17 results on '"Derrick Lau"'
Search Results
2. Negative Staining Transmission Electron Microscopy of HIV Viral Particles Permeabilized with PFO and Capsid Stabilized with IP6
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Derrick Lau, Chantal Márquez, Michael Parker, and Till Böcking
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Biology (General) ,QH301-705.5 - Abstract
The human immunodeficiency virus 1 (HIV-1) consists of a viral membrane surrounding the conical capsid. The capsid is a protein container assembled from approximately 1,500 copies of the viral capsid protein (CA), functioning as a reaction and transport chamber for the viral genome after cell entry. Transmission electron microscopy (TEM) is a widely used technique for characterizing the ultrastructure of isolated viral capsids after removal of the viral membrane, which otherwise hinders negative staining of structures inside the viral particle for TEM. Here, we provide a protocol to permeabilize the membrane of HIV-1 particles using a pore-forming toxin for negative staining of capsids, which are stabilized with inositol hexakisphosphate to prevent premature capsid disassembly. This approach revealed the pleomorphic nature of capsids with a partially intact membrane surrounding them. The permeabilization strategy using pore-forming toxins can be readily applied to visualize the internal architecture of other enveloped viruses using TEM.Graphical abstract:
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- 2022
- Full Text
- View/download PDF
3. Functional analysis of the secondary HIV-1 capsid binding site in the host protein cyclophilin A
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Wang Peng, Jiong Shi, Chantal L. Márquez, Derrick Lau, James Walsh, K. M. Rifat Faysal, Chang H. Byeon, In-Ja L. Byeon, Christopher Aiken, and Till Böcking
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HIV-1 ,Capsid ,Cyclophilin A ,Non-canonical binding site ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Abstract Background Efficient HIV-1 replication depends on interaction of the viral capsid with the host protein cyclophilin A (CypA). CypA, a peptidylprolyl isomerase, binds to an exposed loop in the viral CA protein via the enzyme’s active site. Recent structural analysis of CypA in complex with CA tubes in conjunction with molecular dynamics simulations identified a secondary CA binding site on CypA that allows a bridging interaction with two hexameric subunits of the assembled CA lattice, leading to capsid stabilization (Liu et al. in Nat Commun 7:10714, 2016). Results We performed mutational analysis of residues that have been proposed to mediate CA binding at the secondary binding site on CypA (A25, K27, P29 and K30) and tested the effects of the amino acid substitutions using interaction assays and HIV-1 infection assays in cells. The binding of recombinant CypA to self-assembled CA tubes or native HIV-1 capsids was measured in vitro using a quantitative fluorescence microscopy binding assay revealing that affinity and stoichiometry of CypA to the CA lattice was not affected by the substitutions. To test for functionality of the CypA secondary CA-binding site in HIV-1 infection, mutant CypA proteins were expressed in cells in which endogenous CypA was deleted, and the effects on HIV-1 infection were assayed. In normal HeLa-P4 cells, infection with HIV-1 bearing the A92E substitution in CA is inhibited by endogenous CypA and was inhibited to the same extent by expression of CypA mutants in CypA-null HeLa-P4 cells. Expression of the mutant CypA proteins in CypA-null Jurkat cells restored their permissiveness to infection by wild type HIV-1. Conclusions The amino acid changes at A25, K27, P29 and K30 did not affect the affinity of CypA for the CA lattice and did not impair CypA function in infection assays suggesting that these residues are not part of a secondary CA binding site on CypA.
