The Experts below are selected from a list of 309 Experts worldwide ranked by ideXlab platform
Adam Zlotnick - One of the best experts on this subject based on the ideXlab platform.
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Asymmetrizing an icosahedral Virus Capsid by hierarchical assembly of subunits with designed asymmetry.
Nature communications, 2021Co-Authors: Zhongchao Zhao, Mi Zhang, Nicholas Lyktey, Martin Jarrold, Joseph Che-yen Wang, Stephen C. Jacobson, Adam ZlotnickAbstract:Symmetrical protein complexes are ubiquitous in biology. Many have been re-engineered for chemical and medical applications. Viral Capsids and their assembly are frequent platforms for these investigations. A means to create asymmetric Capsids may expand applications. Here, starting with homodimeric Hepatitis B Virus Capsid protein, we develop a heterodimer, design a hierarchical assembly pathway, and produce asymmetric Capsids. In the heterodimer, the two halves have different growth potentials and assemble into hexamers. These preformed hexamers can nucleate co-assembly with other dimers, leading to Janus-like Capsids with a small discrete hexamer patch. We can remove the patch specifically and observe asymmetric holey Capsids by cryo-EM reconstruction. The resulting hole in the surface can be refilled with fluorescently labeled dimers to regenerate an intact Capsid. In this study, we show how an asymmetric subunit can be used to generate an asymmetric particle, creating the potential for a Capsid with different surface chemistries.
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Asymmetrizing an icosahedral Virus Capsid by hierarchical assembly of subunits with designed asymmetry
2020Co-Authors: Zhongchao Zhao, Joseph Wang, Mi Zhang, Nicholas Lyktey, Martin Jarrold, Stephen Jacobson, Adam ZlotnickAbstract:Abstract Symmetrical protein complexes are ubiquitous in natural biological systems. Many have been reengineered in vitro for chemical and medical applications. Symmetrical viral Capsids and their assembly are frequent platforms for these investigations. Lacking a means to create asymmetric Capsids may limit broader applications. Here, starting with the homodimeric Hepatitis B Virus Capsid protein, we developed a heterodimer, designed a hierarchical assembly pathway, and produced asymmetric Capsids. We showed that the heterodimers assemble into hexamers, and such preformed hexamers can nucleate co-assembly, leading to “Janus” Capsids with two discrete patches. We removed the hexamer patches specifically and observed asymmetric holey Capsids by cryo-EM reconstruction. The resulting holes can be refilled with new engineered dimers. This programmed assembly pathway provides windows for specific engineering and modification inside and outside of the Capsid. This strategy can also be generalized to other Capsid assembly systems.
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detection of late intermediates in Virus Capsid assembly by charge detection mass spectrometry
Journal of the American Chemical Society, 2014Co-Authors: Elizabeth E Pierson, Adam Zlotnick, Joseph Che-yen Wang, David Z Keifer, Lisa Selzer, Lye Siang Lee, Nathan C Contino, Martin JarroldAbstract:The assembly of hundreds of identical proteins into an icosahedral Virus Capsid is a remarkable feat of molecular engineering. How this occurs is poorly understood. Key intermediates have been anticipated at the end of the assembly reaction, but it has not been possible to detect them. In this work we have used charge detection mass spectrometry to identify trapped intermediates from late in the assembly of the hepatitis B Virus T = 4 Capsid, a complex of 120 protein dimers. Prominent intermediates are found with 104/105, 110/111, and 117/118 dimers. Cryo-EM observations indicate the intermediates are incomplete Capsids and, hence, on the assembly pathway. On the basis of their stability and kinetic accessibility we have proposed plausible structures. The prominent trapped intermediate with 104 dimers is attributed to an icosahedron missing two neighboring facets, the 111-dimer species is assigned to an icosahedron missing a single facet, and the intermediate with 117 dimers is assigned to a Capsid missing a ring of three dimers in the center of a facet.
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The thermodynamics of Virus Capsid assembly.
