scholarly journals Kinetics of HIV-1 capsid uncoating revealed by single-molecule analysis

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Chantal L Márquez ◽  
Derrick Lau ◽  
James Walsh ◽  
Vaibhav Shah ◽  
Conall McGuinness ◽  
...  
Keyword(s):  
Single Molecule ◽  
Nuclear Import ◽  
Rate Limiting Step ◽  
Viral Cdna ◽  
Microscopy Method ◽  
Single Molecule Analysis ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Hiv 1

Uncoating of the metastable HIV-1 capsid is a tightly regulated disassembly process required for release of the viral cDNA prior to nuclear import. To understand the intrinsic capsid disassembly pathway and how it can be modulated, we have developed a single-particle fluorescence microscopy method to follow the real-time uncoating kinetics of authentic HIV capsids in vitro immediately after permeabilizing the viral membrane. Opening of the first defect in the lattice is the rate-limiting step of uncoating, which is followed by rapid, catastrophic collapse. The capsid-binding inhibitor PF74 accelerates capsid opening but stabilizes the remaining lattice. In contrast, binding of a polyanion to a conserved arginine cluster in the lattice strongly delays initiation of uncoating but does not prevent subsequent lattice disassembly. Our observations suggest that different stages of uncoating can be controlled independently with the interplay between different capsid-binding regulators likely to determine the overall uncoating kinetics.

scholarly journals Receptor-mediated endocytosis of ovalbumin by two carbohydrate-specific receptors in rat liver cells. The intracellular transport of ovalbumin to lysosomes is faster in liver endothelial cells than in parenchymal cells

1990 ◽  
Vol 270 (1) ◽  
pp. 197-203 ◽  
Author(s):  
G M Kindberg ◽  
S Magnusson ◽  
T Berg ◽  
B Smedsrød
Keyword(s):  
Endothelial Cells ◽  
Intracellular Transport ◽  
Mannose Receptor ◽  
Cell Types ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Parenchymal Cells ◽  
Rate Limiting ◽  
Kinetics Of

1. The uptake of ovalbumin (OVA) in rat liver parenchymal cells (PC) and non-parenchymal cells was studied in vivo and in vitro in order to compare the cellular expression of glycoprotein receptors and the kinetics of intracellular transport of ligand endocytosed by these receptors. 2. Ovalbumin was labelled with 125I or with 125I-tyramine-cellobiose (125I-TC). By using 125I-TC-OVA the labelled degradation products were trapped in the cells. 3. 125I-TC-OVA was rapidly cleared from blood mainly by receptor-mediated uptake in the liver. At 30 min after injection, 50% of the ligand was recovered in the liver. The endothelial cells (EC) and the PC were the predominant cell types responsible for uptake. 4. The uptake in PC was strongly inhibited by asialo-orosomucoid (AOM), but not by mannan, indicating that the uptake in these cells was mediated by the galactose receptor and not by the mannose receptor. This finding is compatible with the observation that a proportion of the OVA contains terminal galactose residues in the carbohydrate moiety. 5. In vitro uptake of OVA in cultured EC was saturable and inhibited by mannan, mannose, fructose, N-acetylglucosamine, EDTA or monensin, but not by galactose or AOM. The uptake of OVA in these cells was therefore mediated by the mannose receptor. 6. To label the organelles involved in endocytosis in PC and EC, 125I-TC-OVA was injected intravenously together with an excess of either AOM or mannan. In this way the labelled ligand could be directed selectively to EC or PC respectively. Subcellular fractionation of total liver in sucrose and Nycodenz gradients revealed that in EC the intracellular transport of OVA is so fast that endocytosed ligand accumulates and thus increases the density of the lysosomes. Conversely, in PC transfer of ligand is slower, with the result that accumulation of undegraded ligand in the lysosomes does not occur. These findings are interpreted to mean that in EC the rate-limiting step of handling of endocytosed ligand is intralysosomal degradation, whereas in PC the rate-limiting step is transport of ligand to the lysosomes. 7. Altogether, these findings suggest that endocytosis of OVA by the liver EC and PC is mediated by mannose and galactose receptors respectively, and that the kinetics of intracellular transport of OVA differ in the two cell types.