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- 2019
- Full Text
- View/download PDF
4. Fluorescence Microscopy Assay to Measure HIV-1 Capsid Uncoating Kinetics in vitro
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Chantal Márquez, Derrick Lau, James Walsh, K. M. Rifat Faysal, Michael Parker, Stuart Turville, and Till Böcking
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Biology (General) ,QH301-705.5 - Abstract
The stability of the HIV-1 capsid and the spatiotemporal control of its disassembly, a process called uncoating, need to be finely tuned for infection to proceed. Biochemical methods for measuring capsid lattice disassembly in bulk are unable to resolve intermediates in the uncoating reaction. We have developed a single-particle fluorescence microscopy method to follow the real-time uncoating kinetics of authentic HIV capsids in vitro. The assay utilizes immobilized viral particles that are permeabilized with the a pore-former protein, and is designed to (1) detect the first defect of the capsid by the release of a solution phase marker (GFP) and (2) visualize the disassembly of the capsid over time by “painting” the capsid lattice with labeled cyclophilin A (CypA), a protein that binds weakly to the outside of the capsid. This novel assay allows the study of dynamic interactions of molecules with hundreds of individual capsids as well as to determine their effect on viral capsid stability, which provides a powerful tool for dissecting uncoating mechanisms and for the development of capsid-binding drugs.
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- 2019
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5. Kinetics of HIV-1 capsid uncoating revealed by single-molecule analysis
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Chantal L Márquez, Derrick Lau, James Walsh, Vaibhav Shah, Conall McGuinness, Andrew Wong, Anupriya Aggarwal, Michael W Parker, David A Jacques, Stuart Turville, and Till Böcking
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HIV-1 ,capsid disassembly ,fluorescence miscoscopy ,single-molecule ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
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.
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- 2018
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6. Single Molecule Fingerprinting Reveals Different Amplification Properties of α-Synuclein Oligomers and Preformed Fibrils in Seeding Assay
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Derrick Lau, Chloé Magnan, Kathryn Hill, Antony Cooper, Yann Gambin, and Emma Sierecki
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Amyloid ,Physiology ,Cognitive Neuroscience ,alpha-Synuclein ,Humans ,Biological Assay ,Parkinson Disease ,Cell Biology ,General Medicine ,Biochemistry ,Biomarkers - Abstract
The quantification of α-synuclein aggregates has emerged as a promising biomarker for synucleinopathies. Assays that amplify and detect such aggregates have revealed the presence of seeding-competent species in biosamples of patients diagnosed with Parkinson's disease. However, multiple species, such as oligomers and amyloid fibrils, are formed during the aggregation of α-synuclein; these species are likely to coexist in biological samples, and thus it remains unclear which species(s) are contributing to the signal detected in seeding assays. To identify individual contributions to the amplification process, recombinant oligomers and preformed fibrils were produced and purified to characterize their individual biochemical and seeding potential. Here, we used single molecule spectroscopy to track the formation and purification of oligomers and fibrils at the single particle level and compare their respective seeding potential in an amplification assay. Single molecule detection validates that size-exclusion chromatography efficiently separates oligomers from fibrils. Oligomers were found to be seeding-competent, but our results reveal that their seeding behavior is very different compared to that of preformed fibrils, in our amplification assay. Overall, our data suggest that even a low number of preformed fibrils present in biosamples is likely to dominate the response in seeding assays.