Methods in enzymology, 2009Co-Authors: Sarah P. Katen, Adam ZlotnickAbstract:Virus Capsid assembly is a critical step in the viral life cycle. The underlying basis of Capsid stability is key to understanding this process. Capsid subunits interact with weak individual contact energies to form a globally stable icosahedral lattice; this structure is ideal for enCapsidating the viral genome and host partners and protecting its contents upon secretion, yet the unique properties of its assembly and inter-subunit contacts allow the Capsid to dissociate upon entering a new host cell. The stability of the Capsid can be analyzed by treating Capsid assembly as an equilibrium polymerization reaction, modified from the traditional polymer model to account for the fact that a separate nucleus is formed for each individual Capsid. From the concentrations of reactants and products in an equilibrated assembly reaction, it is possible to extract the thermodynamic parameters of assembly for a wide array of icosahedral Viruses using well-characterized biochemical and biophysical methods. In this chapter we describe this basic analysis and provide examples of thermodynamic assembly data for several different icosahedral Viruses. These data provide new insights into the assembly mechanisms of spherical Virus Capsids, as well as into the biology of the viral life cycle.
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Theoretical aspects of Virus Capsid assembly.
Journal of molecular recognition : JMR, 2005Co-Authors: Adam ZlotnickAbstract:A Virus Capsid is constructed from many copies of the same protein(s). Molecular recognition is central to Capsid assembly. The Capsid protein must polymerize in order to create a three-dimensional protein polymer. More than structure is required to understand this self-assembly reaction: one must understand how the pieces come together in solution.
Mary K. Estes - One of the best experts on this subject based on the ideXlab platform.
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three dimensional structure of baculoVirus expressed norwalk Virus Capsids
Journal of Virology, 1994Co-Authors: B. V. V. Prasad, R Rothnagel, Xiaofang Jiang, Mary K. EstesAbstract:: The three-dimensional structure of the baculoVirus-expressed Norwalk Virus Capsid has been determined to a resolution of 2.2 nm using electron cryomicroscopy and computer image processing techniques. The empty Capsid, 38.0 nm in diameter, exhibits T = 3 icosahedral symmetry and is composed of 90 dimers of the Capsid protein. The striking features of the Capsid structure are arch-like capsomeres, at the local and strict 2-fold axes, formed by dimers of the Capsid protein and large hollows at the icosahedral 5- and 3-fold axes. Despite its distinctive architecture, the Norwalk Virus Capsid has several similarities with the structures of T = 3 single-stranded RNA (ssRNA) Viruses. The structure of the protein subunit appears to be modular with three distinct domains: the distal globular domain (P2) that appears bilobed, a central stem domain (P1), and a lower shell domain (S). The distal domains of the 2-fold related subunits interact with each other to form the top of the arch. The lower domains of the adjacent subunits associate tightly to form a continuous shell between the radii of 11.0 and 15.0 nm. No significant mass density is observed below the radius of 11.0 mm. It is suspected that the hinge peptide in the adjoining region between the central domain and the shell domain may facilitate the subunits adapting to various quasi-equivalent environments. Architectural similarities between the Norwalk Virus Capsid and the other ssRNA Viruses have suggested a possible domain organization along the primary sequence of the Norwalk Virus Capsid protein. It is suggested that the N-terminal 250 residues constitute the lower shell domain (S) with an eight-strand beta-barrel structure and that the C-terminal residues beyond 250 constitute the protruding (P1+P2) domains. A lack of an N-terminal basic region and the ability of the Norwalk Virus Capsid protein to form empty T = 3 shells suggest that the assembly pathway and the RNA packing mechanisms may be different from those proposed for tomato bushy stunt Virus and southern bean mosaic Virus but similar to that in tymoViruses and comoViruses.
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Subclass-specific serum antibody responses to recombinant Norwalk Virus Capsid antigen (rNV) in adults infected with Norwalk, Snow Mountain, or Hawaii Virus.
