by fluorescence resonance energy transfer.
abstract: A real-time assay system that allows monitoring of intracellular human enterovirus (HEV) protease activity was established using the principle of fluorescence resonance energy transfer (FRET). It was accomplished by engineering cells to constitutively express a genetically encoded FRET probe. The FRET-based probe was designed to contain an enterovirus 71 3C protease (3C(pro)) cleavage motif flanked by the FRET pair composed of green fluorescent protein 2 and red fluorescent protein 2 (DsRed2). Efficient FRET from the stable line was detected in a real-time manner by fluorescence microscopy, and the disruption of FRET was readily monitored upon HEV infection. The level of the repressed FRET was proportional to the input virus titer and the infection duration as measured by the fluorometric method. The FRET biosensor cell line was also responsive to other related HEV serotypes, but not to the phylogenetically distant herpes simplex virus, which was confirmed by Western blot analysis. The FRET biosensor was then utilized to develop a format for the determination of antiviral susceptibility, as the reduced FRET appeared to reflect viral replication. Evaluations of the FRET biosensor system with representative HEV serotypes demonstrated that their susceptibilities to a 3C(pro) inhibitor, rupintrivir, were all accurately determined. In summary, this novel FRET-based system is a means for rapid detection, quantification, and drug susceptibility testing for HEVs, with potential for the development of a high-throughput screening assay.
authors.affiliation: Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taiwan, Republic of China.
authors.forename: Meng-Tian
authors.initials: MT
authors.lastname: Tsai
authors.forename: Yun-Hsiang
authors.initials: YH
authors.lastname: Cheng
authors.forename: Yu-Ning
authors.initials: YN
authors.lastname: Liu
authors.forename: Nien-Chien
authors.initials: NC
authors.lastname: Liao
authors.forename: Wen-Wen
authors.initials: WW
authors.lastname: Lu
authors.forename: Szu-Hao
authors.initials: SH
authors.lastname: Kung
mesh_terms: D000086042:3C Viral Proteases; D000998:Antiviral Agents / Q000494:pharmacology; D015374:Biosensing Techniques; D015153:Blotting, Western; D002459:Cell Fusion; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D029821:Enterovirus A, Human; D004769:Enterovirus Infections / Q000821:virology*; D031541:Fluorescence Resonance Energy Transfer; D005470:Fluorometry; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D018259:Herpesvirus 1, Human / Q000187:drug effects; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007555:Isoxazoles / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D010649:Phenylalanine / Q000031:analogs & derivatives; D010957:Plasmids / Q000235:genetics; D011760:Pyrrolidinones / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014633:Valine / Q000031:analogs & derivatives; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000998:Antiviral Agents; D007555:Isoxazoles; D011760:Pyrrolidinones; D011994:Recombinant Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D010649:Phenylalanine; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases; D014633:Valine; C118874:rupintrivir