99 results on '"Böcking, T"'
Search Results
2. A lysine ring in HIV capsid pores coordinates IP6 to drive mature capsid assembly
- Author
-
Renner, N, Mallery, DL, Faysal, KMR, Peng, W, Jacques, DA, Böcking, T, James, LC, Renner, N, Mallery, DL, Faysal, KMR, Peng, W, Jacques, DA, Böcking, T, and James, LC
- Abstract
The HIV capsid self-assembles a protective conical shell that simultaneously prevents host sensing whilst permitting the import of nucleotides to drive DNA synthesis. This is accomplished through the construction of dynamic, highly charged pores at the centre of each capsid multimer. The clustering of charges required for dNTP import is strongly destabilising and it is proposed that HIV uses the metabolite IP6 to coordinate the pore during assembly. Here we have investigated the role of inositol phosphates in coordinating a ring of positively charged lysine residues (K25) that forms at the base of the capsid pore. We show that whilst IP5, which can functionally replace IP6, engages an arginine ring (R18) at the top of the pore, the lysine ring simultaneously binds a second IP5 molecule. Dose dependent removal of K25 from the pore severely inhibits HIV infection and concomitantly prevents DNA synthesis. Cryo-tomography reveals that K25A virions have a severe assembly defect that inhibits the formation of mature capsid cones. Monitoring both the kinetics and morphology of capsids assembled in vitro reveals that while mutation K25A can still form tubes, the ability of IP6 to drive assembly of capsid cones has been lost. Finally, in single molecule TIRF microscopy experiments, capsid lattices in permeabilised K25 mutant virions are rapidly lost and cannot be stabilised by IP6. These results suggest that the coordination of IP6 by a second charged ring in mature hexamers drives the assembly of conical capsids capable of reverse transcription and infection.
- Published
- 2021
3. A stable immature lattice packages IP6 for HIV capsid maturation
- Author
-
Mallery, DL, Kleinpeter, AB, Renner, N, Rifat Faysal, KM, Novikova, M, Kiss, L, Wilson, MSC, Ahsan, B, Ke, Z, Briggs, JAG, Saiardi, A, Böcking, T, Freed, EO, James, LC, Mallery, DL, Kleinpeter, AB, Renner, N, Rifat Faysal, KM, Novikova, M, Kiss, L, Wilson, MSC, Ahsan, B, Ke, Z, Briggs, JAG, Saiardi, A, Böcking, T, Freed, EO, and James, LC
- Abstract
HIV virion assembly begins with the construction of an immature lattice consisting of Gag hexamers. Upon virion release, protease-mediated Gag cleavage leads to a maturation event in which the immature lattice disassembles and the mature capsid assembles. The cellular metabolite inositiol hexakisphosphate (IP6) and maturation inhibitors (MIs) both bind and stabilize immature Gag hexamers, but whereas IP6 promotes virus maturation, MIs inhibit it. Here we show that HIV is evolutionarily constrained to maintain an immature lattice stability that ensures IP6 packaging without preventing maturation. Replication-deficient mutant viruses with reduced IP6 recruitment display increased infectivity upon treatment with the MI PF46396 (PF96) or the acquisition of second-site compensatory mutations. Both PF96 and second-site mutations stabilise the immature lattice and restore IP6 incorporation, suggesting that immature lattice stability and IP6 binding are interdependent. This IP6 dependence suggests that modifying MIs to compete with IP6 for Gag hexamer binding could substantially improve MI antiviral potency.
- Published
- 2021
4. Rapid HIV-1 capsid interaction screening using fluorescence fluctuation spectroscopy
- Author
-
Böcking, T, Jacques, DA, Lau, D, Walsh, JC, Dickson, CF, Tuckwell, A, Stear, JH, Hunter, DJB, Bhumkar, A, Shah, V, Turville, SG, Sierecki, E, Gambin, Y, Böcking, T, Jacques, DA, Lau, D, Walsh, JC, Dickson, CF, Tuckwell, A, Stear, JH, Hunter, DJB, Bhumkar, A, Shah, V, Turville, SG, Sierecki, E, and Gambin, Y
- 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.
- Published
- 2021
5. Tenth scientific biennial meeting of the australasian virology society - AVS10 2019
- Author
-
Helbig, KJ, Bull, RA, Ambrose, R, Beard, MR, Blanchard, H, Böcking, T, Chua, B, Colmant, AMG, Crosse, KM, Purcell, DFJ, Fraser, J, Hayward, JA, Hamilton, ST, Husain, M, MacDiarmid, R, MacKenzie, JM, Moseley, GW, Nguyen, THO, Quiñones-Mateu, ME, Robinson, K, Rodrigo, C, Rodriguez-Andres, J, Rudd, PA, Werno, A, White, P, Young, P, Speck, P, Hibma, M, Drummer, HE, Tachedjian, G, Helbig, KJ, Bull, RA, Ambrose, R, Beard, MR, Blanchard, H, Böcking, T, Chua, B, Colmant, AMG, Crosse, KM, Purcell, DFJ, Fraser, J, Hayward, JA, Hamilton, ST, Husain, M, MacDiarmid, R, MacKenzie, JM, Moseley, GW, Nguyen, THO, Quiñones-Mateu, ME, Robinson, K, Rodrigo, C, Rodriguez-Andres, J, Rudd, PA, Werno, A, White, P, Young, P, Speck, P, Hibma, M, Drummer, HE, and Tachedjian, G
- Abstract
The Australasian Virology Society (AVS) aims to promote, support and advocate for the discipline of virology in the Australasian region. The society was incorporated in 2011 after 10 years operating as the Australian Virology Group (AVG) founded in 2001, coinciding with the inaugural biennial scientific meeting. AVS conferences aim to provide a forum for the dissemination of all aspects of virology, foster collaboration, and encourage participation by students and post-doctoral researchers. The tenth Australasian Virology Society (AVS10) scientific meeting was held on 2-5 December 2019 in Queenstown, New Zealand. This report highlights the latest research presented at the meeting, which included cutting-edge virology presented by our international plenary speakers Ana Fernandez-Sesma and Benjamin tenOever, and keynote Richard Kuhn. AVS10 honoured female pioneers in Australian virology, Lorena Brown and Barbara Coulson. We report outcomes from the AVS10 career development session on "Successfully transitioning from post-doc to lab head", winners of best presentation awards, and the AVS gender equity policy, initiated in 2013. Plans for the 2021 meeting are underway which will celebrate the 20th anniversary of AVS where it all began, in Fraser Island, Queensland, Australia.
- Published
- 2020
6. Self-Assembly of Fluorescent HIV Capsid Spheres for Detection of Capsid Binders
- Author
-
Lau, D, Walsh, JC, Mousapasandi, A, Ariotti, N, Shah, VB, Turville, S, Jacques, DA, Böcking, T, Lau, D, Walsh, JC, Mousapasandi, A, Ariotti, N, Shah, VB, Turville, S, Jacques, DA, and Böcking, T
- Abstract
© 2020 American Chemical Society. 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
7. Functional analysis of the secondary HIV-1 capsid binding site in the host protein cyclophilin A
- Author
-
Peng, W, Shi, J, Márquez, CL, Lau, D, Walsh, J, Faysal, KMR, Byeon, CH, Byeon, IJL, Aiken, C, Böcking, T, Peng, W, Shi, J, Márquez, CL, Lau, D, Walsh, J, Faysal, KMR, Byeon, CH, Byeon, IJL, Aiken, C, and Böcking, T
- 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.
