scholarly journals Precise triggering and chemical control of single-virus fusion within endosomes

2020 ◽  
Author(s):  
Sourav Haldar ◽  
Kenta Okamoto ◽  
Peter M. Kasson
Keyword(s):  
Protein Composition ◽  
Membrane Curvature ◽  
Endosomal Trafficking ◽  
Viral Fusion ◽  
Restriction Factors ◽  
Membrane Asymmetry ◽  
Synthetic Membranes ◽  
Endosomal Membrane ◽  
Virus Fusion ◽  
Microfluidic Flow

AbstractMany enveloped viruses infect cells within endocytic compartments. The drop in pH that accompanies endosomal maturation, often in conjunction with proteolytic factors, serves as a trigger for viral fusion proteins to insert into the endosomal membrane and drive fusion. The dynamics of this process has been studied by tracking viruses within living cells, which limits the precision with which fusion can be synchronized and controlled, and by reconstituting viral fusion to synthetic membranes, which introduces non-physiological membrane curvature and composition. To overcome these limitations, we have engineered the chemically controllable triggering of single-virus fusion within endosomes. We isolate influenza virus:endosome conjugates from cells prior to fusion, immobilize them in a microfluidic flow cell, and then rapidly and controllably trigger fusion. This platform demonstrates lipid-mixing kinetics that are grossly similar to influenza fusion with model membranes but display some subtle differences. Because it preserves endosomal membrane asymmetry and protein composition, it also provides a means to test how perturbations to endosomal trafficking and cellular restriction factors affect viral membrane fusion.

scholarly journals Precise Triggering and Chemical Control of Single-Virus Fusion within Endosomes

2020 ◽  
Vol 95 (1) ◽  
Author(s):  
Sourav Haldar ◽  
Kenta Okamoto ◽  
Rebecca A. Dunning ◽  
Peter M. Kasson
Keyword(s):  
Influenza Virus ◽  
Membrane Fusion ◽  
Influenza A ◽  
Living Cells ◽  
Membrane Curvature ◽  
Endosomal Trafficking ◽  
Viral Fusion ◽  
Enveloped Viruses ◽  
Endosomal Membrane ◽  
Virus Fusion

ABSTRACT Many enveloped viruses infect cells within endocytic compartments. The pH drop that accompanies endosomal maturation, often in conjunction with proteolysis, triggers viral proteins to insert into the endosomal membrane and drive fusion. Fusion dynamics have been studied by tracking viruses within living cells, which limits the precision with which fusion can be synchronized and controlled, and reconstituting viral fusion to synthetic membranes, which introduces nonphysiological membrane curvature and composition. To overcome these limitations, we report chemically controllable triggering of single-virus fusion within endosomes. We isolated influenza (A/Aichi/68; H3N2) virus:endosome conjugates from cells, immobilized them in a microfluidic flow cell, and rapidly and controllably triggered fusion. Observed lipid-mixing kinetics were surprisingly similar to those of influenza virus fusion with model membranes of opposite curvature: 80% of single-virus events had indistinguishable kinetics. This result suggests that endosomal membrane curvature is not a key permissive feature for viral entry, at least lipid mixing. The assay preserved endosomal membrane asymmetry and protein composition, providing a platform to test how cellular restriction factors and altered endosomal trafficking affect viral membrane fusion. IMPORTANCE Many enveloped viruses infect cells via fusion to endosomes, but controlling this process within living cells has been challenging. We studied the fusion of influenza virus virions to endosomes in a chemically controllable manner. Extracting virus:endosome conjugates from cells and exogenously triggering fusion permits precise study of virus:endosome fusion kinetics. Surprisingly, endosomal curvature does not grossly alter fusion kinetics, although membrane deformability does. This supports a model for influenza virus entry where cells restrict or permit membrane fusion by changing deformability, for instance, using interferon-induced proteins.

scholarly journals HIV envelope tail truncation confers resistance to SERINC5 restriction

2021 ◽  
Vol 118 (21) ◽  
pp. e2101450118
Author(s):  
Tafhima Haider ◽  
Xenia Snetkov ◽  
Clare Jolly
Keyword(s):  
T Cells ◽  
Envelope Glycoprotein ◽  
Cytoplasmic Tail ◽  
Restriction Factor ◽  
Viral Fusion ◽  
Restriction Factors ◽  
Complete Resistance ◽  
Hiv Envelope ◽  
Hiv 1

