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 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 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 The Interplay between HIV-1 Gag Binding to the Plasma Membrane and Env Incorporation

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 548 ◽  
Author(s):  
R. Elliot Murphy ◽  
Jamil S. Saad
Keyword(s):  
Plasma Membrane ◽  
Envelope Glycoprotein ◽  
Virus Production ◽  
Mortality Rates ◽  
Therapeutic Agents ◽  
Structural Level ◽  
Particle Assembly ◽  
Virus Particles ◽  
Molecular Determinants ◽  
Hiv 1

Advancement in drug therapies and patient care have drastically improved the mortality rates of HIV-1 infected individuals. Many of these therapies were developed or improved upon by using structure-based techniques, which underscore the importance of understanding essential mechanisms in the replication cycle of HIV-1 at the structural level. One such process which remains poorly understood is the incorporation of the envelope glycoprotein (Env) into budding virus particles. Assembly of HIV particles is initiated by targeting of the Gag polyproteins to the inner leaflet of the plasma membrane (PM), a process mediated by the N-terminally myristoylated matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). There is strong evidence that formation of the Gag lattice on the PM is a prerequisite for the incorporation of Env into budding particles. It is also suggested that Env incorporation is mediated by an interaction between its cytoplasmic tail (gp41CT) and the MA domain of Gag. In this review, we highlight the latest developments and current efforts to understand the interplay between gp41CT, MA, and the membrane during assembly. Elucidation of the molecular determinants of Gag–Env–membrane interactions may help in the development of new antiviral therapeutic agents that inhibit particle assembly, Env incorporation and ultimately virus production.

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 Monitoring HIV-1 Assembly in Living Cells: Insights from Dynamic and Single Molecule Microscopy

Viruses ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 72 ◽  
Author(s):  
Kaushik Inamdar ◽  
Charlotte Floderer ◽  
Cyril Favard ◽  
Delphine Muriaux
Keyword(s):  
Plasma Membrane ◽  
Host Cell ◽  
Single Molecule ◽  
High Speed ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Living Cells ◽  
Cell Plasma Membrane ◽  
Cell Plasma ◽  
Hiv 1

The HIV-1 assembly process is a multi-complex mechanism that takes place at the host cell plasma membrane. It requires a spatio-temporal coordination of events to end up with a full mature and infectious virus. The molecular mechanisms of HIV-1 assembly have been extensively studied during the past decades, in order to dissect the respective roles of the structural and non-structural viral proteins of the viral RNA genome and of some host cell factors. Nevertheless, the time course of HIV-1 assembly was observed in living cells only a decade ago. The very recent revolution of optical microscopy, combining high speed and high spatial resolution, in addition to improved fluorescent tags for proteins, now permits study of HIV-1 assembly at the single molecule level within living cells. In this review, after a short description of these new approaches, we will discuss how HIV-1 assembly at the cell plasma membrane has been revisited using advanced super resolution microscopy techniques and how it can bridge the study of viral assembly from the single molecule to the entire host cell.

scholarly journals Rendezvous at Plasma Membrane: Cellular Lipids and tRNA Set up Sites of HIV-1 Particle Assembly and Incorporation of Host Transmembrane Proteins

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 842 ◽  
Author(s):  
Dishari Thornhill ◽  
Tomoyuki Murakami ◽  
Akira Ono
Keyword(s):  
Plasma Membrane ◽  
Virus Assembly ◽  
Transmembrane Proteins ◽  
Particle Assembly ◽  
Host Membrane ◽  
The Matrix ◽  
Specific Localization ◽  
Cellular Lipids ◽  
Set Up ◽  
Hiv 1

The HIV-1 structural polyprotein Gag drives the virus particle assembly specifically at the plasma membrane (PM). During this process, the nascent virion incorporates specific subsets of cellular lipids and host membrane proteins, in addition to viral glycoproteins and viral genomic RNA. Gag binding to the PM is regulated by cellular factors, including PM-specific phospholipid PI(4,5)P2 and tRNAs, both of which bind the highly basic region in the matrix domain of Gag. In this article, we review our current understanding of the roles played by cellular lipids and tRNAs in specific localization of HIV-1 Gag to the PM. Furthermore, we examine the effects of PM-bound Gag on the organization of the PM bilayer and discuss how the reorganization of the PM at the virus assembly site potentially contributes to the enrichment of host transmembrane proteins in the HIV-1 particle. Since some of these host transmembrane proteins alter release, attachment, or infectivity of the nascent virions, the mechanism of Gag targeting to the PM and the nature of virus assembly sites have major implications in virus spread.

scholarly journals Visualizing Rous Sarcoma Virus Genomic RNA Dimerization in the Nucleus, Cytoplasm, and at the Plasma Membrane

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 903
Author(s):  
Eunice C. Chen ◽  
Rebecca J. Kaddis Maldonado ◽  
Leslie J. Parent
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Rous Sarcoma Virus ◽  
Genomic Rna ◽  
Virus Particles ◽  
Rna Dimerization ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Genome Dimerization ◽  
Hiv 1

Retroviruses are unique in that they package their RNA genomes as non-covalently linked dimers. Failure to dimerize their genomes results in decreased infectivity and reduced packaging of genomic RNA into virus particles. Two models of retrovirus genome dimerization have been characterized: in murine leukemia virus (MLV), genomic RNA dimerization occurs co-transcriptionally in the nucleus, resulting in the preferential formation of genome homodimers; whereas in human immunodeficiency virus (HIV-1), genomic RNA dimerization occurs in the cytoplasm and at the plasma membrane, with a random distribution of heterodimers and homodimers. Although in vitro studies have identified the genomic RNA sequences that facilitate dimerization in Rous sarcoma virus (RSV), in vivo characterization of the location and preferences of genome dimerization has not been performed. In this study, we utilized three single molecule RNA imaging approaches to visualize genome dimers of RSV in cultured quail fibroblasts. The formation of genomic RNA heterodimers within cells was dependent on the presence of the dimerization initiation site (DIS) sequence in the L3 stem. Subcellular localization analysis revealed that heterodimers were present the nucleus, cytoplasm, and at the plasma membrane, indicating that genome dimers can form in the nucleus. Furthermore, single virion analysis revealed that RSV preferentially packages genome homodimers into virus particles. Therefore, the mechanism of RSV genomic RNA dimer formation appears more similar to MLV than HIV-1.

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