scholarly journals Self assembly of HIV-1 Gag protein on lipid membranes generates PI(4,5)P2/Cholesterol nanoclusters

2016 ◽  
Author(s):  
Naresh Yandrapalli ◽  
Quentin Lubart ◽  
Hanumant S. Tanwar ◽  
Catherine Picart ◽  
Johnson Mak ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Self Assembly ◽  
Lipid Membranes ◽  
Membrane Lipids ◽  
Virus Assembly ◽  
Specific Interaction ◽  
Membrane Lipid ◽  
Gag Protein ◽  
Gag Polyprotein ◽  
Hiv 1

AbstractThe self-assembly of HIV-1 Gag polyprotein at the inner leaflet of the cell host plasma membrane is the key orchestrator of virus assembly. The binding between Gag and the plasma membrane is mediated by specific interaction of the Gag matrix domain and the PI(4,5)P2 lipid (PIP2). It is unknown whether this interaction could lead to local reorganization of the plasma membrane lipids. In this study, using model membranes, we examined the ability of Gag to segregate specific lipids upon self-assembly. We show for the first time that Gag self-assembly is responsible for the formation of PIP2 lipid nanoclusters, enriched in cholesterol but not in sphingomyelin. We also show that Gag mainly partition into liquid-disordered domains of these lipid membranes. Our work strongly suggests that, instead of targeting pre-existing plasma membrane lipid domains, Gag is more prone to generate PIP2/Cholesterol lipid nanodomains at the inner leaflet of the plasma membrane during early events of virus assembly.

scholarly journals Full assembly of HIV-1 particles requires assistance of the membrane curvature factor IRSp53

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Kaushik Inamdar ◽  
Feng-Ching Tsai ◽  
Rayane Dibsy ◽  
Aurore de Poret ◽  
John Manzi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Particle Production ◽  
Virus Assembly ◽  
Membrane Curvature ◽  
Particle Assembly ◽  
Gag Protein ◽  
Cell Plasma ◽  
Curved Membranes ◽  
Hiv 1

During HIV-1 particle formation, the requisite plasma membrane curvature is thought to be solely driven by the retroviral Gag protein. Here, we reveal that the cellular I-BAR protein IRSp53 is required for the progression of HIV-1 membrane curvature to complete particle assembly. SiRNA-mediated knockdown of IRSp53 gene expression induces a decrease in viral particle production and a viral bud arrest at half completion. Single molecule localization microscopy at the cell plasma membrane shows a preferential localization of IRSp53 around HIV-1 Gag assembly sites. In addition, we observe the presence of IRSp53 in purified HIV-1 particles. Finally, HIV-1 Gag protein preferentially localizes to curved membranes induced by IRSp53 I-BAR domain on giant unilamellar vesicles. Overall, our data reveal a strong interplay between IRSp53 I-BAR and Gag at membranes during virus assembly. This highlights IRSp53 as a crucial host factor in HIV-1 membrane curvature and its requirement for full HIV-1 particle assembly.

scholarly journals Self assembly of HIV-1 Gag protein on lipid membranes generates PI(4,5)P2/Cholesterol nanoclusters

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Naresh Yandrapalli ◽  
Quentin Lubart ◽  
Hanumant S. Tanwar ◽  
Catherine Picart ◽  
Johnson Mak ◽  
...  
Keyword(s):  
Self Assembly ◽  
Lipid Membranes ◽  
Gag Protein ◽  
Hiv 1

scholarly journals HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein

2015 ◽  
Vol 113 (2) ◽  
pp. E201-E208 ◽  
Author(s):  
Jianbo Chen ◽  
Sheikh Abdul Rahman ◽  
Olga A. Nikolaitchik ◽  
David Grunwald ◽  
Luca Sardo ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Viral Replication ◽  
Genetic Information ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Virus Assembly ◽  
Viral Population ◽  
Gag Protein ◽  
Hiv 1 ◽  
Dimerization Process

