scholarly journals Application of Advanced Light Microscopy to the Study of HIV and Its Interactions with the Host

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 223
Author(s):  
Saveez Saffarian
Keyword(s):  
Human Immunodeficiency Virus ◽  
Energy Transfer ◽  
High Resolution ◽  
Light Microscopy ◽  
Single Molecule ◽  
Total Internal Reflection ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Immunodeficiency Virus ◽  
Microscopy Techniques

This review highlights the significant observations of human immunodeficiency virus (HIV) assembly, release and maturation made possible with advanced light microscopy techniques. The advances in technology which now enables these light microscopy measurements are discussed with special emphasis on live imaging approaches including Total Internal Reflection Fluorescence (TIRF), high-resolution light microscopy techniques including PALM and STORM and single molecule measurements, including Fluorescence Resonance Energy Transfer (FRET). The review concludes with a discussion on what new insights and understanding can be expected from these measurements.

scholarly journals Quantitative Fluorescence Resonance Energy Transfer Microscopy Analysis of the Human Immunodeficiency Virus Type 1 Gag-Gag Interaction: Relative Contributions of the CA and NC Domains and Membrane Binding

2009 ◽  
Vol 83 (14) ◽  
pp. 7322-7336 ◽  
Author(s):  
Ian B. Hogue ◽  
Adam Hoppe ◽  
Akira Ono
Keyword(s):  
Human Immunodeficiency Virus ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Membrane Binding ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Immunodeficiency Virus

ABSTRACT The human immunodeficiency virus type 1 structural polyprotein Pr55Gag is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated the importance of the capsid C-terminal domain (CA-CTD), nucleocapsid (NC), and membrane association in Gag-Gag interactions, but the relationships between these factors remain unclear. In this study, we systematically altered the CA-CTD, NC, and the ability to bind membrane to determine the relative contributions of, and interplay between, these factors. To directly measure Gag-Gag interactions, we utilized chimeric Gag-fluorescent protein fusion constructs and a fluorescence resonance energy transfer (FRET) stoichiometry method. We found that the CA-CTD is essential for Gag-Gag interactions at the plasma membrane, as the disruption of the CA-CTD has severe impacts on FRET. Data from experiments in which wild-type (WT) and CA-CTD mutant Gag molecules are coexpressed support the idea that the CA-CTD dimerization interface consists of two reciprocal interactions. Mutations in NC have less-severe impacts on FRET between normally myristoylated Gag proteins than do CA-CTD mutations. Notably, when nonmyristoylated Gag interacts with WT Gag, NC is essential for FRET despite the presence of the CA-CTD. In contrast, constitutively enhanced membrane binding eliminates the need for NC to produce a WT level of FRET. These results from cell-based experiments suggest a model in which both membrane binding and NC-RNA interactions serve similar scaffolding functions so that one can functionally compensate for a defect in the other.

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.

Evidence that GPVI is Expressed as a Mixture of Monomers and Dimers, and that the D2 Domain is not Essential for GPVI Activation

2021 ◽  
Author(s):  
Joanne C. Clark ◽  
Raluca A. I. Neagoe ◽  
Malou Zuidscherwoude ◽  
Deirdre M. Kavanagh ◽  
Alexandre Slater ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Glycoprotein Vi ◽  
D2 Domain ◽  
Microscopy Techniques

AbstractCollagen has been proposed to bind to a unique epitope in dimeric glycoprotein VI (GPVI) and the number of GPVI dimers has been reported to increase upon platelet activation. However, in contrast, the crystal structure of GPVI in complex with collagen-related peptide (CRP) showed binding distinct from the site of dimerization. Further fibrinogen has been reported to bind to monomeric but not dimeric GPVI. In the present study, we have used the advanced fluorescence microscopy techniques of single-molecule microscopy, fluorescence correlation spectroscopy (FCS) and bioluminescence resonance energy transfer (BRET), and mutagenesis studies in a transfected cell line model to show that GPVI is expressed as a mixture of monomers and dimers and that dimerization through the D2 domain is not critical for activation. As many of these techniques cannot be applied to platelets to resolve this issue, due to the high density of GPVI and its anucleate nature, we used Förster resonance energy transfer (FRET) to show that endogenous GPVI is at least partially expressed as a dimer on resting and activated platelet membranes. We propose that GPVI may be expressed as a monomer on the cell surface and it forms dimers in the membrane through diffusion, giving rise to a mixture of monomers and dimers. We speculate that the formation of dimers facilitates ligand binding through avidity.

scholarly journals Detection of Human Immunodeficiency Virus Type 1 Nef and CD4 Physical Interaction in Living Human Cells by Using Bioluminescence Resonance Energy Transfer

2005 ◽  
Vol 79 (13) ◽  
pp. 8629-8636 ◽  
Author(s):  
David Cluet ◽  
Christophe Bertsch ◽  
Christian Beyer ◽  
Liliane Gloeckler ◽  
Mathieu Erhardt ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Energy Transfer ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Human Cells ◽  
Bioluminescence Resonance Energy Transfer ◽  
Immunodeficiency Virus

ABSTRACT CD4 down-regulation by human immunodeficiency virus type 1 (HIV-1) Nef protein is a key function for virus virulence. This activity may be mediated by a direct Nef-CD4 interaction. We investigated the formation, in situ, of such a complex between proteins using bioluminescence resonance energy transfer technology and coimmunoprecipitations. Our data clearly demonstrate that Nef and CD4 interact in intact human cells. Moreover, our results clearly indicate that the dileucine motif of the CD4 cytoplasmic domain, critical for the Nef-induced CD4 down-regulation, is not implicated in the Nef/CD4 complex formation in the cellular context.

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