Journal of Clinical Microbiology, 1993Co-Authors: John J. Treanor, H P Madore, Xi Jiang, Mary K. EstesAbstract:Subclass-specific antibody responses to the Norwalk Virus Capsid protein in adults challenged with Norwalk, Snow Mountain, or Hawaii Virus were evaluated by solid-phase enzyme immunoassay using recombinant Norwalk Virus Capsid antigen (rNV). Fourfold or greater serum immunoglobulin G (IgG) antibody responses to rNV were detected in 15 of 20 volunteers challenged with Norwalk Virus, and serum IgA and IgM antibody responses to rNV were seen in almost all subjects who had rNV IgG responses. Serum rNV IgG antibody responses also were detected in 6 of 15 volunteers challenged with Snow Mountain Virus and 2 of 12 volunteers challenged with the Hawaii Virus. However, the magnitude of antibody response and the geometric mean postchallenge rNV IgG antibody titers were lower in subjects challenged with Snow Mountain or Hawaii Virus, and serum IgA and IgM responses generally did not occur.
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expression self assembly and antigenicity of the norwalk Virus Capsid protein
Journal of Virology, 1992Co-Authors: Xi Jiang, David Y. Graham, Min Wang, Mary K. EstesAbstract:Abstract Norwalk Virus Capsid protein was produced by expression of the second and third open reading frames of the Norwalk Virus genome, using a cell-free translation system and baculoVirus recombinants. Analysis of the expressed products showed that the second open reading frame encodes a protein with an apparent molecular weight of 58,000 (58K protein) and that this protein self-assembles to form empty Viruslike particles similar to native Capsids in size and appearance. The antigenicity of these particles was demonstrated by immunoprecipitation and enzyme-linked immunosorbent assays of paired serum samples from volunteers who developed illness following Norwalk Virus challenge. These particles also induced high levels of Norwalk Virus-specific serum antibody in laboratory animals following parenteral inoculation. A minor 34K protein was also found in infected insect cells. Amino acid sequence analysis of the N terminus of the 34K protein indicated that the 34K protein was a cleavage product of the 58K protein. The availability of large amounts of recombinant Norwalk Virus particles will allow the development of rapid, sensitive, and reliable tests for the diagnosis of Norwalk Virus infection as well as the implementation of structural studies.
Dan Endres - One of the best experts on this subject based on the ideXlab platform.
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a theoretical model successfully identifies features of hepatitis b Virus Capsid assembly
Biochemistry, 1999Co-Authors: Adam Zlotnick, Jennifer M Johnson, Paul W Wingfield, Stephen J Stahl, Dan EndresAbstract:The Capsids of most spherical Viruses are icosahedral, an arrangement of multiples of 60 subunits. Though it is a salient point in the life cycle of any Virus, the physical chemistry of Virus Capsid assembly is poorly understood. We have developed general models of Capsid assembly that describe the process in terms of a cascade of low order association reactions. The models predict sigmoidal assembly kinetics, where intermediates approach a low steady state concentration for the greater part of the reaction. Features of the overall reaction can be identified on the basis of the concentration dependence of assembly. In simulations, and on the basis of our understanding of the models, we find that nucleus size and the order of subsequent “elongation” reactions are reflected in the concentration dependence of the extent of the reaction and the rate of the fast phase, respectively. The reaction kinetics deduced for our models of Virus assembly can be related to the assembly of any “spherical” polymer. Using l...