Retroviruses package a dimeric genome comprising two copies of the viral RNA. Each RNA contains all of the genetic information for viral replication. Packaging a dimeric genome allows the recovery of genetic information from damaged RNA genomes during DNA synthesis and promotes frequent recombination to increase diversity in the viral population. Therefore, the strategy of packaging dimeric RNA affects viral replication and viral evolution. Although its biological importance is appreciated, very little is known about the genome dimerization process. HIV-1 RNA genomes dimerize before packaging into virions, and RNA interacts with the viral structural protein Gag in the cytoplasm. Thus, it is often hypothesized that RNAs dimerize in the cytoplasm and the RNA–Gag complex is transported to the plasma membrane for virus assembly. In this report, we tagged HIV-1 RNAs with fluorescent proteins, via interactions of RNA-binding proteins and motifs in the RNA genomes, and studied their behavior at the plasma membrane by using total internal reflection fluorescence microscopy. We showed that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane. Dynamic interactions occur among HIV-1 RNAs, and stabilization of the RNA dimer requires Gag protein. Dimerization often occurs at an early stage of the virus assembly process. Furthermore, the dimerization process is probably mediated by the interactions of two RNA–Gag complexes, rather than two RNAs. These findings advance the current understanding of HIV-1 assembly and reveal important insights into viral replication mechanisms.

scholarly journals HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly

2019 ◽  
Vol 5 (10) ◽  
pp. eaaw8651 ◽  
Author(s):  
C. Favard ◽  
J. Chojnacki ◽  
P. Merida ◽  
N. Yandrapalli ◽  
J. Mak ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Virus Assembly ◽  
Super Resolution ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Gag Protein ◽  
Lipid Dynamics ◽  
Infected Cells ◽  
Lipid Environment ◽  
Hiv 1

HIV-1 Gag protein assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly PI(4,5)P2, at the inner leaflet of this membrane. Here, we address the question whether Gag is able to trap specifically PI(4,5)P2 or other lipids during HIV-1 assembly in the host CD4+ T lymphocytes. Lipid dynamics within and away from HIV-1 assembly sites were determined using super-resolution microscopy coupled with scanning fluorescence correlation spectroscopy in living cells. Analysis of HIV-1–infected cells revealed that, upon assembly, HIV-1 is able to specifically trap PI(4,5)P2 and cholesterol, but not phosphatidylethanolamine or sphingomyelin. Furthermore, our data showed that Gag is the main driving force to restrict the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. This is the first direct evidence highlighting that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol as a membrane nanoplatform for virus assembly.

scholarly journals Full assembly of HIV-1 particles requires assistance of the membrane curvature factor IRSp53

2021 ◽  
Author(s):  
Kaushik Inamdar ◽  
Feng-Ching Tsai ◽  
Aurore de Poret ◽  
Rayane Dibsy ◽  
John Manzi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Particle Production ◽  
Virus Assembly ◽  
Membrane Curvature ◽  
Particle Assembly ◽  
Gag Protein ◽  
Cell Plasma ◽  
Curved Membranes ◽  
Hiv 1

During HIV-1 particle formation, the requisite plasma membrane curvature is thought to be solely driven by the retroviral Gag protein. Here, we reveal that the cellular I-BAR protein IRSp53 is required for the progression of HIV-1 membrane curvature to complete particle assembly. Partial gene editing of IRSp53 induces a decrease in viral particle production and a viral bud arrest at half completion. Single molecule localization microscopy at the cell plasma membrane shows a preferential localization of IRSp53 around HIV-1 Gag assembly sites. In addition, we observe the presence of IRSp53 in purified HIV-1 particles. Finally, HIV-1 Gag protein localizes preferentially to IRSp53 I-BAR domain induced curved membranes on giant unilamellar vesicles. Overall, our data reveal a strong interplay between IRSp53 I-BAR and Gag at membranes during virus assembly. This highlights IRSp53 as a crucial host factor in HIV-1 membrane curvature and its requirement for full HIV-1 particle assembly.

scholarly journals Live single molecule microscopy of HIV-1 assembly in host T cells reveals a spatio-temporal effect of the viral genome

2018 ◽  
Author(s):  
Charlotte Floderer ◽  
Jean-Baptiste Masson ◽  
Elise Boiley ◽  
Sonia Georgeault ◽  
Peggy Merida ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Viral Genome ◽  
Virus Assembly ◽  
Host Cells ◽  
Spherical Particles ◽  
Gag Protein ◽  
Numerous Cell ◽  
Spatio Temporal ◽  
Hiv 1