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- 2022
7. Single‐Molecule Counting Coupled to Rapid Amplification Enables Detection of α‐Synuclein Aggregates in Cerebrospinal Fluid of Parkinson's Disease Patients
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Chloe Magnan, Yann Gambin, Akshay Bhumkar, Derrick Lau, Eugene Soh Wei Jun, Emma Sierecki, and Nicolas Dzamko
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Adult ,Male ,isothermal amplification ,Parkinson's disease ,Amyloid ,Loop-mediated isothermal amplification ,Computational biology ,Protein aggregation ,Single‐Molecule Counting | Hot Paper ,010402 general chemistry ,01 natural sciences ,Catalysis ,Protein Aggregates ,chemistry.chemical_compound ,α-synuclein ,Cerebrospinal fluid ,Limit of Detection ,medicine ,Humans ,single-molecule counting ,Research Articles ,Aged ,Synucleinopathies ,010405 organic chemistry ,Chemistry ,Parkinson Disease ,Single molecule counting ,General Medicine ,General Chemistry ,Middle Aged ,medicine.disease ,Single Molecule Imaging ,0104 chemical sciences ,Biomarker (cell) ,alpha-Synuclein ,Biophysics ,Synuclein ,Female ,Thioflavin ,confocal spectroscopy ,Biomarkers ,Research Article - Abstract
α‐Synuclein aggregation is a hallmark of Parkinson's disease and a promising biomarker for early detection and assessment of disease progression. The prospect of a molecular test for Parkinson's disease is materializing with the recent developments of detection methods based on amplification of synuclein seeds (e.g. RT‐QuIC or PMCA). Here we adapted single‐molecule counting methods for the detection of α‐synuclein aggregates in cerebrospinal fluid (CSF), using a simple 3D printed microscope. Single‐molecule methods enable to probe the early events in the amplification process used in RT‐QuIC and a precise counting of ThT‐positive aggregates. Importantly, the use of single‐molecule counting also allows a refined characterization of the samples and fingerprinting of the protein aggregates present in CSF of patients. The fingerprinting of size and reactivity of individual aggregate shows a unique signature for each PD patients compared to controls and may provide new insights on synucleinopathies in the future., A new single‐molecule method was established for the detection of α‐synuclein fibrils, a promising biomarker of Parkinson's disease (PD). Individual ThT(+) aggregates were fingerprinted and amplified to improve detection limits by 3 orders of magnitude, compared to direct detection. A difference of behaviour was detected between PD and control samples.
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- 2021
8. Rapid HIV-1 Capsid Interaction Screening Using Fluorescence Fluctuation Spectroscopy
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Vaibhav Shah, Stuart Turville, James C. Walsh, Emma Sierecki, Till Böcking, David A. Jacques, Claire F. Dickson, Derrick Lau, Jeffrey H. Stear, Yann Gambin, Akshay Bhumkar, Dominic J. B. Hunter, and Andrew Tuckwell
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Analyte ,Binding Sites ,Chemistry ,viruses ,Ligand binding assay ,010401 analytical chemistry ,Cell ,Human immunodeficiency virus (HIV) ,010402 general chemistry ,medicine.disease_cause ,01 natural sciences ,Fluorescence ,0104 chemical sciences ,Analytical Chemistry ,Fluorescence intensity ,Capsid ,Spectrometry, Fluorescence ,medicine.anatomical_structure ,HIV-1 ,medicine ,Biophysics ,Capsid Proteins ,Binding site ,Spectroscopy - Abstract
The HIV capsid is a multifunctional protein capsule that mediates the delivery of the viral genetic material into the nucleus of the target cell. Host cell proteins bind to a number of repeating binding sites on the capsid to regulate steps in the replication cycle. Here, we develop a fluorescence fluctuation spectroscopy method using self-assembled capsid particles as the bait to screen for fluorescence-labeled capsid-binding analytes ("prey" molecules) in solution. The assay capitalizes on the property of the HIV capsid as a multivalent interaction platform, facilitating high sensitivity detection of multiple prey molecules that have accumulated onto capsids as spikes in fluorescence intensity traces. By using a scanning stage, we reduced the measurement time to 10 s without compromising on sensitivity, providing a rapid binding assay for screening libraries of potential capsid interactors. The assay can also identify interfaces for host molecule binding by using capsids with defects in known interaction interfaces. Two-color coincidence detection using the fluorescent capsid as the bait further allows the quantification of binding levels and determination of binding affinities. Overall, the assay provides new tools for the discovery and characterization of molecules used by the HIV capsid to orchestrate infection. The measurement principle can be extended for the development of sensitive interaction assays, utilizing natural or synthetic multivalent scaffolds as analyte-binding platforms.