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a theoretical model successfully identifies features of hepatitis b Virus Capsid assembly
Biochemistry, 1999Co-Authors: Adam Zlotnick, Jennifer M Johnson, Paul W Wingfield, Stephen J Stahl, Dan EndresAbstract:The Capsids of most spherical Viruses are icosahedral, an arrangement of multiples of 60 subunits. Though it is a salient point in the life cycle of any Virus, the physical chemistry of Virus Capsid assembly is poorly understood. We have developed general models of Capsid assembly that describe the process in terms of a cascade of low order association reactions. The models predict sigmoidal assembly kinetics, where intermediates approach a low steady state concentration for the greater part of the reaction. Features of the overall reaction can be identified on the basis of the concentration dependence of assembly. In simulations, and on the basis of our understanding of the models, we find that nucleus size and the order of subsequent "elongation" reactions are reflected in the concentration dependence of the extent of the reaction and the rate of the fast phase, respectively. The reaction kinetics deduced for our models of Virus assembly can be related to the assembly of any "spherical" polymer. Using light scattering and size exclusion chromatography, we observed polymerization of assembly domain dimers of hepatitis B Virus (HBV) Capsid protein. Empty Capsids assemble at a rate that is a function of protein concentration and ionic strength. The kinetics of Capsid formation were sigmoidal, where the rate of the fast phase had second-power concentration dependence. The extent of assembly had third-power concentration dependence. Simulations based on the models recapitulated the concentration dependences observed for HBV Capsid assembly. These results strongly suggest that in vitro HBV assembly is nucleated by a trimer of dimers and proceeds by the addition of individual dimeric subunits. On the basis of this mechanism, we suggest that HBV Capsid assembly could be an important target for antiviral therapeutics.
Sharon C Glotzer - One of the best experts on this subject based on the ideXlab platform.
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Simulation studies of a phenomenological model for elongated Virus Capsid formation.
Physical review. E Statistical nonlinear and soft matter physics, 2007Co-Authors: Ting Chen, Sharon C GlotzerAbstract:We study a phenomenological model in which the simulated packing of hard, attractive spheres on a prolate spheroid surface with convexity constraints produces structures identical to those of prolate Virus Capsid structures. Our simulation approach combines the traditional Monte Carlo method with a modified method of random sampling on an ellipsoidal surface and a convex hull searching algorithm. Using this approach we identify the minimum physical requirements for nonicosahedral, elongated Virus Capsids, such as two aberrant flock house Virus particles and the prolate prohead of bacteriophage phi29 , and discuss the implication of our simulation results in the context of recent experimental findings. Our predicted structures may also be experimentally realized by the evaporation-driven assembly of colloidal spheres under appropriate conditions.
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Simulation studies of a phenomenological model for elongated Virus Capsid formation.
Physical Review E, 2007Co-Authors: Ting Chen, Sharon C GlotzerAbstract:We study a phenomenological model in which the simulated packing of hard, attractive spheres on a prolate spheroid surface with convexity constraints produces structures identical to those of prolate Virus Capsid structures. Our simulation approach combines the traditional Monte Carlo method with a modified method of random sampling on an ellipsoidal surface and a convex hull searching algorithm. Using this approach we identify the minimum physical requirements for nonicosahedral, elongated Virus Capsids, such as two aberrant flock house Virus particles and the prolate prohead of bacteriophage $\ensuremath{\phi}29$, and discuss the implication of our simulation results in the context of recent experimental findings. Our predicted structures may also be experimentally realized by the evaporation-driven assembly of colloidal spheres under appropriate conditions.
Nuno C. Santos - One of the best experts on this subject based on the ideXlab platform.