Monitoring virus assembly dynamic at the nanoscale level in host cells remains a major challenge. Human Immunodeficiency Virus type 1 (HIV-1) components are addressed to the plasma membrane where they assemble to form spherical particles of 100nm in diameter. HIV-1 Gag protein expression alone is sufficient to produce virus-like particles (VLPs) that resemble immature virus. Here, we monitored Gag assembly in host CD4 T lymphocytes using single molecule dynamics microscopy and energy mapping. A workflow allowing long time recordings of single Gag molecule localization, diffusion and effective energy maps was developed for robust quantitative analysis of HIV assembly and budding. Comparison of numerous cell plasma membrane assembling platforms in cells expressing wild type or assembly-defective Gag proteins showed that VLP formation last 15 minutes, with an assembly time of 5 minutes, and that the nucleocapsid domain is mandatory. Importantly, it reveals that the viral genome coordinates spatio-temporally HIV-1 assembly.

scholarly journals HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly

2019 ◽  
Author(s):  
C. Favard ◽  
J. Chojnacki ◽  
P. Merida ◽  
N. Yandrapalli ◽  
J. Mak ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Virus Assembly ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Gag Protein ◽  
Lipid Dynamics ◽  
Cell Plasma ◽  
Infected Cells ◽  
Lipid Environment ◽  
Hiv 1

HIV-1 Gag protein self-assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly the phosphatidylinositol (4, 5) bisphosphate, at the inner leaflet of this membrane. Here, we address the question whether Gag is able to trap specifically PI(4,5)P2 or other lipids during HIV-1 assembly in the host CD4+ T lymphocytes. Lipid dynamics within and away from HIV-1 assembly sites was determined using super-resolution STED microscopy coupled with scanning Fluorescence Correlation Spectroscopy in living T cells. Analysis of HIV-1 infected cells revealed that, upon assembly, HIV-1 is able to specifically trap PI(4,5)P2, and cholesterol, but not phosphatidylethanolamine or sphingomyelin. Furthermore, our data show that Gag is the main driving force to restrict PI(4,5)P2 and cholesterol mobility at the cell plasma membrane. This is first direct evidence showing that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol, as a membrane nano-platform for virus assembly.

scholarly journals The HIV-1 Nucleocapsid Protein Recruits Negatively Charged Lipids To Ensure Its Optimal Binding to Lipid Membranes

2014 ◽  
Vol 89 (3) ◽  
pp. 1756-1767 ◽  
Author(s):  
Noémie Kempf ◽  
Viktoriia Postupalenko ◽  
Saurabh Bora ◽  
Pascal Didier ◽  
Youri Arntz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Membranes ◽  
Virus Assembly ◽  
Potential Interaction ◽  
The Other ◽  
Free Form ◽  
Nucleic Acid Binding ◽  
Gag Assembly ◽  
Initial Binding ◽  
Hiv 1

ABSTRACTThe HIV-1 Gag polyprotein precursor composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains orchestrates virus assembly via interactions between MA and the cell plasma membrane (PM) on one hand and NC and the genomic RNA on the other hand. As the Gag precursor can adopt a bent conformation, a potential interaction of the NC domain with the PM cannot be excluded during Gag assembly at the PM. To investigate the possible interaction of NC with lipid membranes in the absence of any interference from the other domains of Gag, we quantitatively characterized by fluorescence spectroscopy the binding of the mature NC protein to large unilamellar vesicles (LUVs) used as membrane models. We found that NC, either in its free form or bound to an oligonucleotide, was binding with high affinity (∼107M−1) to negatively charged LUVs. The number of NC binding sites, but not the binding constant, was observed to decrease with the percentage of negatively charged lipids in the LUV composition, suggesting that NC and NC/oligonucleotide complexes were able to recruit negatively charged lipids to ensure optimal binding. However, in contrast to MA, NC did not exhibit a preference for phosphatidylinositol-(4,5)-bisphosphate. These results lead us to propose a modified Gag assembly model where the NC domain contributes to the initial binding of the bent form of Gag to the PM.IMPORTANCEThe NC protein is a highly conserved nucleic acid binding protein that plays numerous key roles in HIV-1 replication. While accumulating evidence shows that NC either as a mature protein or as a domain of the Gag precursor also interacts with host proteins, only a few data are available on the possible interaction of NC with lipid membranes. Interestingly, during HIV-1 assembly, the Gag precursor is thought to adopt a bent conformation where the NC domain may interact with the plasma membrane. In this context, we quantitatively characterized the binding of NC, as a free protein or as a complex with nucleic acids, to lipid membranes and showed that the latter constitute a binding platform for NC. Taken together, our data suggest that the NC domain may play a role in the initial binding events of Gag to the plasma membrane during HIV-1 assembly.