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- 2021
9. Self-Assembly of Fluorescent HIV Capsid Spheres for Detection of Capsid Binders
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Nicholas Ariotti, David A. Jacques, Stuart Turville, Vaibhav Shah, Till Böcking, James C. Walsh, Derrick Lau, and Amir Mousapasandi
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Pentamer ,viruses ,HIV Infections ,Cypa ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Virus ,Cyclophilin A ,Capsid ,Electrochemistry ,Fluorescence microscope ,Humans ,General Materials Science ,Spectroscopy ,biology ,Chemistry ,Surfaces and Interfaces ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,biology.organism_classification ,Fluorescence ,0104 chemical sciences ,HIV-1 ,Biophysics ,Capsid Proteins ,0210 nano-technology ,Macromolecule - Abstract
The human immunodeficiency virus (HIV) capsid is a cone-shaped capsule formed from the viral capsid protein (CA), which is arranged into a lattice of hexamers and pentamers. The capsid comprises multiple binding interfaces for the recruitment of host proteins and macromolecules used by the virus to establish infection. Here, we coassembled CA proteins engineered for pentamer cross-linking and fluorescence labeling, into spherical particles. The CA spheres, which resemble the pentamer-rich structure of the end caps of the native HIV capsid, were immobilized onto surfaces as biorecognition elements for fluorescence microscopy-based quantification of host protein binding. The capsid-binding host protein cyclophilin A (CypA) is bound to CA spheres with the same affinity as CA tubes but at a higher CypA/CA stoichiometry, suggesting that the level of recruitment of CypA to the HIV capsid is dependent on curvature.
- Published
- 2020
10. Recognition of HIV-1 Capsid Licenses Innate Immune Response to Viral Infection
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Sunnie M Yoh, João I. Mamede, Derrick Lau, Narae Ahn, Maria T Sánchez-Aparicio, Joshua Temple, Andrew Tuckwell, Nina V. Fuchs, Gianguido C. Cianci, Laura Riva, Heather Curry, Xin Yin, Stéphanie Gambut, Lacy M. Simons, Judd F. Hultquist, Renate König, Yong Xiong, Adolfo García-Sastre, Till Böcking, Thomas J. Hope, and Sumit K. Chanda
- Abstract
SUMMARYCyclic GMP-AMP synthase (cGAS) is a primary sensor of aberrant DNA that governs an innate immune signaling cascade, leading to the induction of the type-I interferon response. We have previously identified polyglutamine binding protein 1, PQBP1, as an adaptor molecule required for cGAS-mediated innate immune response of lentiviruses, including the human immunodeficiency virus 1 (HIV-1), but dispensable for the recognition of DNA viruses. HIV-1- encoded DNA is synthesized as a single copy from its RNA genome, and is subsequently integrated into the host chromatin. HIV-1 then produces progeny through amplification and packaging of its RNA genome, thus, in contrast to DNA viruses, HIV-1 DNA is both transient and of low abundance. However, the molecular basis for the detection and verification of this low abundance HIV-1 DNA pathogen-associated molecular pattern (PAMP) is not understood. Here, we elucidate a two-factor authentication strategy that is employed by the innate immune surveillance machinery to selectively respond to the low concentration of PAMP, while discerning these species from extranuclear DNA molecules. We find that, upon HIV-1 infection, PQBP1 decorates intact viral capsid, which serves as a primary verification step for the viral nucleic acid cargo. As the reverse transcription and capsid disassembly initiate, cGAS protein is then recruited to the capsid in a PQBP1-dependent manner, enabling cGAS molecules to be co-positioned at the site of PAMP generation. Thus, these data indicate that PQBP1 recognition of the HIV-1 capsid sanctions a robust cGAS-dependent response to a limited abundance and short-lived DNA PAMP. Critically, this illuminates a molecular strategy wherein the modular recruitment of co-factors to germline encoded pattern recognition receptors (PRRs) serves to enhance repertoire of pathogens that can be sensed by the innate immune surveillance machinery.
