In vivo dynamics of enterovirus protease revealed by fluorescence resonance emission transfer (FRET) based on a novel FRET pair.
An in vivo protease assay suitable for analysis by fluorescence resonance energy transfer (FRET) was developed on the basis of a novel FRET pair. The specifically designed fusion substrate consists of green fluorescent protein 2 (GFP2)-peptide-red fluorescent protein 2 (DsRed2), with a cleavage motif for the enterovirus 2A protease (2Apro) embedded within the peptide region. FRET can be readily visualized in real-time from cells expressing the fusion substrate until a proteolytic cleavage by 2Apro from the input virus. The level of FRET decay is a function of the amount and infection duration of the inoculated virus as measured by a fluorometer assay. The FRET biosensor also responded well to other related enteroviruses but not to a phylogenetically distant virus. Western blot analysis confirmed the physical cleavage of the fusion substrate upon the infections. The study provides proof of principle for applying the FRET technology to diagnostics, screening procedures, and cell biological research.
Hsu
Yueh-Ying
YY
Faculty of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan, ROC.
Liu
Yu-Ning
YN
Wang
Wenyen
W
Kao
Fu-Jen
FJ
Kung
Szu-Hao
SH
D015153:Blotting, Western; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004770:Enterovirus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D003546:Cysteine Endopeptidases; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D000097573:Red Fluorescent Protein; C057400:picornain 2A, Picornavirus