- Published
- 2019
8. Molecular integration of the anti-tropomyosin compound ATM-3507 into the coiled coil overlap region of the cancer-associated Tpm3.1
- Author
-
Janco, M, Rynkiewicz, MJ, Li, L, Hook, J, Eiffe, E, Ghosh, A, Böcking, T, Lehman, WJ, Hardeman, EC, Gunning, PW, Janco, M, Rynkiewicz, MJ, Li, L, Hook, J, Eiffe, E, Ghosh, A, Böcking, T, Lehman, WJ, Hardeman, EC, and Gunning, PW
- Abstract
Tropomyosins (Tpm) determine the functional capacity of actin filaments in an isoform-specific manner. The primary isoform in cancer cells is Tpm3.1 and compounds that target Tpm3.1 show promising results as anti-cancer agents both in vivo and in vitro. We have determined the molecular mechanism of interaction of the lead compound ATM-3507 with Tpm3.1-containing actin filaments. When present during co-polymerization of Tpm3.1 with actin, 3H-ATM-3507 is incorporated into the filaments and saturates at approximately one molecule per Tpm3.1 dimer and with an apparent binding affinity of approximately 2 µM. In contrast, 3H-ATM-3507 is poorly incorporated into preformed Tpm3.1/actin co-polymers. CD spectroscopy and thermal melts using Tpm3.1 peptides containing the C-terminus, the N-terminus, and a combination of the two forming the overlap junction at the interface of adjacent Tpm3.1 dimers, show that ATM-3507 shifts the melting temperature of the C-terminus and the overlap junction, but not the N-terminus. Molecular dynamic simulation (MDS) analysis predicts that ATM-3507 integrates into the 4-helix coiled coil overlap junction and in doing so, likely changes the lateral movement of Tpm3.1 across the actin surface resulting in an alteration of filament interactions with actin binding proteins and myosin motors, consistent with the cellular impact of ATM-3507.
- Published
- 2019
9. Parallel assembly of actin and tropomyosin, but not myosin II, during de novo actin filament formation in live mice
- Author
-
Masedunskas, A, Appaduray, MA, Lucas, CA, Cagigas, ML, Heydecker, M, Holliday, M, Meiring, JCM, Hook, J, Kee, A, White, M, Thomas, P, Zhang, Y, Adelstein, RS, Meckel, T, Böcking, T, Weigert, R, Bryce, NS, Gunning, PW, Hardeman, EC, Masedunskas, A, Appaduray, MA, Lucas, CA, Cagigas, ML, Heydecker, M, Holliday, M, Meiring, JCM, Hook, J, Kee, A, White, M, Thomas, P, Zhang, Y, Adelstein, RS, Meckel, T, Böcking, T, Weigert, R, Bryce, NS, Gunning, PW, and Hardeman, EC
- Abstract
Many actin filaments in animal cells are co-polymers of actin and tropomyosin. In many cases, non-muscle myosin II associates with these co-polymers to establish a contractile network. However, the temporal relationship of these three proteins in the de novo assembly of actin filaments is not known. Intravital subcellular microscopy of secretory granule exocytosis allows the visualisation and quantification of the formation of an actin scaffold in real time, with the added advantage that it occurs in a living mammal under physiological conditions. We used this model system to investigate the de novo assembly of actin, tropomyosin Tpm3.1 (a short isoform of TPM3) and myosin IIA (the form of non-muscle myosin II with its heavy chain encoded by Myh9) on secretory granules in mouse salivary glands. Blocking actin polymerization with cytochalasin D revealed that Tpm3.1 assembly is dependent on actin assembly. We used time-lapse imaging to determine the timing of the appearance of the actin filament reporter LifeAct-RFP and of Tpm3.1-mNeonGreen on secretory granules in LifeAct-RFP transgenic, Tpm3.1- mNeonGreen and myosin IIA-GFP (GFP-tagged MYH9) knock-in mice. Our findings are consistent with the addition of tropomyosin to actin filaments shortly after the initiation of actin filament nucleation, followed by myosin IIA recruitment.
- Published
- 2018
10. Ultralow- and Low-Background Surfaces for Single-Molecule Localization Microscopy of Multistep Biointerfaces for Single-Molecule Sensing
- Author
-
Zhao, M, Nicovich, PR, Janco, M, Deng, Q, Yang, Z, Ma, Y, Böcking, T, Gaus, K, Gooding, JJ, Zhao, M, Nicovich, PR, Janco, M, Deng, Q, Yang, Z, Ma, Y, Böcking, T, Gaus, K, and Gooding, JJ
- Abstract
Single-molecule localization microscopy (SMLM) has created the opportunity of pushing fluorescence microscopy from being a biological imaging tool to a surface characterization and possibly even a quantitative analytical tool. The latter could be achieved by molecular counting using pointillist SMLM data sets. However, SMLM is especially sensitive to background fluorescent signals, which influences any subsequent analysis. Therefore, fabricating sensing surfaces that resist nonspecific adsorption of proteins, even after multiple modification steps, has become paramount. Herein is reported two different ways to modify surfaces: dichlorodimethylsilane-biotinylated bovine serum albumin-Tween-20 (DbT20) and poly-l-lysine grafted polyethylene glycol (PLL-PEG) mixed with biotinylated PLL-PEG (PLL-PEG/PEGbiotin). The results show that the ability to resist nonspecific adsorption of DbT20 surfaces deteriorates with an increase in the number of modification steps required after the addition of the DbT20, which limits the applicability of this surface for SMLM. As such, a new surface for SMLM that employs PLL-PEG/PEGbiotin was developed that exhibits ultralow amounts of nonspecific protein adsorption even after many modification steps. The utility of the surface was demonstrated for human influenza hemagglutinin-tagged mEos2, which was directly pulled down from cell lysates onto the PLL-PEG/PEGbiotin surface. The results strongly indicated that the PLL-PEG/PEGbiotin surface satisfies the criteria of SMLM imaging of a negligible background signal and negligible nonspecific adsorption.
- Published
- 2018
11. Interactions of tropomyosin Tpm1.1 on a single actin filament: A method for extraction and processing of high resolution TIRF microscopy data
- Author
-
Janco, M, Böcking, T, He, S, Coster, ACF, Janco, M, Böcking, T, He, S, and Coster, ACF
- Abstract
Skeletal muscle tropomyosin (Tpm1.1) is an elongated, rod-shaped, alpha-helical coiled-coil protein that forms continuous head-to-tail polymers along both sides of the actin filament. In this study we use single molecule fluorescence TIRF microscopy combined with a microfluidic device and fluorescently labelled proteins to measure Tpm1.1 association to and dissociation from single actin filaments. Our experimental setup allows us to clearly resolve Tpm1.1 interactions on both sides of the filaments. Here we provide a semi-automated method for the extraction and quantification of kymograph data for individual actin filaments bound at different Tpm1.1 concentrations. We determine boundaries on the kymograph on each side of the actin filament, based on intensity thresholding, performing fine manual editing of the boundaries (if needed) and extracting user defined kinetic properties of the system. Using our analytical tools we can determine (i) nucleation point(s) and rates, (ii) elongation rates of Tpm1.1, (iii) identify meeting points after the saturation of filament, and when dissociation occurs, (iv) initiation point(s), (v) the final dissociation point(s), as well as (vi) dissociation rates. All of these measurements can be extracted from both sides of the filament, allowing for the determination of possible differences in behaviour on the two sides of the filament, and across concentrations. The robust and repeatable nature of the method allows quantitative, semi-automated analyses to be made of large studies of acto-tropomyosin interactions, as well as for other actin binding proteins or filamentous structures, opening the way for dissection of the dynamics underlying these interactions.
