Abstract
BackgroundFörsters 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.ResultsAmongst 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.ConclusionThe 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.
Highlights
One of the few non-invasive techniques to study protein interactions is Forsters resonance energy transfer (FRET) [1,2]
Our goal was to establish a versatile Fluorescence activated cell sorting (FACS)-based FRET assay using the standard FRET pair CFP/YFP [14]. We evaluated this methodology by investigating interactions between the human and simian immunodeficiency virus (HIV and SIV) Nef and Vpu proteins and various cellular factors [15], as well as HIV Rev multimerization [16]
We gated on living cells according to forward and sideward scatter (FSC/SSC) and adjusted photomultiplier tube (PMT) voltages and compensation for CFP and YFP to assess FRET in double positive cells (Fig. 1a, panel 1)
Summary
One of the few non-invasive techniques to study protein interactions is Forsters resonance energy transfer (FRET) [1,2]. FRET is based upon the transfer of energy from an excited donor fluorophor to a close-by acceptor fluorophor, resulting in enhanced fluorescence emission of the acceptor [3]. This phenomenon only occurs when the distance between donor and acceptor is less than 10 nm and the emission spectra of the donor overlaps with the excitation of the acceptor [3]. 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
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