TY - JOUR
T1 - Application of fluorescence resonance energy transfer resolved by fluorescence lifetime imaging microscopy for the detection of enterovirus 71 infection in cells
AU - Ghukasyan, Vladimir
AU - Hsu, Yueh Ying
AU - Kung, Szu Hao
AU - Kao, Fu Jen
N1 - Funding Information:
This work was financially supported by the Ministry of Education, Taiwan (Aim for the Top University Plan, Grant No. 95A-CT8G01), and National Science Council, Taiwan (under Grants NSC95-2112-M010-001 and NSC95-3112-B-010-015).
PY - 2007/3
Y1 - 2007/3
N2 - Timely and effective virus infection detection is critical for the clinical management and prevention of the disease spread in communities during an outbreak. A range of methods have been developed for this purpose, of which classical serological and viral nucleic acids detection are the most popular. We describe an alternative, imaging-based approach that utilizes fluorescence resonance energy transfer (FRET) resolved by fluorescence lifetime imaging microscopy (FLIM) and demonstrate it on the example of enterovirus 71 (EV71) infection detection. A plasmid construct is developed with the sequence for GFP2 and DsRed2 fluorescent proteins, linked by a 12-amino-acid-long cleavage recognition site for the 2A protease (2Apro), encoded by the EV71 genome and specific for the members of Picornaviridae family. In the construct expressed in HeLa cells, the linker binds the fluorophores within the Förster distance and creates a condition for FRET to occur, thus resulting in shortening of the GFP2 fluorescence lifetime. On cells infection with EV71, viral 2Apro released to the cytoplasm cleaves the recognition site, causing disruption of FRET through separation of the fluorophores. Thus, increased GFP2 lifetime to the native values, manifested by the time-correlated single-photon counting, serves as an efficient and specific indicator of the EV71 virus infection.
AB - Timely and effective virus infection detection is critical for the clinical management and prevention of the disease spread in communities during an outbreak. A range of methods have been developed for this purpose, of which classical serological and viral nucleic acids detection are the most popular. We describe an alternative, imaging-based approach that utilizes fluorescence resonance energy transfer (FRET) resolved by fluorescence lifetime imaging microscopy (FLIM) and demonstrate it on the example of enterovirus 71 (EV71) infection detection. A plasmid construct is developed with the sequence for GFP2 and DsRed2 fluorescent proteins, linked by a 12-amino-acid-long cleavage recognition site for the 2A protease (2Apro), encoded by the EV71 genome and specific for the members of Picornaviridae family. In the construct expressed in HeLa cells, the linker binds the fluorophores within the Förster distance and creates a condition for FRET to occur, thus resulting in shortening of the GFP2 fluorescence lifetime. On cells infection with EV71, viral 2Apro released to the cytoplasm cleaves the recognition site, causing disruption of FRET through separation of the fluorophores. Thus, increased GFP2 lifetime to the native values, manifested by the time-correlated single-photon counting, serves as an efficient and specific indicator of the EV71 virus infection.
KW - Enterovirus 71
KW - Fluorescence lifetime imaging microscopy
KW - Fluorescence resonance energy transfer
KW - Time-correlated single-photon counting
KW - Two-photon excitation
KW - VP1/2A protease
UR - http://www.scopus.com/inward/record.url?scp=34250616219&partnerID=8YFLogxK
U2 - 10.1117/1.2718582
DO - 10.1117/1.2718582
M3 - Article
C2 - 17477731
AN - SCOPUS:34250616219
SN - 1083-3668
VL - 12
JO - Journal of Biomedical Optics
JF - Journal of Biomedical Optics
IS - 2
M1 - 024016
ER -