- Published
- 2018
12. NicoLase - An open-source diode laser combiner, fiber launch, and sequencing controller for fluorescence microscopy
- Author
-
Nicovich, PR, Walsh, J, Böcking, T, Gaus, K, Nicovich, PR, Walsh, J, Böcking, T, and Gaus, K
- Abstract
© 2017 Nicovich et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Modern fluorescence microscopy requires software-controlled illumination sources with high power across a wide range of wavelengths. Diode lasers meet the power requirements and combining multiple units into a single fiber launch expands their capability across the required spectral range. We present the NicoLase, an open-source diode laser combiner, fiber launch, and software sequence controller for fluorescence microscopy and super-resolution microscopy applications. Two configurations are described, giving four or six output wavelengths and one or two single-mode fiber outputs, with all CAD files, machinist drawings, and controller source code openly available.
- Published
- 2017
13. Huntingtin Inclusions Trigger Cellular Quiescence, Deactivate Apoptosis, and Lead to Delayed Necrosis
- Author
-
Ramdzan, YM, Trubetskov, MM, Ormsby, AR, Newcombe, EA, Sui, X, Tobin, MJ, Bongiovanni, MN, Gras, SL, Dewson, G, Miller, JML, Finkbeiner, S, Moily, NS, Niclis, J, Parish, CL, Purcell, AW, Baker, MJ, Wilce, JA, Waris, S, Stojanovski, D, Böcking, T, Ang, CS, Ascher, DB, Reid, GE, Hatters, DM, Ramdzan, YM, Trubetskov, MM, Ormsby, AR, Newcombe, EA, Sui, X, Tobin, MJ, Bongiovanni, MN, Gras, SL, Dewson, G, Miller, JML, Finkbeiner, S, Moily, NS, Niclis, J, Parish, CL, Purcell, AW, Baker, MJ, Wilce, JA, Waris, S, Stojanovski, D, Böcking, T, Ang, CS, Ascher, DB, Reid, GE, and Hatters, DM
- Abstract
© 2017 The Authors Competing models exist in the literature for the relationship between mutant Huntingtin exon 1 (Httex1) inclusion formation and toxicity. In one, inclusions are adaptive by sequestering the proteotoxicity of soluble Httex1. In the other, inclusions compromise cellular activity as a result of proteome co-aggregation. Using a biosensor of Httex1 conformation in mammalian cell models, we discovered a mechanism that reconciles these competing models. Newly formed inclusions were composed of disordered Httex1 and ribonucleoproteins. As inclusions matured, Httex1 reconfigured into amyloid, and other glutamine-rich and prion domain-containing proteins were recruited. Soluble Httex1 caused a hyperpolarized mitochondrial membrane potential, increased reactive oxygen species, and promoted apoptosis. Inclusion formation triggered a collapsed mitochondrial potential, cellular quiescence, and deactivated apoptosis. We propose a revised model where sequestration of soluble Httex1 inclusions can remove the trigger for apoptosis but also co-aggregate other proteins, which curtails cellular metabolism and leads to a slow death by necrosis.
- Published
- 2017
14. Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence
- Author
-
Sierecki, E, Giles, N, Bowden, Q, Polinkovsky, ME, Steinbeck, J, Arrioti, N, Rahman, D, Bhumkar, A, Nicovich, PR, Ross, I, Parton, RG, Böcking, T, Gambin, Y, Sierecki, E, Giles, N, Bowden, Q, Polinkovsky, ME, Steinbeck, J, Arrioti, N, Rahman, D, Bhumkar, A, Nicovich, PR, Ross, I, Parton, RG, Böcking, T, and Gambin, Y
- Abstract
Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer's and Parkinson's disease. Parkinson's disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson's disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils.
- Published
- 2016
15. The impact of tropomyosins on actin filament assembly is isoform specific
- Author
-
Janco, M, Bonello, TT, Byun, A, Coster, ACF, Lebhar, H, Dedova, I, Gunning, PW, Böcking, T, Janco, M, Bonello, TT, Byun, A, Coster, ACF, Lebhar, H, Dedova, I, Gunning, PW, and Böcking, T
- Abstract
Tropomyosin (Tpm) is an α helical coiled-coil dimer that forms a co-polymer along the actin filament. Tpm is involved in the regulation of actin's interaction with binding proteins as well as stabilization of the actin filament and its assembly kinetics. Recent studies show that multiple Tpm isoforms also define the functional properties of distinct actin filament populations within a cell. Subtle structural variations within well conserved Tpm isoforms are the key to their functional specificity. Therefore, we purified and characterized a comprehensive set of 8 Tpm isoforms (Tpm1.1, Tpm1.12, Tpm1.6, Tpm1.7, Tpm1.8, Tpm2.1, Tpm3.1, and Tpm4.2), using well-established actin co-sedimentation and pyrene fluorescence polymerization assays. We observed that the apparent affinity (Kd(app)) to filamentous actin varied in all Tpm isoforms between ∼0.1–5 μM with similar values for both, skeletal and cytoskeletal actin filaments. The data did not indicate any correlation between affinity and size of Tpm molecules, however high molecular weight (HMW) isoforms Tpm1.1, Tpm1.6, Tpm1.7 and Tpm2.1, showed ∼3-fold higher cooperativity compared to low molecular weight (LMW) isoforms Tpm1.12, Tpm1.8, Tpm3.1, and Tpm4.2. The rate of actin filament elongation in the presence of Tpm2.1 increased, while all other isoforms decreased the elongation rate by 27–85 %. Our study shows that the biochemical properties of Tpm isoforms are finely tuned and depend on sequence variations in alternatively spliced regions of Tpm molecules.
- Published
- 2016
16. Effect of surface chemistry on tropomyosin binding to actin filaments on surfaces
- Author
-
Nicovich, PR, Janco, M, Sobey, T, Gajwani, M, Obeidy, P, Whan, R, Gaus, K, Gunning, PW, Coster, ACF, Böcking, T, Nicovich, PR, Janco, M, Sobey, T, Gajwani, M, Obeidy, P, Whan, R, Gaus, K, Gunning, PW, Coster, ACF, and Böcking, T
- Abstract
Reconstitution of actin filaments on surfaces for observation of filament-associated protein dynamics by fluorescence microscopy is currently an exciting field in biophysics. Here we examine the effects of attaching actin filaments to surfaces on the binding and dissociation kinetics of a fluorescence-labeled tropomyosin, a rod-shaped protein that forms continuous strands wrapping around the actin filament. Two attachment modalities of the actin to the surface are explored: where the actin filament is attached to the surface at multiple points along its length; and where the actin filament is attached at one end and aligned parallel to the surface by buffer flow. To facilitate analysis of actin-binding protein dynamics, we have developed a software tool for the viewing, tracing and analysis of filaments and co-localized species in noisy fluorescence timelapse images. Our analysis shows that the interaction of tropomyosin with actin filaments is similar for both attachment modalities. © 2016 Wiley Periodicals, Inc.