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- 2022
11. Recognition of HIV-1 capsid by PQBP1 licenses an innate immune sensing of nascent HIV-1 DNA
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Sunnie M. Yoh, João I. Mamede, Derrick Lau, Narae Ahn, Maria T. Sánchez-Aparicio, Joshua Temple, Andrew Tuckwell, Nina V. Fuchs, Gianguido C. Cianci, Laura Riva, Heather Curry, Xin Yin, Stéphanie Gambut, Lacy M. Simons, Judd F. Hultquist, Renate König, Yong Xiong, Adolfo García-Sastre, Till Böcking, Thomas J. Hope, and Sumit K. Chanda
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DNA-Binding Proteins ,Capsid ,Pathogen-Associated Molecular Pattern Molecules ,HIV-1 ,Humans ,Cell Biology ,DNA ,Molecular Biology ,Nucleotidyltransferases ,Immunity, Innate - Abstract
We have previously described polyglutamine-binding protein 1 (PQBP1) as an adapter required for the cyclic GMP-AMP synthase (cGAS)-mediated innate response to the human immunodeficiency virus 1 (HIV-1) and other lentiviruses. Cytoplasmic HIV-1 DNA is a transient and low-abundance pathogen-associated molecular pattern (PAMP), and the mechanism for its detection and verification is not fully understood. Here, we show a two-factor authentication strategy by the innate surveillance machinery to selectively respond to the low concentration of HIV-1 DNA, while distinguishing these species from extranuclear DNA molecules. We find that, upon HIV-1 infection, PQBP1 decorates the intact viral capsid, and this serves as a primary verification step for the viral nucleic acid cargo. As reverse transcription and capsid disassembly initiate, cGAS is recruited to the capsid in a PQBP1-dependent manner. This positions cGAS at the site of PAMP generation and sanctions its response to a low-abundance DNA PAMP.
- Published
- 2021
12. Single molecule fingerprinting reveals different amplification properties of α-synuclein oligomers and preformed fibrils in seeding assay
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Kathryn J. Hill, Emma Sierecki, Antony A. Cooper, Yann Gambin, Derrick Lau, and Chloe Magnan
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Synucleinopathies ,Chemistry ,law ,Biophysics ,Recombinant DNA ,food and beverages ,Molecule ,α synuclein ,Seeding ,Multiple species ,Fibril ,Single Molecule Spectroscopy ,law.invention - Abstract
The quantification of α-synuclein (α-syn) aggregates has emerged as a promising biomarker for synucleinopathies. Assays that amplify and detect such aggregates have revealed the presence of seeding-competent species in biosamples of patients diagnosed with Parkinson’s disease. However, multiple species such as oligomers and amyloid fibrils, are formed during the aggregation of α-synuclein and these species are likely to co-exist in biological samples and thus it remains unclear which species(s) are contributing to the signal detected in seeding assays. To identify which species can be detected in seeding assays, recombinant oligomers and preformed fibrils were produced and purified to characterise their individual biochemical and seeding potential. Here, we used single molecule spectroscopy to track the formation and purification of oligomers and fibrils at the single particle level and compare their respective seeding potential in an amplification assay. Single molecule detection validates that size-exclusion chromatography efficiently separates oligomers from fibrils. Oligomers were found to be seeding-competent but our results reveal that their seeding behaviour is very different compared to preformed fibrils in our amplification assay. Overall, our data suggest that even a low number of preformed fibrils present in biosamples are likely to dominate the response in seeding assays.
