A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells

• Published in: PloS one Vol. 5; no. 2; p. e9344
• Main Authors: Banning, Carina, Votteler, Jörg, Hoffmann, Dirk, Koppensteiner, Herwig, Warmer, Martin, Reimer, Rudolph, Kirchhoff, Frank, Schubert, Ulrich, Hauber, Joachim, Schindler, Michael
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 22.02.2010; Public Library of Science (PLoS)
• Subjects: Animals; Antigens, CD - genetics; Antigens, CD - metabolism; Assaying; Binding Sites - genetics; Biotechnology/Protein Chemistry and Proteomics; Cell Biology; Cell Line; Cells (biology); Cloning; Cytometry; Energy transfer; Flow cytometry; Flow Cytometry - methods; Fluorescence resonance energy transfer; Fluorescence Resonance Energy Transfer - methods; Gene Products, nef - genetics; Gene Products, nef - metabolism; GPI-Linked Proteins; Health aspects; HeLa Cells; HIV; HIV-1 - metabolism; Human immunodeficiency virus; Human Immunodeficiency Virus Proteins - genetics; Human Immunodeficiency Virus Proteins - metabolism; Humans; Immunology; Immunoprecipitation; Infectious Diseases/HIV Infection and AIDS; Jurkat Cells; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Medical treatment; Membrane Glycoproteins - genetics; Membrane Glycoproteins - metabolism; Microscopy; Microscopy, Confocal; Mutation; Nef protein; Protein Binding; Protein interaction; Protein Interaction Mapping - methods; Protein-protein interactions; Proteins; Proteomics; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Simian Immunodeficiency Virus - metabolism; Studies; Target recognition; Transfection; Viral Proteins - genetics; Viral Proteins - metabolism; Viral Regulatory and Accessory Proteins - genetics; Viral Regulatory and Accessory Proteins - metabolism; Virology; Virology/Virulence Factors and Mechanisms; Viruses; Germany
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0009344

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. 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. 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.