scholarly journals Specific Lipid Recruitment by the Retroviral Gag Protein upon HIV-1 Assembly: From Model Membranes to Infected Cells

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 102
Author(s):  
Cyril Favard ◽  
Jakub Chojnacki ◽  
Naresh Yandrapalli ◽  
Johnson Mak ◽  
Christian Eggeling ◽  
...  
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
In Silico ◽  
Cd4 T Cells ◽  
Virus Assembly ◽  
Gag Protein ◽  
Infected Cells ◽  
Specific Lipid ◽  
Hiv 1

The retroviral Gag protein targets the plasma membrane of infected cells for viral particle formation and release. The matrix domain (MA) of Gag is myristoylated for membrane anchoring but also contains a highly basic region that recognizes acidic phospholipids. Gag targets lipid molecules at the inner leaflet of the plasma membrane including phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) and cholesterol. Here, we addressed the question whether HIV-1 Gag was able to trap PI(4,5)P2 and/or other lipids during HIV-1 assembly in silico, in vitro on reconstituted membranes and in cellulo at the plasma membrane of the host CD4+ T cells. In silico, we could observe the first PI(4,5)P2 preferential recruitment by HIV-1 MA or Gag while protein docked on artificial membranes. In vitro, using biophysical techniques, we observed the specific trapping of PI(4,5)P2, and, to a lesser extent, cholesterol and the exclusion of sphingomyelin, during HIV-1 myr(-)Gag self-assembly on LUVs and SLBs. Finally, in infected living CD4+ T cells, we measured lipid dynamics within and away from HIV-1 assembly sites using super-resolution stimulated emission depletion (STED) microscopy coupled with scanning Fluorescence Correlation Spectroscopy (sSTED-FCS). The analysis of HIV-1 infected CD4+ T lymphocytes revealed that, upon virus assembly, HIV-1 is able to specifically trap PI(4,5)P2, and cholesterol but not phosphatidylethanolamine (PE) or sphingomyelin (SM) at the cellular membrane. Furthermore, analyzing CD4+ T cells expressing only HIV-1 Gag protein showed that Gag is the main driving force restricting the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. Our data provide the first direct evidence showing that HIV-1 Gag creates its own specific lipid environment for virus assembly by selectively recruiting lipids to generate PI(4,5)P2/cholesterol-enriched nanodomains favoring virus assembly, and that HIV-1 does not assemble on pre-existing lipid domains.

scholarly journals Impact of Membrane Lipids on UapA and AzgA Transporter Subcellular Localization and Activity in Aspergillus nidulans

2021 ◽  
Vol 7 (7) ◽  
pp. 514
Author(s):  
Mariangela Dionysopoulou ◽  
George Diallinas
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Aspergillus Nidulans ◽  
Membrane Lipids ◽  
Membrane Lipid ◽  
Lipid Biosynthesis ◽  
Transport Kinetics ◽  
Negative Effect ◽  
And Function

Recent biochemical and biophysical evidence have established that membrane lipids, namely phospholipids, sphingolipids and sterols, are critical for the function of eukaryotic plasma membrane transporters. Here, we study the effect of selected membrane lipid biosynthesis mutations and of the ergosterol-related antifungal itraconazole on the subcellular localization, stability and transport kinetics of two well-studied purine transporters, UapA and AzgA, in Aspergillus nidulans. We show that genetic reduction in biosynthesis of ergosterol, sphingolipids or phosphoinositides arrest A. nidulans growth after germling formation, but solely blocks in early steps of ergosterol (Erg11) or sphingolipid (BasA) synthesis have a negative effect on plasma membrane (PM) localization and stability of transporters before growth arrest. Surprisingly, the fraction of UapA or AzgA that reaches the PM in lipid biosynthesis mutants is shown to conserve normal apparent transport kinetics. We further show that turnover of UapA, which is the transporter mostly sensitive to membrane lipid content modification, occurs during its trafficking and by enhanced endocytosis, and is partly dependent on autophagy and Hect-type HulARsp5 ubiquitination. Our results point out that the role of specific membrane lipids on transporter biogenesis and function in vivo is complex, combinatorial and transporter-dependent.

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