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A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells.
- Source :
-
PloS one [PLoS One] 2010 Feb 22; Vol. 5 (2), pp. e9344. Date of Electronic Publication: 2010 Feb 22. - Publication Year :
- 2010
-
Abstract
- Background: Försters resonance energy transfer (FRET) microscopy is widely used for the analysis of protein interactions in intact cells. However, FRET microscopy is technically challenging and does not allow assessing interactions in large cell numbers. To overcome these limitations we developed a flow cytometry-based FRET assay and analysed interactions of human and simian immunodeficiency virus (HIV and SIV) Nef and Vpu proteins with cellular factors, as well as HIV Rev multimer-formation.<br />Results: Amongst others, we characterize the interaction of Vpu with CD317 (also termed Bst-2 or tetherin), a host restriction factor that inhibits HIV release from infected cells and demonstrate that the direct binding of both is mediated by the Vpu membrane-spanning region. Furthermore, we adapted our assay to allow the identification of novel protein interaction partners in a high-throughput format.<br />Conclusion: The presented combination of FRET and FACS offers the precious possibility to discover and define protein interactions in living cells and is expected to contribute to the identification of novel therapeutic targets for treatment of human diseases.
- Subjects :
- Animals
Antigens, CD genetics
Antigens, CD metabolism
Binding Sites genetics
Cell Line
GPI-Linked Proteins
Gene Products, nef genetics
Gene Products, nef metabolism
HIV-1 metabolism
HeLa Cells
Human Immunodeficiency Virus Proteins genetics
Human Immunodeficiency Virus Proteins metabolism
Humans
Immunoprecipitation
Jurkat Cells
Luminescent Proteins genetics
Luminescent Proteins metabolism
Membrane Glycoproteins genetics
Membrane Glycoproteins metabolism
Microscopy, Confocal
Mutation
Protein Binding
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins metabolism
Simian Immunodeficiency Virus metabolism
Transfection
Viral Proteins genetics
Viral Regulatory and Accessory Proteins genetics
Viral Regulatory and Accessory Proteins metabolism
Flow Cytometry methods
Fluorescence Resonance Energy Transfer methods
Protein Interaction Mapping methods
Viral Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1932-6203
- Volume :
- 5
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- PloS one
- Publication Type :
- Academic Journal
- Accession number :
- 20179761
- Full Text :
- https://doi.org/10.1371/journal.pone.0009344