- Published
- 2016
17. A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics
- Author
-
Bonello, TT, Janco, M, Hook, J, Byun, A, Appaduray, M, Dedova, I, Hitchcock-DeGregori, S, Hardeman, EC, Stehn, JR, Böcking, T, Gunning, PW, Bonello, TT, Janco, M, Hook, J, Byun, A, Appaduray, M, Dedova, I, Hitchcock-DeGregori, S, Hardeman, EC, Stehn, JR, Böcking, T, and Gunning, PW
- Abstract
The tropomyosin family of proteins form end-to-end polymers along the actin filament. Tumour cells rely on specific tropomyosin-containing actin filament populations for growth and survival. To dissect out the role of tropomyosin in actin filament regulation we use the small molecule TR100 directed against the C terminus of the tropomyosin isoform Tpm3.1. TR100 nullifies the effect of Tpm3.1 on actin depolymerisation but surprisingly Tpm3.1 retains the capacity to bind F-actin in a cooperative manner. In vivo analysis also confirms that, in the presence of TR100, fluorescently tagged Tpm3.1 recovers normally into stress fibers. Assembling end-to-end along the actin filament is thereby not sufficient for tropomyosin to fulfil its function. Rather, regulation of F-actin stability by tropomyosin requires fidelity of information communicated at the barbed end of the actin filament. This distinction has significant implications for perturbing tropomyosin-dependent actin filament function in the context of anti-cancer drug development.
- Published
- 2016
18. Binding of transcription factor GabR to DNA requires recognition of DNA shape at a location distinct from its cognate binding site
- Author
-
Al-Zyoud, WA, Hynson, RMG, Ganuelas, LA, Coster, ACF, Duff, AP, Baker, MAB, Stewart, AG, Giannoulatou, E, Ho, JWK, Gaus, K, Liu, D, Lee, LK, Böcking, T, Al-Zyoud, WA, Hynson, RMG, Ganuelas, LA, Coster, ACF, Duff, AP, Baker, MAB, Stewart, AG, Giannoulatou, E, Ho, JWK, Gaus, K, Liu, D, Lee, LK, and Böcking, T
- Abstract
Mechanisms for transcription factor recognition of specific DNA base sequences are well characterized and recent studies demonstrate that the shape of these cognate binding sites is also important. Here, we uncover a new mechanism where the transcription factor GabR simultaneously recognizes two cognate binding sites and the shape of a 29 bp DNA sequence that bridges these sites. Small- Angle X-ray scattering and multi- Angle laser light scattering are consistent with a model where the DNA undergoes a conformational change to bend around GabR during binding. In silico predictions suggest that the bridging DNA sequence is likely to be bendable in one direction and kinetic analysis of mutant DNA sequences with biolayer interferometry, allowed the independent quantification of the relative contribution of DNA base and shape recognition in the GabR- DNA interaction. These indicate that the two cognate binding sites as well as the bendability of the DNA sequence in between these sites are required to form a stable complex. The mechanism of GabR-DNA interaction provides an example where the correct shape of DNA, at a clearly distinct location fromthe cognate binding site, is required for transcription factor binding and has implications for bioinformatics searches for novel binding sites.
- Published
- 2016
19. Probing Biomimetic Molecular Structures on Gold and Silicon(111) with Electrical Impedance Spectroscopy
- Author
-
Chilcott, T.C., primary, Wong, E.L.S., additional, Böcking, T., additional, and Coster, H.G.L., additional
- Full Text
- View/download PDF
20. Creating adhesive and soluble gradients for imaging cell migration with fluorescence microscopy.
- Author
-
Ngalim, SH, Magenau, A, Zhu, Y, Tønnesen, L, Fairjones, Z, Gooding, JJ, Böcking, T, Gaus, K, Ngalim, SH, Magenau, A, Zhu, Y, Tønnesen, L, Fairjones, Z, Gooding, JJ, Böcking, T, and Gaus, K
- Abstract
Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.
- Published
- 2013
21. Electrical characterizations of biomimetic molecular layers on gold and silicon substrates
- Author
-
Chilcott, T C, primary, Wong, E L S, additional, Böcking, T, additional, and Coster, H G L, additional
- Published
- 2008
- Full Text
- View/download PDF
22. Forming Antifouling Organic Multilayers on Porous Silicon Rugate Filters Towards In Vivo/Ex Vivo Biophotonic Devices
- Author
-
Kilian, K. A., primary, Böcking, T., additional, Ilyas, S., additional, Gaus, K., additional, Jessup, W., additional, Gal, M., additional, and Gooding, J. J., additional
- Published
- 2007
- Full Text
- View/download PDF
23. Porous silicon based narrow line-width rugate filters
- Author
-
Ilyas, S., primary, Böcking, T., additional, Kilian, K., additional, Reece, P.J., additional, Gooding, J., additional, Gaus, K., additional, and Gal, M., additional
- Published
- 2007
- Full Text
- View/download PDF
24. Probing Biomimetic Molecular Structures on Gold and Silicon(111) with Electrical Impedance Spectroscopy.
- Author
-
Scharfetter, Hermann, Merwa, Robert, Chilcott, T. C., Wong, E. L. S., Böcking, T., and Coster, H. G. L.
- Abstract
Self-assembled monolayers (SAMs) offer an approach for engineering molecular interfaces that mimic biological structures and their processes on solid substrates. Here we demonstrate the ability of electrical impedance spectroscopy to monitor the biological process of DNA recognition in a monolayer comprised of single stranded DNA assembled on a gold substrate and characterize the structure of a hybrid bimolecular lipid membrane (BLM) on an atomically flat silicon surface. These biomimetic examples demonstrate the crucial importance of the impedance phase in being able to distinguish between electrical conductive and capacitive properties of the molecular interfaces. [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
25. Probing biomimetic molecular structures on gold and silicon(111) with electrical impedance spectroscopy
- Author
-
Terry Chilcott, Wong, E. L. S., Böcking, T., and Coster, H. G. L.
26. Bey der ehelichen Verbindung des Hochwürdigen und Hochgelahrten Herrn, Herrn Johann August Nösselt, der heiligen Schrift Docktor und der Gottesgelahrtheit ordentlichen öffentlichen Lehrers auf der Friedrichsuniversität Halle mit der Wohlgebohrnen Demoiselle Demoiselle Dorothea Conradina Conerus, die den 12ten Jenner 1768. durch feyerliche Einsegnung vollzogen wurde bezeugten ihre Hochachtung nachstehende Zuhörer
- Author
-
Böcking, T. T., Buchholz, P. C., Matthiessen, G. C., Mellmann, J. B. T., Middendorf, T. H. Z., Steinweg, W. G., Beurhaus, J. H. F., Böcking, T. T., Buchholz, P. C., Matthiessen, G. C., Mellmann, J. B. T., Middendorf, T. H. Z., Steinweg, W. G., and Beurhaus, J. H. F.