scholarly journals Single-particle kinetics of influenza virus membrane fusion

2008 ◽  
Vol 105 (40) ◽  
pp. 15382-15387 ◽  
Author(s):  
Daniel L. Floyd ◽  
Justin R. Ragains ◽  
John J. Skehel ◽  
Stephen C. Harrison ◽  
Antoine M. van Oijen
Keyword(s):  
Membrane Fusion ◽  
Pore Formation ◽  
Enveloped Viruses ◽  
Virus Particles ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Multiple Copies ◽  
Rate Limiting ◽  
Kinetics Of

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays are generally limited to observation of ensembles of multiple fusion events, confounding more detailed analysis of the sequence of the molecular steps involved. We have developed anin vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support. Analysis of lipid- and content-mixing trajectories on a particle-by-particle basis provides evidence for multiple, long-lived kinetic intermediates leading to hemifusion, followed by a single, rate-limiting step to pore formation. We interpret the series of intermediates preceding hemifusion as a result of the requirement that multiple copies of the trimeric hemagglutinin fusion protein be activated to initiate the fusion process.

scholarly journals KIF3A accelerates KIF3C within the kinesin-2 heterodimer to generate symmetrical phosphate release rates for each processive step

2020 ◽  
pp. jbc.RA120.015272
Author(s):  
Sean M. Quinn ◽  
Troy Vargason ◽  
Nilisha Pokhrel ◽  
Edwin Antony ◽  
Juergen Hahn ◽  
...  
Keyword(s):  
Single Molecule ◽  
Slow Step ◽  
Phosphate Release ◽  
Intrinsic Kinetics ◽  
The Central Nervous System ◽  
Atp Binding And Hydrolysis ◽  
Future Work ◽  
Rate Limiting ◽  
Kinetics Of

Heterodimeric KIF3AC is a mammalian kinesin-2 that is highly expressed in the central nervous system and is associated with vesicles in neurons. KIF3AC is an intriguing member of the kinesin-2 family because the intrinsic kinetics of KIF3A and KIF3C when expressed as homodimers and analyzed in vitro are distinctively different from each other. For example, the single-molecule velocities of the engineered homodimers KIF3AA and KIF3CC are 293 nm/s and 7.5 nm/s, respectively, whereas KIF3AC has a velocity of 186 nm/s. These results led us to hypothesize that heterodimerization alters the intrinsic catalytic properties of the two heads, and an earlier computational analysis predicted that processive steps would alternate between a fast step for KIF3A followed by a slow step for KIF3C resulting in asymmetric stepping. To test this hypothesis directly, we measured the presteady-state kinetics of phosphate release for KIF3AC, KIF3AA, and KIF3CC followed by computational modeling of the KIF3AC phosphate release transients. The results reveal that KIF3A and KIF3C retain their intrinsic ATP binding and hydrolysis kinetics. Yet within KIF3AC, KIF3A activates the rate of phosphate release for KIF3C such that the coupled steps of phosphate release and dissociation from the microtubule become more similar for KIF3A and KIF3C. These coupled steps are the rate-limiting transition for the ATPase cycle suggesting that within KIF3AC, the stepping kinetics are similar for each head during the processive run. Future work will be directed to define how these properties enable KIF3AC to achieve its physiological functions.

scholarly journals Author response: Kinetics of HIV-1 capsid uncoating revealed by single-molecule analysis

2018 ◽  
Author(s):  
Chantal L Márquez ◽  
Derrick Lau ◽  
James Walsh ◽  
Vaibhav Shah ◽  
Conall McGuinness ◽  
...  
Keyword(s):  
Single Molecule ◽  
Author Response ◽  
Single Molecule Analysis ◽  
Kinetics Of ◽  
Hiv 1

Kinetics of cyclization of S-ethoxycarbonylmethylisothiouronium chloride to 2-imino-4-thiazolidone

1979 ◽  
Vol 44 (3) ◽  
pp. 912-917 ◽  
Author(s):  
Vladimír Macháček ◽  
Said A. El-bahai ◽  
Vojeslav Štěrba
Keyword(s):  
Hydrochloric Acid ◽  
Dilute Hydrochloric Acid ◽  
Base Catalysis ◽  
General Base ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Kinetics Of Formation ◽  
Acid Catalyzed ◽  
Rate Limiting ◽  
Kinetics Of

Kinetics of formation of 2-imino-4-thiazolidone from S-ethoxycarbonylmethylisothiouronium chloride has been studied in aqueous buffers and dilute hydrochloric acid. The reaction is subject to general base catalysis, the β value being 0.65. Its rate limiting step consists in acid-catalyzed splitting off of ethoxide ion from dipolar tetrahedral intermediate. At pH < 2 formation of this intermediate becomes rate-limiting; rate constant of its formation is 2 . 104 s-1.

