The advent of nanoscience and nanotechnology and the development of atomic force microscopy and other single-molecule techniques are leading to a renewed look at viruses from the point of view of the ...physical sciences. As any other solid-state object, virus particles are endowed with mechanical properties such as elasticity or brittleness. Emerging studies on virus mechanics may facilitate the engineering of the physical properties of viruses to improve their potential application in nanotechnology, and may be also relevant to understand virus biology. Viruses are subject to internal and external forces, and as evolving entities they may have selectively adapted their mechanical behavior to resist, or even use, those forces. This article adopts the perspective of structural and molecular virology to review the results obtained to date, using the atomic force microscope, on the mechanical properties of virus particles, their molecular determinants, and possible biological implications.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Direct visualization of pathways followed by single molecules while they spontaneously self-assemble into supramolecular biological machines may provide fundamental knowledge to guide molecular ...therapeutics and the bottom-up design of nanomaterials and nanodevices. Here, high-speed atomic force microscopy is used to visualize self-assembly of the bidimensional lattice of protein molecules that constitutes the framework of the mature human immunodeficiency virus capsid. By real-time imaging of the assembly reaction, individual transient intermediates and reaction pathways followed by single molecules could be revealed. As when assembling a jigsaw puzzle, the capsid protein lattice is randomly built. Lattice patches grow independently from separate nucleation events whereby individual molecules follow different paths. Protein subunits can be added individually, while others form oligomers before joining a lattice or are occasionally removed from the latter. Direct real-time imaging of supramolecular self-assembly has revealed a complex, chaotic process involving multiple routes followed by individual molecules that are inaccessible to bulk (averaging) techniques.
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Chemically and/or genetically engineered viruses, viral capsids and viral-like particles carry the promise of important and diverse applications in biomedicine, biotechnology and nanotechnology. ...Potential uses include new vaccines, vectors for gene therapy and targeted drug delivery, contrast agents for molecular imaging and building blocks for the construction of nanostructured materials and electronic nanodevices. For many of the contemplated applications, the improvement of the physical stability of viral particles may be critical to adequately meet the demanding physicochemical conditions they may encounter during production, storage and/or medical or industrial use. The first part of this review attempts to provide an updated general overview of the fast-moving, interdisciplinary virus engineering field; the second part focuses specifically on the modification of the physical stability of viral particles by protein engineering, an emerging subject that has not been reviewed before.
This study investigates whether the biochemical and antiviral effects of organic compounds that bind different sites in the mature human immunodeficiency virus capsid may be related to the modulation ...of different mechanical properties of the protein lattice from which the capsid is built. Mechanical force was used as a probe to quantify, in atomic force microscopy experiments at physiological pH and ionic strength, ligand-mediated changes in capsid lattice elasticity, breathing, strength against local dislocation by mechanical stress, and resistance to material fatigue. The results indicate that the effects of the tested compounds on assembly or biochemical stability can be linked, from a physics-based perspective, to their interference with the mechanical behavior of the viral capsid framework. The antivirals CAP-1 and CAI-55 increased the intrinsic elasticity and breathing of the capsid protein lattice and may entropically decrease the probability of the capsid protein to assemble into a functionally competent conformation. Antiviral PF74 increased the resistance of the capsid protein lattice to disruption by mechanical stress and material fatigue and may enthalpically strengthen the basal capsid lattice against breakage and disintegration. This study provides proof of concept that the interrogation of the mechanical properties of the nanostructured protein material that makes a virus capsid may provide fundamental insights into the biophysical action of capsid-binding antiviral agents. The implications for drug design by specifically targeting the biomechanics of viruses are discussed.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Self-assembling, protein-based bidimensional lattices are being developed as functionalizable, highly ordered biocoatings for multiple applications in nanotechnology and nanomedicine. Unfortunately, ...protein assemblies are soft materials that may be too sensitive to mechanical disruption, and their intrinsic conformational dynamism may also influence their applicability. Thus, it may be critically important to characterize, understand and manipulate the mechanical features and dynamic behavior of protein assemblies in order to improve their suitability as nanomaterials. In this study, the capsid protein of the human immunodeficiency virus was induced to self-assemble as a continuous, single layered, ordered nanocoating onto an inorganic substrate. Atomic force microscopy (AFM) was used to quantify the mechanical behavior and the equilibrium dynamics ("breathing") of this virus-based, self-assembled protein lattice in close to physiological conditions. The results uniquely provided: (i) evidence that AFM can be used to directly visualize in real time and quantify slow breathing motions leading to dynamic disorder in protein nanocoatings and viral capsid lattices; (ii) characterization of the dynamics and mechanics of a viral capsid lattice and protein-based nanocoating, including flexibility, mechanical strength and remarkable self-repair capacity after mechanical damage; (iii) proof of principle that chemical additives can modify the dynamics and mechanics of a viral capsid lattice or protein-based nanocoating, and improve their applied potential by increasing their mechanical strength and elasticity. We discuss the implications for the development of mechanically resistant and compliant biocoatings precisely organized at the nanoscale, and of novel antiviral agents acting on fundamental physical properties of viruses.