- Abstract
Autopsie nach Ex. der ULB Sachsen-Anhalt, Vorlageform des Erscheinungsvermerks: Halle, gedruckt bey Johann Gottfried Trampe.
27. Bey der ehelichen Verbindung des Hochwürdigen und Hochgelahrten Herrn, Herrn Johann August Nösselt, der heiligen Schrift Docktor und der Gottesgelahrtheit ordentlichen öffentlichen Lehrers auf der Friedrichsuniversität Halle mit der Wohlgebohrnen Demoiselle Demoiselle Dorothea Conradina Conerus, die den 12ten Jenner 1768. durch feyerliche Einsegnung vollzogen wurde bezeugten ihre Hochachtung nachstehende Zuhörer
- Author
-
Böcking, T. T., Buchholz, P. C., Matthiessen, G. C., Mellmann, J. B. T., Middendorf, T. H. Z., Steinweg, W. G., Beurhaus, J. H. F., Böcking, T. T., Buchholz, P. C., Matthiessen, G. C., Mellmann, J. B. T., Middendorf, T. H. Z., Steinweg, W. G., and Beurhaus, J. H. F.
- Abstract
Autopsie nach Ex. der ULB Sachsen-Anhalt, Vorlageform des Erscheinungsvermerks: Halle, gedruckt bey Johann Gottfried Trampe.
28. Pharmacologic hyperstabilisation of the HIV-1 capsid lattice induces capsid failure.
- Author
-
Faysal KMR, Walsh JC, Renner N, Márquez CL, Shah VB, Tuckwell AJ, Christie MP, Parker MW, Turville SG, Towers GJ, James LC, Jacques DA, and Böcking T
- Subjects
- Humans, Capsid, Capsid Proteins, HIV-1, Anti-HIV Agents pharmacology, HIV Infections
- Abstract
The HIV-1 capsid has emerged as a tractable target for antiretroviral therapy. Lenacapavir, developed by Gilead Sciences, is the first capsid-targeting drug approved for medical use. Here, we investigate the effect of lenacapavir on HIV capsid stability and uncoating. We employ a single particle approach that simultaneously measures capsid content release and lattice persistence. We demonstrate that lenacapavir's potent antiviral activity is predominantly due to lethal hyperstabilisation of the capsid lattice and resultant loss of compartmentalisation. This study highlights that disrupting capsid metastability is a powerful strategy for the development of novel antivirals., Competing Interests: KF, JW, NR, CM, VS, AT, MC, MP, ST, GT, LJ, DJ, TB No competing interests declared, (© 2024, Faysal, Walsh et al.)
- Published
- 2024
- Full Text
- View/download PDF
29. HIV-1 binds dynein directly to hijack microtubule transport machinery.
- Author
-
Badieyan S, Lichon D, Andreas MP, Gillies JP, Peng W, Shi J, DeSantis ME, Aiken CR, Böcking T, Giessen TW, Campbell EM, and Cianfrocco MA
- Abstract
Viruses exploit host cytoskeletal elements and motor proteins for trafficking through the dense cytoplasm. Yet the molecular mechanism that describes how viruses connect to the motor machinery is unknown. Here, we demonstrate the first example of viral microtubule trafficking from purified components: HIV-1 hijacking microtubule transport machinery. We discover that HIV-1 directly binds to the retrograde microtubule-associated motor, dynein, and not via a cargo adaptor, as previously suggested. Moreover, we show that HIV-1 motility is supported by multiple, diverse dynein cargo adaptors as HIV-1 binds to dynein light and intermediate chains on dynein's tail. Further, we demonstrate that multiple dynein motors tethered to rigid cargoes, like HIV-1 capsids, display reduced motility, distinct from the behavior of multiple motors on membranous cargoes. Our results introduce a new model of viral trafficking wherein a pathogen opportunistically 'hijacks' the microtubule transport machinery for motility, enabling multiple transport pathways through the host cytoplasm., Competing Interests: Declaration of interests Authors declare that they have no competing interests.
- Published
- 2023
- Full Text
- View/download PDF
30. HIV-1 is dependent on its immature lattice to recruit IP6 for mature capsid assembly.
- Author
-
Renner N, Kleinpeter A, Mallery DL, Albecka A, Rifat Faysal KM, Böcking T, Saiardi A, Freed EO, and James LC
- Subjects
- Capsid metabolism, Virus Assembly, Capsid Proteins metabolism, Phytic Acid metabolism, Virion, HIV-1 metabolism
- Abstract
HIV-1 Gag metamorphoses inside each virion, from an immature lattice that forms during viral production to a mature capsid that drives infection. Here we show that the immature lattice is required to concentrate the cellular metabolite inositol hexakisphosphate (IP6) into virions to catalyze mature capsid assembly. Disabling the ability of HIV-1 to enrich IP6 does not prevent immature lattice formation or production of the virus. However, without sufficient IP6 molecules inside each virion, HIV-1 can no longer build a stable capsid and fails to become infectious. IP6 cannot be replaced by other inositol phosphate (IP) molecules, as substitution with other IPs profoundly slows mature assembly kinetics and results in virions with gross morphological defects. Our results demonstrate that while HIV-1 can become independent of IP6 for immature assembly, it remains dependent upon the metabolite for mature capsid formation., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2023
- Full Text
- View/download PDF
31. Cholesterol-dependent cytolysins: The outstanding questions.
- Author
-
Johnstone BA, Joseph R, Christie MP, Morton CJ, McGuiness C, Walsh JC, Böcking T, Tweten RK, and Parker MW
- Subjects
- Cholesterol metabolism, Bacteria metabolism, Cell Membrane metabolism, Bacterial Proteins metabolism, Cytotoxins metabolism, Bacterial Toxins genetics
- Abstract
The cholesterol-dependent cytolysins (CDCs) are a major family of bacterial pore-forming proteins secreted as virulence factors by Gram-positive bacterial species. CDCs are produced as soluble, monomeric proteins that bind specifically to cholesterol-rich membranes, where they oligomerize into ring-shaped pores of more than 30 monomers. Understanding the details of the steps the toxin undergoes in converting from monomer to a membrane-spanning pore is a continuing challenge. In this review we summarize what we know about CDCs and highlight the remaining outstanding questions that require answers to obtain a complete picture of how these toxins kill cells., (© 2022 The Authors. IUBMB Life published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)
- Published
- 2022
- Full Text
- View/download PDF
32. Single-molecule analysis of the entire perfringolysin O pore formation pathway.
- Author
-
McGuinness C, Walsh JC, Bayly-Jones C, Dunstone MA, Christie MP, Morton CJ, Parker MW, and Böcking T
- Subjects
- Animals, Clostridium perfringens metabolism, Liposomes metabolism, Mammals metabolism, Bacterial Toxins metabolism, Hemolysin Proteins metabolism
- Abstract
The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings (~35 mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled., Competing Interests: CM, JW, CB, MD, MC, CM, MP, TB No competing interests declared, (© 2022, Mc Guinness, Walsh et al.)