Kinetics and mechanism of cyclization of N-(2-methoxycarbonylphenyl)-N’-methylsulphonamide to 3-methyl-(1H)-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide

1991 ◽  
Vol 56 (8) ◽  
pp. 1701-1710 ◽  
Author(s):  
Jaromír Kaválek ◽  
Vladimír Macháček ◽  
Miloš Sedlák ◽  
Vojeslav Štěrba
Keyword(s):  
Potassium Hydroxide ◽  
Acid Catalysis ◽  
Potassium Hydroxide Solution ◽  
Hydroxide Solution ◽  
General Base ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Cyclization Kinetics ◽  
Rate Limiting ◽  
Kinetics Of

The cyclization kinetics of N-(2-methylcarbonylphenyl)-N’-methylsulfonamide (IIb) into 3-methyl-(1H)-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (Ib) has been studied in ethanolamine, morpholine, and butylamine buffers and in potassium hydroxide solution. The cyclization is subject to general base and general acid catalysis. The value of the Bronsted coefficient β is about 0.1, which indicates that splitting off of the proton from negatively charged tetrahedral intermediate represents the rate-limiting and thermodynamically favourable step. In the solutions of potassium hydroxide the cyclization of dianion of the starting ester IIb probably becomes the rate-limiting step.

High field 1H NMR Studies of Sol-Gel Kinetics

1986 ◽  
Vol 73 ◽  
Author(s):  
Bruce D. Kay ◽  
Roger A. Assink
Keyword(s):  
Equilibrium Distribution ◽  
Sol Gel ◽  
Nmr Studies ◽  
H Nmr ◽  
Rate Limiting Step ◽  
Rate Of Hydrolysis ◽  
Acid Catalyzed ◽  
Product Species ◽  
Rate Limiting ◽  
Kinetics Of

ABSTRACTHigh resolution 1H NMR spectroscopy at high magnetic fields is employed to study the reaction kinetics of the Si(OCH3)4:CH3OH:H2O sol-gel system. Both the overall extent of reaction as a function of time and the equilibrium distribution of species are measured. In acid catalyzed solution, condensation is the rate limiting step while in base catalyzed solution, hydrolysis becomes rate limiting. A kinetic model in which the rate of hydrolysis is assumed to be independent of the adjacent functional groups is presented. This model correctly predicts the distribution of product species during the initial stages of the sol-gel reaction.

scholarly journals Exploitation of the IDO Pathway in the Therapy of Rheumatoid Arthritis

2013 ◽  
Vol 6s1 ◽  
pp. IJTR.S11737 ◽  
Author(s):  
Richard O. Williams
Keyword(s):  
Rheumatoid Arthritis ◽  
Anthranilic Acid ◽  
Kynurenine Pathway ◽  
Gene Encoding ◽  
Rate Limiting Step ◽  
Tryptophan Metabolites ◽  
Synthetic Derivative ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Draining Lymph Nodes

Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting step along the kynurenine pathway and is thought to play a key role in immune homeostasis through depletion of tryptophan and accumulation of kynurenines. In this review we summarize recent research into the possibility of harnessing the IDO pathway for the therapy of rheumatoid arthritis. Inhibition of IDO activity, or knockout of the gene encoding IDO, was shown to cause an increase in the severity of collagen-induced arthritis, an animal model of rheumatoid arthritis. The increased severity of disease was associated with elevated numbers of pathogenic Th1 and Th17 cells in the joints and draining lymph nodes. In another study, analysis of the kinetics of expression of downstream kynurenine pathway enzymes during the course of arthritis revealed a potential role for tryptophan metabolites in resolution of arthritis. Furthermore, the therapeutic administration of L-kynurenine or [3,4-dimethoxycinnamonyl]-anthranilic acid (a synthetic derivative of 3-hydroxy-anthranilic acid) significantly reduced both clinical and histological progression of experimental arthritis. These findings raise the possibility of exploiting the IDO pathway for the therapy of autoimmune disease.

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