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- 2021
13. Functional analysis of the secondary HIV-1 capsid binding site in the host protein cyclophilin A
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Chang H Byeon, Christopher Aiken, Derrick Lau, K. M. Rifat Faysal, James C. Walsh, Wang Peng, Chantal L. Márquez, Jiong Shi, In-Ja L. Byeon, and Till Böcking
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lcsh:Immunologic diseases. Allergy ,Cypa ,Non-canonical binding site ,Virus Replication ,03 medical and health sciences ,Cyclophilin A ,Jurkat Cells ,Protein structure ,Capsid ,Virology ,Humans ,Binding site ,Amino Acids ,030304 developmental biology ,Peptidylprolyl isomerase ,0303 health sciences ,Binding Sites ,biology ,Host Microbial Interactions ,030306 microbiology ,Chemistry ,Ligand binding assay ,Research ,Wild type ,Virion ,biology.organism_classification ,3. Good health ,Cell biology ,Infectious Diseases ,HIV-1 ,Capsid Proteins ,lcsh:RC581-607 ,HeLa Cells ,Protein Binding - Abstract
Background Efficient HIV-1 replication depends on interaction of the viral capsid with the host protein cyclophilin A (CypA). CypA, a peptidylprolyl isomerase, binds to an exposed loop in the viral CA protein via the enzyme’s active site. Recent structural analysis of CypA in complex with CA tubes in conjunction with molecular dynamics simulations identified a secondary CA binding site on CypA that allows a bridging interaction with two hexameric subunits of the assembled CA lattice, leading to capsid stabilization (Liu et al. in Nat Commun 7:10714, 2016). Results We performed mutational analysis of residues that have been proposed to mediate CA binding at the secondary binding site on CypA (A25, K27, P29 and K30) and tested the effects of the amino acid substitutions using interaction assays and HIV-1 infection assays in cells. The binding of recombinant CypA to self-assembled CA tubes or native HIV-1 capsids was measured in vitro using a quantitative fluorescence microscopy binding assay revealing that affinity and stoichiometry of CypA to the CA lattice was not affected by the substitutions. To test for functionality of the CypA secondary CA-binding site in HIV-1 infection, mutant CypA proteins were expressed in cells in which endogenous CypA was deleted, and the effects on HIV-1 infection were assayed. In normal HeLa-P4 cells, infection with HIV-1 bearing the A92E substitution in CA is inhibited by endogenous CypA and was inhibited to the same extent by expression of CypA mutants in CypA-null HeLa-P4 cells. Expression of the mutant CypA proteins in CypA-null Jurkat cells restored their permissiveness to infection by wild type HIV-1. Conclusions The amino acid changes at A25, K27, P29 and K30 did not affect the affinity of CypA for the CA lattice and did not impair CypA function in infection assays suggesting that these residues are not part of a secondary CA binding site on CypA. Electronic supplementary material The online version of this article (10.1186/s12977-019-0471-4) contains supplementary material, which is available to authorized users.
- Published
- 2019
14. Fluorescence biosensor for real-time interaction dynamics of host proteins with HIV-1 capsid tubes
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Derrick Lau, Stuart Turville, David A. Jacques, Vaibhav Shah, Till Böcking, Wang Peng, and James C. Walsh
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Materials science ,viruses ,Human immunodeficiency virus (HIV) ,Biosensing Techniques ,medicine.disease_cause ,03 medical and health sciences ,Capsid ,0302 clinical medicine ,Viral life cycle ,Lab-On-A-Chip Devices ,Microscopy ,medicine ,Humans ,General Materials Science ,Interaction dynamics ,030223 otorhinolaryngology ,Host protein ,030304 developmental biology ,0303 health sciences ,Total internal reflection fluorescence microscope ,Chemistry ,Fluorescence biosensor ,Single-molecule experiment ,Fluorescence ,Small molecule ,3. Good health ,Microscopy, Fluorescence ,HIV-1 ,Biophysics ,Biosensor ,030217 neurology & neurosurgery - Abstract
The human immunodeficiency virus 1 (HIV-1) capsid serves as a binding platform for proteins and small molecules from the host cell that regulate various steps in the virus life cycle. However, there are currently no quantitative methods that use assembled capsid lattices for measuring host-pathogen interaction dynamics. Here we developed a single molecule fluorescence biosensor using self-assembled capsid tubes as biorecognition elements and imaged capsid binders using total internal reflection fluorescence microscopy in a microfluidic setup. The method is highly sensitive in its ability to observe and quantify binding, obtain dissociation constants, extract kinetics with an extended application of using more complex analytes that can accelerate characterisation of novel capsid binders.
