Measuring protein‐protein interactions inside living cells using single color fluorescence correlation spectroscopy. Application to human immunodeficiency virus type 1 integrase and LEDGF/p75

2005 ◽  
Vol 19 (8) ◽  
pp. 1039-1041 ◽  
Author(s):  
Goedele Maertens ◽  
Jo Vercammen ◽  
Zeger Debyser ◽  
Yves Engelborghs
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Protein Interactions ◽  
Fluorescence Correlation Spectroscopy ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Protein Protein Interactions ◽  
Fluorescence Correlation ◽  
Immunodeficiency Virus

scholarly journals Vif and the p55Gag Polyprotein of Human Immunodeficiency Virus Type 1 Are Present in Colocalizing Membrane-Free Cytoplasmic Complexes

1999 ◽  
Vol 73 (4) ◽  
pp. 2667-2674 ◽  
Author(s):  
James H. M. Simon ◽  
Elise A. Carpenter ◽  
Ron A. M. Fouchier ◽  
Michael H. Malim
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Protein Interactions ◽  
Subcellular Fractionation ◽  
Current Data ◽  
Protein Protein Interactions ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The Vif protein of human immunodeficiency virus type 1 (HIV-1) is a potent regulator of viral infectivity. Current data posit that Vif functions late in replication to modulate assembly, budding, and/or maturation. Consistent with this model, earlier indirect immunofluorescence analyses of HIV-1-infected cells demonstrated that Vif and Gag colocalize to a substantial degree (J. H. M. Simon, R. A. M. Fouchier, T. E. Southerling, C. B. Guerra, C. K. Grant, and M. H. Malim, J. Virol. 71:5259–5267, 1997). Here, we describe a series of subcellular fractionation studies which indicate that Vif and the p55Gag polyprotein are present in membrane-free cytoplasmic complexes that copurify in sucrose density gradients and are stable in nonionic detergents. Both Vif and Gag are targeted to these complexes independent of each other, and their association with them appears to be mediated by protein-protein interactions. We propose that these complexes may represent viral assembly intermediates and that Vif is appropriately localized to influence the final stages of the viral life cycle and, therefore, the infectivity of progeny virions.

scholarly journals Myristoylation Is Required for Human Immunodeficiency Virus Type 1 Gag-Gag Multimerization in Mammalian Cells

2007 ◽  
Vol 81 (23) ◽  
pp. 12899-12910 ◽  
Author(s):  
Hua Li ◽  
Jun Dou ◽  
Lingmei Ding ◽  
Paul Spearman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Protein Interactions ◽  
Myristic Acid ◽  
Mammalian Cells ◽  
Protein Protein Interactions ◽  
Gag Protein ◽  
Gag Proteins ◽  
Immunodeficiency Virus

ABSTRACT The Gag protein of human immunodeficiency virus type 1 directs the virion assembly process. Gag proteins must extensively multimerize during the formation of the spherical immature virion shell. In vitro, virus-like particles can be generated from Gag proteins that lack the N-terminal myristic acid modification or the nucleocapsid (NC) protein. The precise requirements for Gag-Gag multimerization under conditions present in mammalian cells, however, have not been fully elucidated. In this study, a Gag-Gag multimerization assay measuring fluorescence resonance energy transfer was employed to define the Gag domains that are essential for homomultimerization. Three essential components were identified: protein-protein interactions contributed by residues within both the N- and C-terminal domains of capsid (CA), basic residues in NC, and the presence of myristic acid. The requirement of myristic acid for multimerization was reproduced using the heterologous myristoylation sequence from v-src. Only when a leucine zipper dimerization motif was placed in the position of NC was a nonmyristoylated Gag protein able to multimerize. These results support a three-component model for Gag-Gag multimerization that includes membrane interactions mediated by the myristoylated N terminus of Gag, protein-protein interactions between CA domains, and NC-RNA interactions.

scholarly journals Targeting RNA–Protein Interactions within the Human Immunodeficiency Virus Type 1 Lifecycle

Biochemistry ◽  
2013 ◽  
Vol 52 (51) ◽  
pp. 9269-9274 ◽  
Author(s):  
Neil M. Bell ◽  
Anne L’Hernault ◽  
Pierre Murat ◽  
James E. Richards ◽  
Andrew M. L. Lever ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Protein Interactions ◽  
Immunodeficiency Virus

scholarly journals Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein

2015 ◽  
Vol 26 (11) ◽  
pp. 2054-2066 ◽  
Author(s):  
Yinghua Guan ◽  
Matthias Meurer ◽  
Sarada Raghavan ◽  
Aleksander Rebane ◽  
Jake R. Lindquist ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fluorescence Correlation Spectroscopy ◽  
Fluorescent Protein ◽  
Stokes Shift ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Quantitative Detection ◽  
Multiphoton Excitation ◽  
Fluorescence Correlation ◽  
Large Stokes Shift

We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (∼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein–protein interactions. We also use MPE-FCCS to detect drug–protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells.

Self-diffusion of a highly concentrated monoclonal antibody by fluorescence correlation spectroscopy: insight into protein–protein interactions and self-association

Soft Matter ◽  
2019 ◽  
Vol 15 (33) ◽  
pp. 6660-6676 ◽  
Author(s):  
Jessica J. Hung ◽  
Wade F. Zeno ◽  
Amjad A. Chowdhury ◽  
Barton J. Dear ◽  
Kishan Ramachandran ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Protein Interactions ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Protein Protein Interactions ◽  
Fluorescence Correlation ◽  
Self Diffusion ◽  
Self Association ◽  
Insight Into

Measurement and interpretation of self-diffusion of a highly concentrated mAb with different formulations in context of viscosity and protein self-interactions.

scholarly journals A Novel Fluorescence Resonance Energy Transfer Assay Demonstrates that the Human Immunodeficiency Virus Type 1 Pr55Gag I Domain Mediates Gag-Gag Interactions

2004 ◽  
Vol 78 (3) ◽  
pp. 1230-1242 ◽  
Author(s):  
Aaron Derdowski ◽  
Lingmei Ding ◽  
Paul Spearman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Energy Transfer ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Protein Interactions ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) assembly takes place at the plasma membrane of cells and is directed by the Pr55Gag polyprotein (Gag). One of the essential steps in the assembly process is the multimerization of Gag. We have developed a novel fluorescence resonance energy transfer (FRET) assay for the detection of protein-protein interactions between Gag molecules. We demonstrate that Gag multimerization takes place primarily on cellular membranes, with the majority of these interactions occurring on the plasma membrane. However, distinct sites of Gag-Gag interaction are also present at punctate intracellular locations. The I domain is a functional assembly domain within the nucleocapsid region of Gag that affects particle density, the subcellular localization of Gag, and the formation of detergent-resistant Gag protein complexes. Results from this study provide evidence that the I domain mediates Gag-Gag interactions. Using Gag-fluorescent protein fusion constructs that were previously shown to define the minimal I domain within HIV-1 Pr55Gag, we show by FRET techniques that protein-protein interactions are greatly diminished when Gag proteins lacking the I domain are expressed. Gag-Tsg101 interactions are also seen in living cells and result in a shift of Tsg101 to the plasma membrane. The results within this study provide direct evidence that the I domain mediates protein-protein interactions between Gag molecules. Furthermore, this study establishes FRET as a powerful tool for the detection of protein-protein interactions involved in retrovirus assembly.

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