Viruses and their protein capsids can be regarded as biologically evolved nanomachines able to perform multiple, complex biological functions through coordinated mechano-chemical actions during the ...infectious cycle. The advent of nanoscience and nanotechnology has opened up, in the last 10 years or so, a vast number of novel possibilities to exploit engineered viral capsids as protein-based nanoparticles for multiple biomedical, biotechnological or nanotechnological applications. This chapter attempts to provide a broad, updated overview on the self-assembly and engineering of virus capsids, and on applications of virus-based nanoparticles. Different sections provide outlines on: (i) the structure, functions and properties of virus capsids; (ii) general approaches for obtaining assembled virus particles; (iii) basic principles and events related to virus capsid self-assembly; (iv) genetic and chemical strategies for engineering virus particles; (v) some applications of engineered virus particles being developed; and (vi) some examples on the engineering of virus particles to modify their physical properties, in order to improve their suitability for different uses.
Understanding the fundamental principles underlying supramolecular self-assembly may facilitate many developments, from novel antivirals to self-organized nanodevices. Icosahedral virus particles ...constitute paradigms to study self-assembly using a combination of theory and experiment. Unfortunately, assembly pathways of the structurally simplest virus capsids, those more accessible to detailed theoretical studies, have been difficult to study experimentally. We have enabled the in vitro self-assembly under close to physiological conditions of one of the simplest virus particles known, the minute virus of mice (MVM) capsid, and experimentally analyzed its pathways of assembly and disassembly. A combination of electron microscopy and high-resolution atomic force microscopy was used to structurally characterize and quantify a succession of transient assembly and disassembly intermediates. The results provided an experiment-based model for the reversible self-assembly pathway of a most simple (T = 1) icosahedral protein shell. During assembly, trimeric capsid building blocks are sequentially added to the growing capsid, with pentamers of building blocks and incomplete capsids missing one building block as conspicuous intermediates. This study provided experimental verification of many features of self-assembly of a simple T = 1 capsid predicted by molecular dynamics simulations. It also demonstrated atomic force microscopy imaging and automated analysis, in combination with electron microscopy, as a powerful single-particle approach to characterize at high resolution and quantify transient intermediates during supramolecular self-assembly/disassembly reactions. Finally, the efficient in vitro self-assembly achieved for the oncotropic, cell nucleus-targeted MVM capsid may facilitate its development as a drug-encapsidating nanoparticle for anticancer targeted drug delivery.