- Published
- 2019
15. Kinetics of HIV-1 capsid uncoating revealed by single-molecule analysis
- Author
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David A. Jacques, Vaibhav Shah, Conall McGuinness, Andrew Wong, Stuart Turville, Chantal L. Márquez, James C. Walsh, Derrick Lau, Anupriya Aggarwal, Till Böcking, and Michael W. Parker
- Subjects
0301 basic medicine ,QH301-705.5 ,Science ,viruses ,Protein subunit ,030106 microbiology ,Plasma protein binding ,General Biochemistry, Genetics and Molecular Biology ,Virus ,03 medical and health sciences ,Biology (General) ,General Immunology and Microbiology ,Chemistry ,General Neuroscience ,HEK 293 cells ,capsid disassembly ,General Medicine ,biochemical phenomena, metabolism, and nutrition ,Virology ,Small molecule ,Reverse transcriptase ,3. Good health ,030104 developmental biology ,Capsid ,Structural biology ,HIV-1 ,fluorescence miscoscopy ,Medicine ,single-molecule - Abstract
Viruses need to enter their host’s cells in order to replicate their genetic material and produce more copies of the virus. A protein shell called a capsid protects the virus during this journey. But the structure of the capsid presents a mystery. How can this protein shell be strong enough to remain intact as it enters a host cell, and yet quickly open up to release the viral genome after replication? Unlike the capsids of many other viruses, those of HIV have irregular structures that rapidly fall apart once removed from the virus. This has thwarted attempts to study intact HIV capsids in order to understand how they work. However, we do know that HIV hijacks a range of molecules produced by the invaded host cell. Dissecting their effects on the capsid is key to understanding how the capsid disassembles. Marquez et al. have now developed a method that can visualize individual HIV capsids – and how they disassemble – in real time using single-molecule microscopy. This revealed that capsids differ widely in their stability. The shell remains closed for a variable period of time and then collapses catastrophically as soon as it loses its first subunit. Using the new technique, Marquez et al. also found that a small molecule drug called PF74 causes the capsid to crack open rapidly, but the remaining shell is then stabilized against further disassembly. These observations reconcile seemingly contradictory observations made by different research groups about how this drug affects the stability of the capsid. The method developed by Marquez et al. enables researchers to measure how molecules produced by host cells interact with the viral capsid, a structure that is fundamental for the virus to establish an infection. It could also be used to test the effects of antiviral drugs that have been designed to target the capsid. The new technique has already been instrumental in related research by Mallery et al., which identifies a molecule found in host cells that stabilizes the capsid of HIV.
- Published
- 2018
16. Author response: Kinetics of HIV-1 capsid uncoating revealed by single-molecule analysis
- Author
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David A. Jacques, Michael W. Parker, Stuart Turville, James C. Walsh, Derrick Lau, Conall McGuinness, Chantal L. Márquez, Vaibhav Shah, Anupriya Aggarwal, Till Böcking, and Andrew Wong
- Subjects
Capsid ,Chemistry ,Kinetics ,Human immunodeficiency virus (HIV) ,medicine ,Molecule ,medicine.disease_cause ,Virology - Published
- 2018
17. Negative Staining Transmission Electron Microscopy of HIV Viral Particles Permeabilized with PFO and Capsid Stabilized with IP6
- Author
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Derrick Lau, Chantal Márquez, Michael Parker, and Till Böcking
- Subjects
General Immunology and Microbiology ,General Neuroscience ,Methods Article ,Plant Science ,General Biochemistry, Genetics and Molecular Biology - Abstract
The human immunodeficiency virus 1 (HIV-1) consists of a viral membrane surrounding the conical capsid. The capsid is a protein container assembled from approximately 1,500 copies of the viral capsid protein (CA), functioning as a reaction and transport chamber for the viral genome after cell entry. Transmission electron microscopy (TEM) is a widely used technique for characterizing the ultrastructure of isolated viral capsids after removal of the viral membrane, which otherwise hinders negative staining of structures inside the viral particle for TEM. Here, we provide a protocol to permeabilize the membrane of HIV-1 particles using a pore-forming toxin for negative staining of capsids, which are stabilized with inositol hexakisphosphate to prevent premature capsid disassembly. This approach revealed the pleomorphic nature of capsids with a partially intact membrane surrounding them. The permeabilization strategy using pore-forming toxins can be readily applied to visualize the internal architecture of other enveloped viruses using TEM. Graphical abstract.
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