- Published
- 2022
- Full Text
- View/download PDF
33. Recognition of HIV-1 capsid by PQBP1 licenses an innate immune sensing of nascent HIV-1 DNA.
- Author
-
Yoh SM, Mamede JI, Lau D, Ahn N, Sánchez-Aparicio MT, Temple J, Tuckwell A, Fuchs NV, Cianci GC, Riva L, Curry H, Yin X, Gambut S, Simons LM, Hultquist JF, König R, Xiong Y, García-Sastre A, Böcking T, Hope TJ, and Chanda SK
- Subjects
- Capsid metabolism, DNA metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Humans, Immunity, Innate, Nucleotidyltransferases metabolism, Pathogen-Associated Molecular Pattern Molecules metabolism, HIV-1 genetics
- 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., Competing Interests: Declaration of interests The A.G.-S. laboratory has received research support from Pfizer, Senhwa Biosciences, Kenall Manufacturing, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, Pharmamar, ImmunityBio, Accurius, Nanocomposix, Hexamer, N-fold LLC, Model Medicines, Atea Pharma, Applied Biological Laboratories and Merck, outside of the reported work. A.G.-S. has consulting agreements for the following companies involving cash and/or stock: Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Vaxalto, Pagoda, Accurius, Esperovax, Farmak, Applied Biological Laboratories, Pharmamar, Paratus, CureLab Oncology, CureLab Veterinary, Synairgen, and Pfizer, outside of the reported work. A.G.-S. has been an invited speaker in meeting events organized by Sequirus, Janssen, and Astrazeneca outside of the reported work. A.G.-S. is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York, outside of the reported work. The S.K.C. laboratory has received research support from Eli Lilly & Co., Boehringer Ingelheim, Merck & Co., Cidara Therapeutics, outside of the reported work. S.K.C. has consulting agreements for the following companies involving cash and/or stock: Pagoda Genomics, Cidara Therapeutics, and Samsara Biocapital, outside of the reported work. S.K.C. is inventor on patents and patent applications on the use of antivirals, adjuvants, and immunotherapies for the treatment and prevention of virus infections and cancer, owned by Sanford Burnham Prebys Medical Discovery Institute and Scripps Research, outside of the reported work., (Copyright © 2022 Elsevier Inc. All rights reserved.)
- Published
- 2022
- Full Text
- View/download PDF
34. Rapid Exchange of Stably Bound Protein and DNA Cargo on a DNA Origami Receptor.
- Author
-
Brown JWP, Alford RG, Walsh JC, Spinney RE, Xu SY, Hertel S, Berengut JF, Spenkelink LM, van Oijen AM, Böcking T, Morris RG, and Lee LK
- Subjects
- DNA chemistry, DNA, Single-Stranded, Nucleic Acid Conformation, Nanotechnology, Nanostructures chemistry
- Abstract
Biomolecular complexes can form stable assemblies yet can also rapidly exchange their subunits to adapt to environmental changes. Simultaneously allowing for both stability and rapid exchange expands the functional capacity of biomolecular machines and enables continuous function while navigating a complex molecular world. Inspired by biology, we design and synthesize a DNA origami receptor that exploits multivalent interactions to form stable complexes that are also capable of rapid subunit exchange. The system utilizes a mechanism first outlined in the context of the DNA replisome, known as multisite competitive exchange, and achieves a large separation of time scales between spontaneous subunit dissociation, which requires days, and rapid subunit exchange, which occurs in minutes. In addition, we use the DNA origami receptor to demonstrate stable interactions with rapid exchange of both DNA and protein subunits, thus highlighting the applicability of our approach to arbitrary molecular cargo, an important distinction with canonical toehold exchange between single-stranded DNA. We expect this study to benefit future studies that use DNA origami structures to exploit multivalent interactions for the design and synthesis of a wide range of possible kinetic behaviors.
- Published
- 2022
- Full Text
- View/download PDF
35. Biomolecular Binding under Confinement: Statistical Predictions of Steric Influence in Absence of Long-Distance Interactions.
- Author
-
Longatte G, Lisi F, Bakthavathsalam P, Böcking T, Gaus K, Tilley RD, and Gooding JJ
- Subjects
- DNA chemistry, Models, Theoretical
- Abstract
We propose a theoretical model for the influence of confinement on biomolecular binding at the single-molecule scale at equilibrium, based on the change of the number of microstates (localization and orientation) upon reaction. Three cases are discussed: DNA sequences shorter and longer than the single strain DNA Kuhn length and spherical proteins, confined into a spherical container (liposome, droplet, etc.). The influence of confinement is found to be highly dependent on the molecular structure and significant for large molecules (relative to container size)., (© 2021 Wiley-VCH GmbH.)
- Published
- 2022
- Full Text
- View/download PDF
36. Insights into HIV uncoating from single-particle imaging techniques.
- Author
-
Zhang MJ, Stear JH, Jacques DA, and Böcking T
- Abstract
Human immunodeficiency virus (HIV) is the most extensively researched human pathogen. Despite this massive scientific endeavour, several fundamental viral processes remain enigmatic. One such critical process is uncoating-the event that releases the viral genome from the proteinaceous shell of the capsid during infection. While this process is conceptually simple, the molecular underpinnings, timing, regulation, and cellular location of uncoating remain contentious. This review describes the hurdles that have limited our understanding in this area and presents recently deployed in vitro and in cellulo techniques that have been developed expressly with the aim of directly visualising capsid uncoating at the single-particle level and understanding the mechanics behind this essential aspect of HIV infection., Competing Interests: Conflict of interestThe authors declare no competing interests., (© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2021.)
- Published
- 2022
- Full Text
- View/download PDF
37. Structure of native HIV-1 cores and their interactions with IP6 and CypA.
- Author
-
Ni T, Zhu Y, Yang Z, Xu C, Chaban Y, Nesterova T, Ning J, Böcking T, Parker MW, Monnie C, Ahn J, Perilla JR, and Zhang P
- Abstract
The viral capsid plays essential roles in HIV replication and is a major platform engaging host factors. To overcome challenges in study native capsid structure, we used the perfringolysin O to perforate the membrane of HIV-1 particles, thus allowing host proteins and small molecules to access the native capsid while improving cryo–electron microscopy image quality. Using cryo–electron tomography and subtomogram averaging, we determined the structures of native capsomers in the presence and absence of inositol hexakisphosphate (IP6) and cyclophilin A and constructed an all-atom model of a complete HIV-1 capsid. Our structures reveal two IP6 binding sites and modes of cyclophilin A interactions. Free energy calculations substantiate the two binding sites at R18 and K25 and further show a prohibitive energy barrier for IP6 to pass through the pentamer. Our results demonstrate that perfringolysin O perforation is a valuable tool for structural analyses of enveloped virus capsids and interactions with host cell factors.