SERINC5 is a potent lentiviral restriction factor that gets incorporated into nascent virions and inhibits viral fusion and infectivity. The envelope glycoprotein (Env) is a key determinant for SERINC restriction, but many aspects of this relationship remain incompletely understood, and the mechanism of SERINC5 restriction remains unresolved. Here, we have used mutants of HIV-1 and HIV-2 to show that truncation of the Env cytoplasmic tail (ΔCT) confers complete resistance of both viruses to SERINC5 and SERINC3 restriction. Critically, fusion of HIV-1 ΔCT virus was not inhibited by SERINC5 incorporation into virions, providing a mechanism to explain how EnvCT truncation allows escape from restriction. Neutralization and inhibitor assays showed ΔCT viruses have an altered Env conformation and fusion kinetics, suggesting that EnvCT truncation dysregulates the processivity of entry, in turn allowing Env to escape targeting by SERINC5. Furthermore, HIV-1 and HIV-2 ΔCT viruses were also resistant to IFITMs, another entry-targeting family of restriction factors. Notably, while the EnvCT is essential for Env incorporation into HIV-1 virions and spreading infection in T cells, HIV-2 does not require the EnvCT. Here, we reveal a mechanism by which human lentiviruses can evade two potent Env-targeting restriction factors but show key differences in the capacity of HIV-1 and HIV-2 to exploit this. Taken together, this study provides insights into the interplay between HIV and entry-targeting restriction factors, revealing viral plasticity toward mechanisms of escape and a key role for the long lentiviral EnvCT in regulating these processes.

Viral Fusion Peptides: A Tool Set to Disrupt and Connect Biological Membranes

2000 ◽  
Vol 20 (6) ◽  
pp. 501-518 ◽  
Author(s):  
Lukas K. Tamm ◽  
Xing Han
Keyword(s):  
Lipid Bilayers ◽  
Biological Membrane ◽  
Membrane Curvature ◽  
Current Evidence ◽  
Viral Fusion ◽  
Fusion Peptides ◽  
Membrane Surfaces ◽  
Self Association ◽  
Α Helix ◽  
And Function

The structure and function of viral fusion peptides are reviewed. The fusion peptides of influenza virus hemagglutinin and human immunodeficiency virus are used as paradigms. Fusion peptides associated with lipid bilayers are conformationally polymorphic. Current evidence suggests that the fusion-promoting state is the obliquely inserted α-helix. Fusion peptides also have a tendency to self-associate into γ-sheets at membrane surfaces. Although the conformational conversion between α- and γ-states is reversible under controlled conditions, its physiological relevance is not yet known. The energetics of peptide insertion and self-association could be measured recently using more soluble “second generation” fusion peptides. Fusion peptides have been reported to change membrane curvature and the state of hydration of membrane surfaces. The combined results are built into a model for the mechanism by which fusion peptides are proposed to assist in biological membrane fusion.

scholarly journals Connecdenn 3/DENND1C binds actin linking Rab35 activation to the actin cytoskeleton

2012 ◽  
Vol 23 (1) ◽  
pp. 163-175 ◽  
Author(s):  
Andrea L. Marat ◽  
Maria S. Ioannou ◽  
Peter S. McPherson
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Small Gtpase ◽  
Actin Binding ◽  
Binding Motif ◽  
Endosomal Trafficking ◽  
Nucleotide Exchange ◽  
Endosomal Membrane ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

The small GTPase Rab35 regulates endosomal membrane trafficking but also recruits effectors that modulate actin assembly and organization. Differentially expressed in normal and neoplastic cells (DENN)–domain proteins are a newly identified class of Rab guanine-nucleotide exchange factors (GEFs) that are grouped into eight families, each activating a common Rab. The members of one family, connecdenn 1–3/DENND1A–C, are all GEFs for Rab35. Why Rab35 requires multiple GEFs is unknown. We demonstrate that connecdenn 3 uses a unique C-terminal motif, a feature not found in connecdenn 1 or 2, to directly bind actin. This interaction couples Rab35 activation to the actin cytoskeleton, resulting in dramatic changes in cell shape, notably the formation of protrusive membrane extensions. These alterations are specific to Rab35 activated by connecdenn 3 and require both the actin-binding motif and N-terminal DENN domain, which harbors the GEF activity. It was previously demonstrated that activated Rab35 recruits the actin-bundling protein fascin to actin, but the relevant GEF for this activity was unknown. We demonstrate that connecdenn 3 and Rab35 colocalize with fascin and actin filaments, suggesting that connecdenn 3 is the relevant GEF. Thus, whereas connecdenn 1 and 2 activate Rab35 for endosomal trafficking, connecdenn 3 uniquely activates Rab35 for its role in actin regulation.

scholarly journals The phosphoinositide-associated protein Rush hour regulates endosomal trafficking in Drosophila