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Emerging studies at the nanoscale on the relationships between the structure, mechanical properties and infectivity of virus particles are revealing important physics-based foundations of virus ...biology that may have biomedical and nanotechnological applications. Human rhinovirus (HRV) is the major causative agent of common colds leading to important economic losses, and is also associated with more severe diseases. There is renewed interest in developing effective anti-HRV drugs, but none have been approved so far. We have chosen HRV to explore a possible link between virus mechanics and infectivity and the antiviral effect of certain drugs. In particular, we have investigated a suggestion that the antiviral action of drugs that bind to capsid cavities (pockets) may be related to changes in virus stiffness. Mechanical analysis using atomic force microscopy shows that filling the pockets with drugs or genetically introducing bulkier amino acid side chains into the pockets stiffen HRV virions to different extents. Drug-mediated stiffening affected some regions distant from the pockets and involved in genome uncoating, and may be caused by a subtle structural rearrangement of the virus particle. The results also revealed for HRV a quantitative, logarithmic relationship between mechanical stiffening, achieved either by drug binding or introducing bulkier amino acid side chains into the pockets, and reduced infectivity. From a fundamental physics perspective, these drugs may exert their biological effect by decreasing the deformability of the virion, thus impairing its equilibrium dynamics. The results encourage the design of novel antiviral drugs that inhibit infection by mechanically stiffening the viral particles.
Human rhinovirus (HRV), one of the most frequent human pathogens, is the major causative agent of common colds. HRVs also cause or exacerbate severe respiratory diseases, such as asthma or chronic ...obstructive pulmonary disease. Despite the biomedical and socioeconomic importance of this virus, no anti-HRV vaccines or drugs are available yet. Protein-protein interfaces in virus capsids have increasingly been recognized as promising virus-specific targets for the development of antiviral drugs. However, the specific structural elements and residues responsible for the biological functions of these extended capsid regions are largely unknown. In this study, we performed a thorough mutational analysis to determine which particular residues along the capsid interpentamer interfaces are relevant to HRV infection as well as the stage(s) in the viral cycle in which they are involved. The effect on the virion infectivity of the individual mutation to alanine of 32 interfacial residues that, together, removed most of the interpentamer interactions was analyzed. Then, a representative sample that included many of those 32 single mutants were tested for capsid and virion assembly as well as virion conformational stability. The results indicate that most of the interfacial residues, and the interactions they establish, are biologically relevant, largely because of their important roles in virion assembly and/or stability. The HRV interpentamer interface is revealed as an atypical protein-protein interface, in which infectivity-determining residues are distributed at a high density along the entire interface. Implications for a better understanding of the relationship between the molecular structure and function of HRV and the development of novel capsid interface-binding anti-HRV agents are discussed.
The rising concern about the serious medical and socioeconomic consequences of respiratory infections by HRV has elicited a renewed interest in the development of anti-HRV drugs. The conversion into effective drugs of compounds identified via screening, as well as antiviral drug design, rely on the acquisition of fundamental knowledge about the targeted viral elements and their roles during specific steps of the infectious cycle. The results of this study provide a detailed view on structure-function relationships in a viral capsid protein-protein interface, a promising specific target for antiviral intervention. The high density and scattering of the interfacial residues found to be involved in HRV assembly and/or stability support the possibility that any compound designed to bind any particular site at the interface will inhibit infection by interfering with virion morphogenesis or stabilization of the functional virion conformation.
The hollow protein capsids from a number of different viruses are being considered for multiple biomedical or nanotechnological applications. In order to improve the applied potential of a given ...viral capsid as a nanocarrier or nanocontainer, specific conditions must be found for achieving its faithful and efficient assembly in vitro. The small size, adequate physical properties and specialized biological functions of the capsids of parvoviruses such as the minute virus of mice (MVM) make them excellent choices as nanocarriers and nanocontainers. In this study we analyzed the effects of protein concentration, macromolecular crowding, temperature, pH, ionic strength, or a combination of some of those variables on the fidelity and efficiency of self-assembly of the MVM capsid in vitro. The results revealed that the in vitro reassembly of the MVM capsid is an efficient and faithful process. Under some conditions, up to ~40% of the starting virus capsids were reassembled in vitro as free, non aggregated, correctly assembled particles. These results open up the possibility of encapsidating different compounds in VP2-only capsids of MVM during its reassembly in vitro, and encourage the use of virus-like particles of MVM as nanocontainers.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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