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.
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.
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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Antigens, CD / genetics
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Antigens, CD / metabolism
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Binding Sites / genetics
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Cell Line
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Flow Cytometry / methods*
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Fluorescence Resonance Energy Transfer / methods*
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GPI-Linked Proteins
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Gene Products, nef / genetics
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Gene Products, nef / metabolism
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HIV-1 / metabolism
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HeLa Cells
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Human Immunodeficiency Virus Proteins / genetics
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Human Immunodeficiency Virus Proteins / metabolism
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Humans
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Immunoprecipitation
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Jurkat Cells
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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Membrane Glycoproteins / genetics
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Membrane Glycoproteins / metabolism
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Microscopy, Confocal
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Mutation
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Protein Binding
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Protein Interaction Mapping / methods*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Simian Immunodeficiency Virus / metabolism
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Transfection
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Viral Proteins / genetics
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Viral Proteins / metabolism*
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Viral Regulatory and Accessory Proteins / genetics
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Viral Regulatory and Accessory Proteins / metabolism
Substances
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Antigens, CD
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BST2 protein, human
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GPI-Linked Proteins
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Gene Products, nef
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Human Immunodeficiency Virus Proteins
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Luminescent Proteins
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Membrane Glycoproteins
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Recombinant Fusion Proteins
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Viral Proteins
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Viral Regulatory and Accessory Proteins
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vpu protein, Human immunodeficiency virus 1