- Published
- 2021
- Full Text
- View/download PDF
38. A stable immature lattice packages IP 6 for HIV capsid maturation.
- Author
-
Mallery DL, Kleinpeter AB, Renner N, Faysal KMR, Novikova M, Kiss L, Wilson MSC, Ahsan B, Ke Z, Briggs JAG, Saiardi A, Böcking T, Freed EO, and James LC
- Abstract
HIV virion assembly begins with the construction of an immature lattice consisting of Gag hexamers. Upon virion release, protease-mediated Gag cleavage leads to a maturation event in which the immature lattice disassembles and the mature capsid assembles. The cellular metabolite inositiol hexakisphosphate (IP
6 ) and maturation inhibitors (MIs) both bind and stabilize immature Gag hexamers, but whereas IP6 promotes virus maturation, MIs inhibit it. Here we show that HIV is evolutionarily constrained to maintain an immature lattice stability that ensures IP6 packaging without preventing maturation. Replication-deficient mutant viruses with reduced IP6 recruitment display increased infectivity upon treatment with the MI PF46396 (PF96) or the acquisition of second-site compensatory mutations. Both PF96 and second-site mutations stabilise the immature lattice and restore IP6 incorporation, suggesting that immature lattice stability and IP6 binding are interdependent. This IP6 dependence suggests that modifying MIs to compete with IP6 for Gag hexamer binding could substantially improve MI antiviral potency., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)- Published
- 2021
- Full Text
- View/download PDF
39. Rapid HIV-1 Capsid Interaction Screening Using Fluorescence Fluctuation Spectroscopy.
- Author
-
Lau D, Walsh JC, Dickson CF, Tuckwell A, Stear JH, Hunter DJB, Bhumkar A, Shah V, Turville SG, Sierecki E, Gambin Y, Böcking T, and Jacques DA
- Subjects
- Binding Sites, Capsid Proteins, Spectrometry, Fluorescence, Capsid, HIV-1
- 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.
- Published
- 2021
- Full Text
- View/download PDF
40. A lysine ring in HIV capsid pores coordinates IP6 to drive mature capsid assembly.
- Author
-
Renner N, Mallery DL, Faysal KMR, Peng W, Jacques DA, Böcking T, and James LC
- Subjects
- Cell Line, DNA, Viral biosynthesis, HIV-1 genetics, HIV-1 metabolism, Humans, Microscopy, Fluorescence, Nucleotides metabolism, Capsid metabolism, HIV-1 physiology, Lysine metabolism, Phytic Acid metabolism, Virus Assembly physiology
- Abstract
The HIV capsid self-assembles a protective conical shell that simultaneously prevents host sensing whilst permitting the import of nucleotides to drive DNA synthesis. This is accomplished through the construction of dynamic, highly charged pores at the centre of each capsid multimer. The clustering of charges required for dNTP import is strongly destabilising and it is proposed that HIV uses the metabolite IP6 to coordinate the pore during assembly. Here we have investigated the role of inositol phosphates in coordinating a ring of positively charged lysine residues (K25) that forms at the base of the capsid pore. We show that whilst IP5, which can functionally replace IP6, engages an arginine ring (R18) at the top of the pore, the lysine ring simultaneously binds a second IP5 molecule. Dose dependent removal of K25 from the pore severely inhibits HIV infection and concomitantly prevents DNA synthesis. Cryo-tomography reveals that K25A virions have a severe assembly defect that inhibits the formation of mature capsid cones. Monitoring both the kinetics and morphology of capsids assembled in vitro reveals that while mutation K25A can still form tubes, the ability of IP6 to drive assembly of capsid cones has been lost. Finally, in single molecule TIRF microscopy experiments, capsid lattices in permeabilised K25 mutant virions are rapidly lost and cannot be stabilised by IP6. These results suggest that the coordination of IP6 by a second charged ring in mature hexamers drives the assembly of conical capsids capable of reverse transcription and infection., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2021
- Full Text
- View/download PDF
41. Dynamics of Tpm1.8 domains on actin filaments with single-molecule resolution.
- Author
-
Bareja I, Wioland H, Janco M, Nicovich PR, Jégou A, Romet-Lemonne G, Walsh J, and Böcking T
- Subjects
- Actin Cytoskeleton physiology, Actins metabolism, Kinetics, Microfluidics methods, Microscopy, Fluorescence methods, Protein Binding, Protein Domains, Protein Isoforms metabolism, Tropomyosin physiology, Actin Cytoskeleton metabolism, Tropomyosin metabolism
- Abstract
Tropomyosins regulate the dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of the fluorescently labeled tropomyosin isoform Tpm1.8 to unlabeled actin filaments in real time. This approach, in conjunction with mathematical modeling, enabled us to quantify the nucleation, assembly, and disassembly kinetics of Tpm1.8 on single filaments and at the single-molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilized by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented toward the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.
- Published
- 2020
- Full Text
- View/download PDF
42. Corneal Confocal Microscopy Demonstrates Corneal Nerve Loss in Patients With Trigeminal Neuralgia.
- Author
-
Lee JI, Böcking T, Holle-Lee D, Malik RA, Kieseier BC, Hartung HP, Guthoff R, Kleinschnitz C, and Stettner M
- Abstract
Background: The diagnosis of trigeminal neuralgia (TN) is challenging due to the lack of objective diagnostics. Corneal confocal microscopy (CCM) is a non-invasive ophthalmic imaging technique, which allows quantification of corneal nerve fibers arising from the trigeminal ganglion and may allow the assessment of neurodegeneration in TN. Methods: CCM was undertaken in 11 patients with TN and 11 age-matched healthy controls. Corneal nerve fiber density (CNFD), corneal nerve branch density, corneal nerve fiber length (CNFL), corneal nerve fiber width, corneal nerve fiber area, and dendritic cell and non-dendritic cell density with or without nerve fiber contact were quantified. Results: Patients with TN had significantly lower CNFD and CNFL but no difference for any other corneal nerve or dendritic cell parameter in the ipsilateral and the contralateral cornea compared to the control group. There was no significant difference in corneal nerve and cell parameters between patients with TN with and without involvement of the ophthalmic nerve (V1) or with nerve vessel conflict. Conclusion: Corneal confocal microscopy is a rapid non-invasive imaging technique that identifies symmetrical corneal nerve loss in patients with TN., (Copyright © 2020 Lee, Böcking, Holle-Lee, Malik, Kieseier, Hartung, Guthoff, Kleinschnitz and Stettner.)
- Published
- 2020
- Full Text
- View/download PDF
43. Tenth Scientific Biennial Meeting of the Australasian Virology Society-AVS10 2019.
- Author
-
Helbig KJ, Bull RA, Ambrose R, Beard MR, Blanchard H, Böcking T, Chua B, Colmant AMG, Crosse KM, Purcell DFJ, Fraser J, Hayward JA, Hamilton ST, Husain M, MacDiarmid R, Mackenzie JM, Moseley GW, Nguyen THO, Quiñones-Mateu ME, Robinson K, Rodrigo C, Rodriguez-Andres J, Rudd PA, Werno A, White P, Young P, Speck P, Hibma M, Drummer HE, and Tachedjian G
- Subjects
- Australia, Awards and Prizes, Group Processes, Societies, Scientific, Virology organization & administration
- Abstract
The Australasian Virology Society (AVS) aims to promote, support and advocate for the discipline of virology in the Australasian region. The society was incorporated in 2011 after 10 years operating as the Australian Virology Group (AVG) founded in 2001, coinciding with the inaugural biennial scientific meeting. AVS conferences aim to provide a forum for the dissemination of all aspects of virology, foster collaboration, and encourage participation by students and post-doctoral researchers. The tenth Australasian Virology Society (AVS10) scientific meeting was held on 2-5 December 2019 in Queenstown, New Zealand. This report highlights the latest research presented at the meeting, which included cutting-edge virology presented by our international plenary speakers Ana Fernandez-Sesma and Benjamin tenOever, and keynote Richard Kuhn. AVS10 honoured female pioneers in Australian virology, Lorena Brown and Barbara Coulson. We report outcomes from the AVS10 career development session on "Successfully transitioning from post-doc to lab head", winners of best presentation awards, and the AVS gender equity policy, initiated in 2013. Plans for the 2021 meeting are underway which will celebrate the 20th anniversary of AVS where it all began, in Fraser Island, Queensland, Australia., Competing Interests: The authors declare no conflict of interest. The conference sponsors had no role in the decision to publish this report.
