Dataset Information

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.

SUBMITTER: Tsai MT

PROVIDER: S-EPMC2630644 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Real-time monitoring of human enterovirus (HEV)-infected cells and anti-HEV 3C protease potency by fluorescence resonance energy transfer.

Tsai Meng-Tian MT Cheng Yun-Hsiang YH Liu Yu-Ning YN Liao Nien-Chien NC Lu Wen-Wen WW Kung Szu-Hao SH

Antimicrobial agents and chemotherapy 20081117 2

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). E ...[more]

PMID: 19015331

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Project description:BACKGROUND:Engineering microorganisms to improve metabolite flux requires detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Fluorescence resonance energy transfer sensors represent a promising technology for measuring metabolite levels and corresponding rate changes in live cells. These sensors have been applied successfully in mammalian and plant cells but potentially could also be used to monitor steady-state levels of metabolites in microorganisms using fluorimetric assays. Sensors for hexose and pentose carbohydrates could help in the development of fermentative microorganisms, for example, for biofuels applications. Arabinose is one of the carbohydrates to be monitored during biofuels production from lignocellulose, while maltose is an important degradation product of starch that is relevant for starch-derived biofuels production. RESULTS:An Escherichia coli expression vector compatible with phage lambda recombination technology was constructed to facilitate sensor construction and was used to generate a novel fluorescence resonance energy transfer sensor for arabinose. In parallel, a strategy for improving the sensor signal was applied to construct an improved maltose sensor. Both sensors were expressed in the cytosol of E. coli and sugar accumulation was monitored using a simple fluorimetric assay of E. coli cultures in microtiter plates. In the case of both nanosensors, the addition of the respective ligand led to concentration-dependent fluorescence resonance energy transfer responses allowing quantitative analysis of the intracellular sugar levels at given extracellular supply levels as well as accumulation rates. CONCLUSION:The nanosensor destination vector combined with the optimization strategy for sensor responses should help to accelerate the development of metabolite sensors. The new carbohydrate fluorescence resonance energy transfer sensors can be used for in vivo monitoring of sugar levels in prokaryotes, demonstrating the potential of such sensors as reporter tools in the development of metabolically engineered microbial strains or for real-time monitoring of intracellular metabolite during fermentation.

Dataset Information

Real-time monitoring of human enterovirus (HEV)-infected cells and anti-HEV 3C protease potency by fluorescence resonance energy transfer.

Publications

Real-time monitoring of human enterovirus (HEV)-infected cells and anti-HEV 3C protease potency by fluorescence resonance energy transfer.

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