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West Nile Virus Capsid Protein Interacts With Biologically Relevant Host Lipid Systems
Frontiers Media S.A., 2019Co-Authors: Ana S. Martins, Filomena A. Carvalho, André F. Faustino, Ivo C. Martins, Nuno C. SantosAbstract:West Nile and dengue Viruses are closely related flaviViruses, originating mosquito-borne viral infections for which there are no effective and specific treatments. Their Capsid proteins sequence and structure are particularly similar, forming highly superimposable α-helical homodimers. Measuring protein-ligand interactions at the single-molecule level yields detailed information of biological and biomedical relevance. In this work, such an approach was successfully applied on the characterization of the West Nile Virus Capsid protein interaction with host lipid systems, namely intracellular lipid droplets (an essential step for dengue Virus replication) and blood plasma lipoproteins. Dynamic light scattering measurements show that West Nile Virus Capsid protein binds very low-density lipoproteins, but not low-density lipoproteins, and this interaction is dependent of potassium ions. Zeta potential experiments show that the interaction with lipid droplets is also dependent of potassium ions as well as surface proteins. The forces involved on the binding of the Capsid protein with lipid droplets and lipoproteins were determined using atomic force microscopy-based force spectroscopy, proving that these interactions are K+-dependent rather than a general dependence of ionic strength. The Capsid protein interaction with host lipid systems may be targeted in future therapeutic strategies against different flaviViruses. The biophysical and nanotechnology approaches employed in this study may be applied to characterize the interactions of other important proteins from different Viruses, in order to understand their life cycles, as well as to find new strategies to inhibit them
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Dengue Virus Capsid Protein Delivers Nucleic Acids Intracellularly
Biophysical Journal, 2014Co-Authors: Miguel A. R. B. Castanho, Nuno C. Santos, João M. Freire, A. Salomé Veiga, Thaís M. Conceição, Wioleta Kowalczyk, Ronaldo Mohana Borges, David Andreu, Andrea T. Da PoianAbstract:Supercharged proteins are a recently identified class of proteins that have the ability to deliver functional macromolecules into mammalian cells very efficiently. They were first known as bioengineering products but were later found in the human proteome. In this work we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among Capsid proteins. In particular, the Capsid proteins of Viruses from of the Flaviviridae family have all a very high net charge/molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral Capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue Virus Capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains from this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. It is possible that the ability to penetrate cells is part of the native biological functions of these proteins.
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Intracellular nucleic acid delivery by the supercharged dengue Virus Capsid protein
PLOS ONE, 2013Co-Authors: João M. Freire, Nuno C. Santos, Ronaldo Mohana-borges, Thaís M. Conceição, Wioleta Kowalczyk, David Andreu, Andrea T. Da Poian, Ana Salomé Veiga, Miguel A. R. B. CastanhoAbstract:Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among Capsid proteins. In particular, the Capsid proteins of Viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral Capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue Virus Capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral Capsid proteins.
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Characterization of the Interaction of the Dengue Virus Capsid Protein with Lipid Droplets
Biophysical Journal, 2011Co-Authors: Ivo C. Martins, Filomena A. Carvalho, André F. Faustino, Nuno C. Santos, Fabiana A. Carneiro, Ronaldo Mohana-borges, Renata M. S. Pereira, Miguel A. R. B. Castanho, Fabio C. L. Almeida, Andrea T. DapoianAbstract:Dengue Virus affects 100 million people yearly, but this number may grow since Aedes spp. mosquitoes, the disease vectors, are spreading to temperate climates, including in the USA. No effective vaccines are available. A poor understanding of the viral life cycle is to blame, especially regarding the viral assembly and enCapsidation process, mediated by Dengue Virus Capsid Protein (DVCP). DVCP is a symmetric homodimmer α-helical protein that must interact with intracellular lipid droplets during viral enCapsidation. DVCP charge distribution suggests that its α2-α2’ nonpolar region may interact with lipids and the α4-α4’ positive charged region could interact with viral RNA. By employing biophysical techniques combined with bioinformatics tools, we found this hypothesis correct.Nuclear magnetic resonance (NMR) shows a strong interaction with lipid droplets on the N-terminus and the α2-α2’ region of DVCP and points to a conformational change transmitted to the α4-α4’ region (C-terminus) via specific residues located in the α2-α2’ region. Aligning DVCP sequence with 16 FlaviVirus spp. Capsid proteins demonstrates that the residues identified by NMR as important for the lipid droplets interaction are conserved in the genus. Moreover, Dengue and West-Nile Virus Capsid protein structures super-impose in the α2, α3 and α4 helices, pointing to a fold conservation among FlaviVirus spp. DVCP α4-helices superimpose with oligonucleotide binding motifs, being therefore likely to bind RNA. Upon interaction with DVCP, the zeta potential of lipid droplets progressively shifts from negative to positive values, suggesting the positive α4-helices exposure on the surface of the lipid droplet-DVCP conjugate.Concluding, DVCP specifically interacts with lipid droplets via its N-terminus and the α2-α2’ region, resulting in conformational changes in the α4-α4’ region and, finally, the DVCP-RNA binding. These regions could thus be targeted in future dengue drug development strategies.