- Published
- 2020
- Full Text
- View/download PDF
44. Self-Assembly of Fluorescent HIV Capsid Spheres for Detection of Capsid Binders.
- Author
-
Lau D, Walsh JC, Mousapasandi A, Ariotti N, Shah VB, Turville S, Jacques DA, and Böcking T
- Subjects
- Capsid Proteins, Cyclophilin A, Humans, Capsid, HIV Infections, HIV-1
- 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
- Full Text
- View/download PDF
45. Cellular IP 6 Levels Limit HIV Production while Viruses that Cannot Efficiently Package IP 6 Are Attenuated for Infection and Replication.
- Author
-
Mallery DL, Faysal KMR, Kleinpeter A, Wilson MSC, Vaysburd M, Fletcher AJ, Novikova M, Böcking T, Freed EO, Saiardi A, and James LC
- Subjects
- Capsid chemistry, HIV Infections metabolism, HIV Infections pathology, HeLa Cells, Humans, Protein Conformation, Capsid metabolism, HIV Infections virology, HIV-1 physiology, Host-Pathogen Interactions, Phytic Acid metabolism, Virus Assembly, Virus Replication
- Abstract
HIV-1 hijacks host proteins to promote infection. Here we show that HIV is also dependent upon the host metabolite inositol hexakisphosphate (IP
6 ) for viral production and primary cell replication. HIV-1 recruits IP6 into virions using two lysine rings in its immature hexamers. Mutation of either ring inhibits IP6 packaging and reduces viral production. Loss of IP6 also results in virions with highly unstable capsids, leading to a profound loss of reverse transcription and cell infection. Replacement of one ring with a hydrophobic isoleucine core restores viral production, but IP6 incorporation and infection remain impaired, consistent with an independent role for IP6 in stable capsid assembly. Genetic knockout of biosynthetic kinases IPMK and IPPK reveals that cellular IP6 availability limits the production of diverse lentiviruses, but in the absence of IP6 , HIV-1 packages IP5 without loss of infectivity. Together, these data suggest that IP6 is a critical cofactor for HIV-1 replication., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2019
- Full Text
- View/download PDF
46. Fluorescence Biosensor for Real-Time Interaction Dynamics of Host Proteins with HIV-1 Capsid Tubes.
- Author
-
Lau D, Walsh JC, Peng W, Shah VB, Turville S, Jacques DA, and Böcking T
- Subjects
- Humans, Microscopy, Fluorescence, Biosensing Techniques, Capsid chemistry, Capsid metabolism, HIV-1 chemistry, HIV-1 metabolism, Lab-On-A-Chip Devices
- 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 to measure 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, to obtain dissociation constants, and to extract kinetics with an extended application of using more complex analytes that can accelerate characterization of novel capsid binders.
- Published
- 2019
- Full Text
- View/download PDF
47. Building Complexity: Making and Breaking Synthetic Subunits of the HIV Capsid.
- Author
-
James LC and Böcking T
- Subjects
- Adaptor Proteins, Signal Transducing, Capsid Proteins, Kinesins, Capsid, HIV-1
- Abstract
In this issue of Cell Host & Microbe, Summers et al. (2019) use protein engineering to generate a toolbox of HIV-1 capsid oligomers. In an accompanying Cell Reports paper, Huang et al. (2019) use these oligomers to determine how the capsid engages the kinesin-1 adaptor protein FEZ1., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
48. Molecular integration of the anti-tropomyosin compound ATM-3507 into the coiled coil overlap region of the cancer-associated Tpm3.1.
- Author
-
Janco M, Rynkiewicz MJ, Li L, Hook J, Eiffe E, Ghosh A, Böcking T, Lehman WJ, Hardeman EC, and Gunning PW
- Subjects
- Antineoplastic Agents chemistry, Antineoplastic Agents therapeutic use, Circular Dichroism, Crystallography, X-Ray, Humans, Molecular Dynamics Simulation, Neoplasms drug therapy, Protein Conformation, alpha-Helical drug effects, Protein Domains genetics, Protein Isoforms antagonists & inhibitors, Protein Isoforms genetics, Protein Isoforms metabolism, Protein Isoforms ultrastructure, Protein Multimerization drug effects, Protein Multimerization genetics, Recombinant Proteins genetics, Recombinant Proteins metabolism, Recombinant Proteins ultrastructure, Structure-Activity Relationship, Thermodynamics, Tropomyosin metabolism, Tropomyosin ultrastructure, Actin Cytoskeleton metabolism, Antineoplastic Agents pharmacology, Tropomyosin antagonists & inhibitors
- Abstract
Tropomyosins (Tpm) determine the functional capacity of actin filaments in an isoform-specific manner. The primary isoform in cancer cells is Tpm3.1 and compounds that target Tpm3.1 show promising results as anti-cancer agents both in vivo and in vitro. We have determined the molecular mechanism of interaction of the lead compound ATM-3507 with Tpm3.1-containing actin filaments. When present during co-polymerization of Tpm3.1 with actin,
3 H-ATM-3507 is incorporated into the filaments and saturates at approximately one molecule per Tpm3.1 dimer and with an apparent binding affinity of approximately 2 µM. In contrast,3 H-ATM-3507 is poorly incorporated into preformed Tpm3.1/actin co-polymers. CD spectroscopy and thermal melts using Tpm3.1 peptides containing the C-terminus, the N-terminus, and a combination of the two forming the overlap junction at the interface of adjacent Tpm3.1 dimers, show that ATM-3507 shifts the melting temperature of the C-terminus and the overlap junction, but not the N-terminus. Molecular dynamic simulation (MDS) analysis predicts that ATM-3507 integrates into the 4-helix coiled coil overlap junction and in doing so, likely changes the lateral movement of Tpm3.1 across the actin surface resulting in an alteration of filament interactions with actin binding proteins and myosin motors, consistent with the cellular impact of ATM-3507.- Published
- 2019
- Full Text
- View/download PDF
49. Fluorescence Microscopy Assay to Measure HIV-1 Capsid Uncoating Kinetics in vitro .
- Author
-
Márquez CL, Lau D, Walsh J, Faysal KMR, Parker MW, Turville SG, and Böcking T
- 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., Competing Interests: Competing interestsThere are no conflicts of interest or competing interest., (©Copyright Márquez et al.)
- Published
- 2019
- Full Text
- View/download PDF
50. Editorial: Special issue of Biophysical Reviews dedicated to the joint 10th Asian Biophysics Association Symposium and 42nd Australian Society for Biophysics Meeting, Melbourne, Australia, December 2-6, 2018.
- Author
-
Battle AR, Norton RS, Böcking T, Noji H, Kim KK, and Nagayama K
- Published
- 2019
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.