A Flow Cytometry-Based FRET Assay to Identify and Analyse Protein-Protein Interactions in Living Cells

被引：123

作者：

Banning, Carina ^{[1
]}

Votteler, Joerg ^{[2
]}

Hoffmann, Dirk ^{[1
]}

Koppensteiner, Herwig ^{[1
]}

Warmer, Martin ^{[1
]}

Reimer, Rudolph ^{[1
]}

Kirchhoff, Frank ^{[3
]}

Schubert, Ulrich ^{[2
]}

Hauber, Joachim ^{[1
]}

Schindler, Michael ^{[1
]}

机构：

[1] Univ Hamburg, Heinrich Pette Inst Expt Virol & Immunol, D-2000 Hamburg, Germany

[2] Univ Erlangen Nurnberg, Inst Clin & Mol Virol, Erlangen, Germany

[3] Univ Ulm, Inst Virol, Ulm, Germany

来源：

PLOS ONE | 2010年 / 5卷 / 02期

关键词：

RESONANCE ENERGY-TRANSFER; VIRUS TYPE-1 NEF; VPU PROTEIN; FLUORESCENT PROTEIN; DOWN-MODULATION; HIV-1; VPU; ACTIVATION; DEGRADATION; EXPRESSION; BINDS;

D O I：

10.1371/journal.pone.0009344

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

Background: Forsters 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.

引用

页数：10