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 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

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 Super-Resolution STED Microscopy-Based Mobility Studies of the Viral Env Protein at HIV-1 Assembly Sites of Fully Infected T-Cells

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 608
Author(s):  
Jakub Chojnacki ◽  
Christian Eggeling
Keyword(s):  
Molecular Dynamics ◽  
Plasma Membrane ◽  
Molecular Mobility ◽  
Virus Assembly ◽  
Methodological Approach ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Model Systems ◽  
Hiv 1

The ongoing threat of human immunodeficiency virus (HIV-1) requires continued, detailed investigations of its replication cycle, especially when combined with the most physiologically relevant, fully infectious model systems. Here, we demonstrate the application of the combination of stimulated emission depletion (STED) super-resolution microscopy with beam-scanning fluorescence correlation spectroscopy (sSTED-FCS) as a powerful tool for the interrogation of the molecular dynamics of HIV-1 virus assembly on the cell plasma membrane in the context of a fully infectious virus. In this process, HIV-1 envelope glycoprotein (Env) becomes incorporated into the assembling virus by interacting with the nascent Gag structural protein lattice. Molecular dynamics measurements at these distinct cell surface sites require a guiding strategy, for which we have used a two-colour implementation of sSTED-FCS to simultaneously target individual HIV-1 assembly sites via the aggregated Gag signal. We then compare the molecular mobility of Env proteins at the inside and outside of the virus assembly area. Env mobility was shown to be highly reduced at the assembly sites, highlighting the distinct trapping of Env as well as the usefulness of our methodological approach to study the molecular mobility of specifically targeted sites at the plasma membrane, even under high-biosafety conditions.

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 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 Single-molecule imaging of HIV-1 envelope glycoprotein dynamics and Gag lattice association exposes determinants responsible for virus incorporation

2019 ◽  
Vol 116 (50) ◽  
pp. 25269-25277 ◽  
Author(s):  
Nairi Pezeshkian ◽  
Nicholas S. Groves ◽  
Schuyler B. van Engelenburg
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Envelope Glycoprotein ◽  
Virus Assembly ◽  
Single Molecule Tracking ◽  
Virus Particles ◽  
Cell Plasma ◽  
The Matrix ◽  
Resolution Single ◽  
Hiv 1

The HIV-1 envelope glycoprotein (Env) is sparsely incorporated onto assembling virus particles on the host cell plasma membrane in order for the virus to balance infectivity and evade the immune response. Env becomes trapped in a nascent particle on encounter with the polymeric viral protein Gag, which forms a dense protein lattice on the inner leaflet of the plasma membrane. While Env incorporation efficiency is readily measured biochemically from released particles, very little is known about the spatiotemporal dynamics of Env trapping events. Herein, we demonstrate, via high-resolution single-molecule tracking, that retention of Env trimers within single virus assembly sites requires the Env cytoplasmic tail (CT) and the L12 residue in the matrix (MA) domain of Gag but does not require curvature of the viral lattice. We further demonstrate that Env trimers are confined to subviral regions of a budding Gag lattice, supporting a model where direct interactions and/or steric corralling between the Env-CT and a lattice of MA trimers promote Env trapping and infectious HIV-1 assembly.

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 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 Lipid Composition but not Curvature Is the Determinant Factor for the Low Molecular Mobility Observed on the Membrane of Virus-Like Vesicles

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 415 ◽  
Author(s):  
Iztok Urbančič ◽  
Juliane Brun ◽  
Dilip Shrestha ◽  
Dominic Waithe ◽  
Christian Eggeling ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Composition ◽  
Molecular Mobility ◽  
Lipid Membrane ◽  
Dynamic Properties ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Membrane Composition ◽  
Hiv 1

Human Immunodeficiency Virus type-1 (HIV-1) acquires its lipid membrane from the plasma membrane of the infected cell from which it buds out. Previous studies have shown that the HIV-1 envelope is an environment of very low mobility, with the diffusion of incorporated proteins two orders of magnitude slower than in the plasma membrane. One of the reasons for this difference is thought to be the HIV-1 membrane composition that is characterised by a high degree of rigidity and lipid packing, which has, until now, been difficult to assess experimentally. To further refine the model of the molecular mobility on the HIV-1 surface, we herein investigated the relative importance of membrane composition and curvature in simplified model membrane systems, large unilamellar vesicles (LUVs) of different lipid compositions and sizes (0.1–1 µm), using super-resolution stimulated emission depletion (STED) microscopy-based fluorescence correlation spectroscopy (STED-FCS). Establishing an approach that is also applicable to measurements of molecule dynamics in virus-sized particles, we found, at least for the 0.1–1 µm sized vesicles, that the lipid composition and thus membrane rigidity, but not the curvature, play an important role in the decreased molecular mobility on the vesicles’ surface. This observation suggests that the composition of the envelope rather than the particle geometry contributes to the previously described low mobility of proteins on the HIV-1 surface. Our vesicle-based study thus provides further insight into the dynamic properties of the surface of individual HIV-1 particles, as well as paves the methodological way towards better characterisation of the properties and function of viral lipid envelopes in general.

scholarly journals Lipid composition but not curvature is a determinant of a low molecular mobility within HIV-1 lipid envelope

2018 ◽  
Author(s):  
Iztok Urbančič ◽  
Juliane Brun ◽  
Dilip Shrestha ◽  
Dominic Waithe ◽  
Christian Eggeling ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Composition ◽  
Molecular Mobility ◽  
Lipid Membrane ◽  
Dynamic Properties ◽  
Super Resolution ◽  
Membrane Curvature ◽  
Correlation Spectroscopy ◽  
Membrane Composition ◽  
Hiv 1

AbstractHuman Immunodeficiency Virus type-1 (HIV-1) acquires its lipid membrane from the plasma membrane of the infected cell from where it buds out. Previous studies have shown that the HIV-1 envelope is a very low mobility environment with the diffusion of incorporated proteins two orders of magnitude slower than in plasma membrane. One of the reasons for this difference is thought to be due to HIV-1 membrane composition that is characterised by a high degree of rigidity and lipid packing. To further refine the model of the molecular mobility on HIV-1 surface, we here investigated the relative importance of membrane composition and curvature in Large Unilamellar Vesicles of different composition and size (0.2–1 μm) by super-resolution STED microscopy-based fluorescence correlation spectroscopy (STED-FCS) analysis. We find that lipid composition and its rigidity but not membrane curvature play an important role in the decreased molecular mobility on vesicle surface thus confirming that this factor is an essential determinant of HIV-1 low surface mobility. Our results provide further insight into the dynamic properties of the surface of individual HIV-1 particles.

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