2012 ◽  
Vol 23 (3) ◽  
pp. 433-447 ◽  
Author(s):  
Ieva Gailite ◽  
Diane Egger-Adam ◽  
Andreas Wodarz
Keyword(s):  
Cell Polarity ◽  
Protein Composition ◽  
Genetic Interactions ◽  
Rab Proteins ◽  
Endosomal Trafficking ◽  
Intercellular Signaling ◽  
Cellular Processes ◽  
Late Endosomes ◽  
Direct Binding ◽  
Gdp Dissociation Inhibitor

Endocytosis regulates multiple cellular processes, including the protein composition of the plasma membrane, intercellular signaling, and cell polarity. We have identified the highly conserved protein Rush hour (Rush) and show that it participates in the regulation of endocytosis. Rush localizes to endosomes via direct binding of its FYVE (Fab1p, YOTB, Vac1p, EEA1) domain to phosphatidylinositol 3-phosphate. Rush also directly binds to Rab GDP dissociation inhibitor (Gdi), which is involved in the activation of Rab proteins. Homozygous rush mutant flies are viable but show genetic interactions with mutations in Gdi, Rab5, hrs, and carnation, the fly homologue of Vps33. Overexpression of Rush disrupts progression of endocytosed cargo and increases late endosome size. Lysosomal marker staining is decreased in Rush-overexpressing cells, pointing to a defect in the transition between late endosomes and lysosomes. Rush also causes formation of endosome clusters, possibly by affecting fusion of endosomes via an interaction with the class C Vps/homotypic fusion and vacuole protein-sorting (HOPS) complex. These results indicate that Rush controls trafficking from early to late endosomes and from late endosomes to lysosomes by modulating the activity of Rab proteins.

Membrane Curvature Induced by Sugar and Polymer Solutions

1997 ◽  
Vol 489 ◽  
Author(s):  
H.-G. Döbereiner ◽  
A. Lehmann ◽  
W. Goedel ◽  
O. Selchow ◽  
R. Lipowsky
Keyword(s):  
Polymer Solutions ◽  
Lipid Vesicles ◽  
Membrane Curvature ◽  
Elastic Membrane ◽  
Spontaneous Curvature ◽  
Membrane Asymmetry ◽  
Bending Modulus ◽  
Bending Elasticity ◽  
Cellular Organelles

AbstractWe monitor the effect of transversal membrane asymmetry on the morphology of giant uni-lamellar vesicles in sugar and polymer solutions. The shapes of fluid lipid vesicles are governed by the bending elasticity of their membrane which is characterized by the bending modulus and the spontaneous curvature of the bilayer. We present a recently developed technique for the measurement of the spontaneous curvature using quantitative phase contrast microscopy. Different mechanisms for elastic membrane asymmetry and the role of the bending energy concept for the morphology of cellular organelles are discussed.

scholarly journals Lipid flip-flop and desorption from supported lipid bilayers is independent of curvature

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244460
Author(s):  
Haoyuan Jing ◽  
Yanbin Wang ◽  
Parth Rakesh Desai ◽  
Kumaran S. Ramamurthi ◽  
Siddhartha Das
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Md Simulations ◽  
Packing Fraction ◽  
Membrane Curvature ◽  
Supported Lipid Bilayer ◽  
Flip Flop ◽  
Lipid Molecule ◽  
Membrane Asymmetry ◽  
Cell Shapes

Flip-flop of lipids of the lipid bilayer (LBL) constituting the plasma membrane (PM) plays a crucial role in a myriad of events ranging from cellular signaling and regulation of cell shapes to cell homeostasis, membrane asymmetry, phagocytosis, and cell apoptosis. While extensive research has been conducted to probe the lipid flip flop of planar lipid bilayers (LBLs), less is known regarding lipid flip-flop for highly curved, nanoscopic LBL systems despite the vast importance of membrane curvature in defining the morphology of cells and organelles and in maintaining a variety of cellular functions, enabling trafficking, and recruiting and localizing shape-responsive proteins. In this paper, we conduct molecular dynamics (MD) simulations to study the energetics, structure, and configuration of a lipid molecule undergoing flip-flop and desorption in a highly curved LBL, represented as a nanoparticle-supported lipid bilayer (NPSLBL) system. We compare our findings against those of a planar substrate supported lipid bilayer (PSSLBL). Our MD simulation results reveal that despite the vast differences in the curvature and other curvature-dictated properties (e.g., lipid packing fraction, difference in the number of lipids between inner and outer leaflets, etc.) between the NPSLBL and the PSSLBL, the energetics of lipid flip-flop and lipid desorption as well as the configuration of the lipid molecule undergoing lipid flip-flop are very similar for the NPSLBL and the PSSLBL. In other words, our results establish that the curvature of the LBL plays an insignificant role in lipid flip-flop and desorption.

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