The use of fluorescence microscopy to visualise homotypic interactions of tomato spotted wilt virus nucleocapsid protein in living cells.
Fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) were employed to study homotypic protein-protein interactions in living cells. To this end, the nucleocapsid (N) protein of tomato spotted wilt virus (TSWV) was expressed as a fusion protein with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP). Co-expression experiments of the two fusion proteins were carried out in baby hamster kidney (BHK21) cells. Both the wild type and the fusion proteins showed a peri-nuclear localisation pattern and were observed to form aggregates. In sensitised emission experiments, energy transfer was observed to take place from CFP to YFP when the two fluorophores were fused to TSWV N protein, indicating strongly homotypic interaction of the N proteins. This was confirmed by acceptor photobleaching studies as well as by FLIM experiments. All three methods showed interactions taking place, not only in the aggregates in the peri-nuclear region, but also throughout the cytoplasm. These experiments clearly demonstrated the potential of these fluorescence methods for studying the interactions of viral proteins in living cells.
Snippe
Marjolein
M
Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands.
Borst
Jan Willem
JW
Goldbach
Rob
R
Kormelink
Richard
R
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D006224:Cricetinae; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism*; D038761:Photobleaching; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017865:Tospovirus / Q000502:physiology*; D014162:Transfection; D019065:Virus Assembly
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D019590:Nucleocapsid Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins