Self-assembly Of Capsids Research Articles

Strategy toward In-Cell Self-Assembly of an Artificial Viral Capsid from a Fluorescent Protein-Modified β-Annulus Peptide.

In-cell self-assembly of natural viral capsids is an event that can be visualized under transmission electron microscopy (TEM) observations. By mimicking the self-assembly of natural viral capsids, various artificial protein- and peptide-based nanocages were developed; however, few studies have reported the in-cell self-assembly of such nanocages. Our group developed a β-Annulus peptide that can form a nanocage called artificial viral capsid in vitro, but in-cell self-assembly of the capsid has not been achieved. Here, we designed an artificial viral capsid decorated with a fluorescent protein, StayGold, to visualize in-cell self-assembly. Fluorescence anisotropy measurements and fluorescence resonance energy transfer imaging, in addition to TEM observations of the cells and super-resolution microscopy, revealed that StayGold-conjugated β-Annulus peptides self-assembled into the StayGold-decorated artificial viral capsid in a cell. Using these techniques, we achieved the in-cell self-assembly of an artificial viral capsid.

Point mutation in a virus-like capsid drives symmetry reduction to form tetrahedral cages

Protein capsids are a widespread form of compartmentalization in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximizes the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of unique symmetry-reduced structures. Starting with the encapsulin from Myxococcus xanthus, a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryoelectron microscopy, we determine the structures of a precedented 60-mer icosahedral assembly and an unexpected 36-mer tetrahedron that features significant geometric rearrangements around a new interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple-point mutation to various amino acids and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent a unique example of tetrahedral geometry when surveying all characterized encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in the protein sequence.

A multiscale coarse-grained model of HIV-1 capsid self-assembly

A multiscale coarse-grained model of HIV-1 capsid self-assembly

Biotechnological Frontiers of DNA Nanomaterials Continue to Expand: Bacterial Infection using Virus-Inspired Capsids.

The elegant geometry of viruses has inspired bio-engineers to synthetically explore the self-assembly of polyhedral capsids employed to protect new cargo or change an enzymatic microenvironment. Recently, Yang and co-workers used DNA nanotechnology to revisit the icosahedral capsid structure of the phiX174 bacteriophage and reloaded the original viral genome as cargo into their fully synthetic architecture. Surprisingly, when using a favorable combination of structural rigidity and dynamic multivalent cargo entrapment, the synthetic particles were able to infect non-competent bacterial cells and produce the original phiX174 bacteriophage. This work presents an exciting new direction of DNA nanotech for bio-engineering applications which involve bacterial interactions.

Self-assembly of a fluorescent virus-like particle for imaging in tissues with high autofluorescence.

Virus-like particles (VLPs) are engineered nanoparticles that mimic the properties of viruses-like high tolerance to heat and proteases-but lack a viral genome, making them non-infectious. They are easily modified chemically and genetically, making them useful in drug delivery, enhancing vaccine efficacy, gene delivery, and cancer immunotherapy. One such VLP is Qβ, which has an affinity towards an RNA hairpin structure found in its viral RNA that drives the self-assembly of the capsid. It is possible to usurp the native way infectious Qβ self-assembles to encapsidate its RNA to place enzymes inside the VLP's lumen as a protease-resistant cage. Further, using RNA templates that mimic the natural self-assembly of the native capsid, fluorescent proteins (FPs) have been placed inside VLPs in a "one pot" expression system. Autofluorescence in tissues can lead to misinterpretation of results and unreliable science, so we created a single-pot expression system that uses the fluorescent protein smURFP, which avoids autofluorescence and has spectral properties compatible with standard commercial filter sets on confocal microscopes. In this work, we were able to simplify the existing "one-pot" expression system while creating high-yielding fluorescent VLP nanoparticles that could easily be imaged inside lung epithelial tissue.

Self-Assembly, Self-Folding, and Origami: Comparative Design Principles

Self-assembly is usually considered a parallel process while self-folding and origami are usually considered to be serial processes. We believe that these distinctions do not hold in actual experiments. Based upon our experience with 4D printing, we have developed three additional hybrid classes: (1) templated-assisted (tethered) self-assembly: e.g., when RNA is bound to viral capsomeres, the subunits are constricted in their interactions to have aspects of self-folding as well; (2) self-folding can depend upon interactions with the environment; for example, a protein synthesized on a ribosome will fold as soon as peptides enter the intracellular environment in a serial process whereas if denatured complete proteins are put into solution, parallel folding can occur simultaneously; and, (3) in turbulent environments, chaotic conditions continuously alternate processes. We have examined the 43,380 Dürer nets of dodecahedra and 43,380 Dürer nets of icosahedra and their corresponding duals: Schlegel diagrams. In order to better understand models of self-assembly of viral capsids, we have used both geometric (radius of gyration, convex hulls, angles) and topological (vertex connections, leaves, spanning trees, cutting trees, and degree distributions) perspectives to develop design principles for 4D printing experiments. Which configurations fold most rapidly? Which configurations lead to complete polyhedra most of the time? By using Hamiltonian circuits of the vertices of Dürer nets and Eulerian paths of cutting trees of polyhedra unto Schlegel diagrams, we have been able to develop a systematic sampling procedure to explore the 86,760 configurations, models of a T1 viral capsid with 60 subunits and to test alternatives with 4D printing experiments, use of MagformsTM, and origami models to demonstrate via movies the five processes described above.

Spanning tree model and the assembly kinetics of RNA viruses.

Single-stranded RNA (ssRNA) viruses self-assemble spontaneously in solutions that contain the viral RNA genome molecules and viral capsid proteins. The self-assembly of empty capsids can be understood on the basis of free energy minimization. However, during the self-assembly of complete viral particles in the cytoplasm of an infected cell, the viral genome molecules must be selected from a large pool of very similar host messenger RNA molecules and it is not known whether this also can be understood by free energy minimization. We address this question using a simple mathematical model, the spanning tree model, that was recently proposed for the assembly of small ssRNA viruses. We present a statistical physics analysis of the properties of this model. RNA selection takes place via a kinetic mechanism that operates during the formation of the nucleation complex and that is related to Hopfield kinetic proofreading.

Enveloped artificial viral capsids self-assembled from anionic β-annulus peptide and cationic lipid bilayer.

Anionic artificial viral capsids were self-assembled from β-annulus-EE peptide, then complexed with lipid-bilayer-containing cationic lipids via electrostatic interaction to form enveloped artificial viral capsids. The critical aggregation concentration of the enveloped artificial viral capsid was significantly lower than that of the uncomplexed artificial viral capsid, indicating that the lipid bilayer stabilised the capsid structure.

Hot Spots and Their Contribution to the Self-Assembly of the Viral Capsid: In Silico Prediction and Analysis.

The viral capsid is a macromolecular complex formed by a defined number of self-assembled proteins, which, in many cases, are biopolymers with an identical amino acid sequence. Specific protein–protein interactions (PPI) drive the capsid self-assembly process, leading to several distinct protein interfaces. Following the PPI hot spot hypothesis, we present a conservation-based methodology to identify those interface residues hypothesized to be crucial elements on the self-assembly and thermodynamic stability of the capsid. We validate the predictions through a rigorous physical framework which integrates molecular dynamics simulations and free energy calculations by Umbrella sampling and the potential of mean force using an all-atom molecular representation of the capsid proteins of an icosahedral virus in an explicit solvent. Our results show that a single mutation in any of the structure-conserved hot spots significantly perturbs the quaternary protein–protein interaction, decreasing the absolute value of the binding free energy, without altering the protein’s secondary nor tertiary structure. Our conservation-based hot spot prediction methodology can lead to strategies to rationally modulate the capsid’s thermodynamic properties.

A Multiscale Model for the Self-Assembly of Coat Proteins in Bacteriophage MS2.

The self-assembly of viral capsids is an essential step to the formation of infectious viruses. Elucidating the kinetic mechanisms of how a capsid or virus-like particle assembles could advance our knowledge about the viral lifecycle, as well as the general principles in self-assembly of biomaterials. However, current understanding of capsid assembly remains incomplete for many viruses due to the fact that the transient intermediates along the assembling pathways are experimentally difficult to be detected. In this paper, we constructed a new multiscale computational framework to simulate the self-assembly of virus-like particles. We applied our method to the coat proteins of bacteriophage MS2 as a specific model system. This virus-like particle of bacteriophage MS2 has a unique feature that its 90 sequence-identical dimers can be classified into two structurally various groups: one is the symmetric CC dimer, and the other is the asymmetric AB dimer. The homotypic interactions between AB dimers result in a 5-fold symmetric contact, while the heterotypic interactions between AB and CC dimers result in 6-fold symmetric contact. We found that the assembly can be described as a physical process of phase transition that is regulated by various factors such as concentration and specific stoichiometry between AB and CC dimers. Our simulations also demonstrate that heterotypic and homotypic interfaces play distinctive roles in modulating the assembling kinetics. The interaction between AB and CC dimers is much more dynamic than that between two AB dimers. We therefore suggest that the alternate growth of viral capsid through the heterotypic dimer interactions dominates the assembling pathways. This is, to the best of our knowledge, the first multiscale model to simulate the assembling process of coat proteins in bacteriophage MS2. The generality of this approach opens the door to its further applications in assembly of other viral capsids, virus-like particles, and novel drug delivery systems.

Evolution of Intermediates during Capsid Assembly of Hepatitis B Virus with Phenylpropenamide-Based Antivirals.

Self-assembly of virus capsids is a potential target for antivirals due to its importance in the virus lifecycle. Here, we investigate the effect of phenylpropenamide derivatives B-21 and AT-130 on the assembly of hepatitis B virus (HBV) core protein. Phenylpropenamides are widely believed to yield assembly of spherical particles resembling native, empty HBV capsids. Because the details of assembly can be overlooked with ensemble measurements, we performed resistive-pulse sensing on nanofluidic devices with four pores in series to characterize the size distributions of the products in real time. With its single particle sensitivity and compatibility with typical assembly buffers, resistive-pulse sensing is well-suited for analyzing virus assembly in vitro. We observed that assembly with B-21 and AT-130 produced a large fraction of partially complete virus particles that may be on-path, off-path, or trapped. For both B-21 and AT-130, capsid assembly was more sensitive to disruption under conditions where the interprotein association energy was low at lower salt concentrations. Dilution of the reaction solutions led to the rearrangement of the incomplete particles and demonstrated that these large intermediates may be on-path, but are labile, and exist in a frustrated dynamic equilibrium. During capsid assembly, phenylpropenamide molecules modestly increase the association energy of dimers, prevent intermediates from dissociating, and lead to kinetic trapping where the formation of too many capsids has been initiated, which results in both empty and incomplete particles.

Nanoparticle-Templated Self-Assembly of Viral Capsids Probed by Time-Resolved Absorbance Spectroscopy and X-Ray Scattering

Viral capsid proteins have the remarkable ability to self-assemble around a cargo core, whether their genome, a polyelectrolyte or an inorganic nanoparticle. Although the equilibrium properties of such composite objects have been broadly investigated, little is known about the kinetic pathways leading to their assembly. By exploiting the plasmon resonance effect of gold nanoparticles, we show that the adsorption of viral proteins can be monitored through the redshift of the resonance wavelength. Kinetic measurements reveal that the process occurs in less than a second. The spatial arrangement of nanoparticles is probed by time-resolved small-angle X-ray scattering, and we observe the formation of transient aggregates with a lifetime in the hour range. The biocompatibility of gold nanoparticles induced by adsorbed proteins is of great importance for their use in biomedicine and the reported results should help better understand the processes of formation.

Identification of Factors Promoting HBV Capsid Self-Assembly by Assembly-Promoting Antivirals.

Around 270 million individuals currently live with hepatitis B virus (HBV) infection. Heteroaryldihydropyrimidines (HAPs) are a family of antivirals that target the HBV capsid protein and induce aberrant self-assembly. The capsids formed resemble the native capsid structure but are unable to propagate the virus progeny because of a lack of RNA/DNA. Under normal conditions, self-assembly is initiated by the viral genome. The mode of action of HAPs, however, remains largely unknown. In this work, using molecular dynamics simulations, we attempted to understand the action of HAP by comparing the dynamics of capsid proteins with and without HAPs. We found that the inhibitor is more stable in higher oligomers. It retains its stability in the hexamer throughout 1 μs of simulation. Our results also show that the inhibitor might help in stabilizing the C-terminus, the HBc 149-183 arginine-rich domain of the capsid protein. The C-termini of dimers interact with each other, assisted by the HAP inhibitor. During capsid assembly, the termini are supposed to directly interact with the viral genome, thereby suggesting that the viral genome might work in a similar way to stabilize the capsid protein. Our results may help in understanding the underlying molecular mechanism of HBV capsid self-assembly, which should be crucial for exploring new drug targets and structure-based drug design.

Reversible Supracolloidal Self-Assembly of Cobalt Nanoparticles to Hollow Capsids and Their Superstructures.

The synthesis and spontaneous, reversible supracolloidal hydrogen bond-driven self-assembly of cobalt nanoparticles (CoNPs) into hollow shell-like capsids and their directed assembly to higher order superstructures is presented. CoNPs and capsids form in one step upon mixing dicobalt octacarbonyl (Co2 CO8 ) and p-aminobenzoic acid (pABA) in 1,2-dichlorobenzene using heating-up synthesis without additional catalysts or stabilizers. This leads to pABA capped CoNPs (core ca. 5 nm) with a narrow size distribution. They spontaneously assemble into tunable spherical capsids (d≈50-200 nm) with a few-layered shells, as driven by inter-nanoparticle hydrogen bonds thus warranting supracolloidal self-assembly. The capsids can be reversibly disassembled and reassembled by controlling the hydrogen bonds upon heating or solvent exchanges. The superparamagnetic nature of CoNPs allows magnetic-field-directed self-assembly of capsids to capsid chains due to an interplay of induced dipoles and inter-capsid hydrogen bonds. Finally, self-assembly on air-water interface furnishes lightweight colloidal framework films.

Incorporation of the Kaposi’s sarcoma-associated herpesvirus capsid vertex-specific component (CVSC) into self-assembled capsids

Self-assembly of herpesvirus capsids can be accomplished in heterologous expression systems provided all six capsid proteins are present. We have demonstrated the assembly of icosahedral Kaposi’s sarcoma-associated herpesvirus (KSHV) capsids in insect cells using the baculovirus expression system. Using this self-assembly system we investigated whether we could add additional capsid associated proteins and determine their incorporation into the assembled capsid. We chose the capsid vertex-specific component (CVSC) proteins encoded by open reading frames (ORFs) 19 and 32 to test this. This complex sits on the capsid vertex and is important for capsid maturation in herpesvirus-infected cells. Co-immunoprecipitation assays were used to initially confirm a bi-molecular interaction between ORF19 and ORF32. Both proteins also precipitated the triplex proteins of the capsid shell (ORF26 and ORF62) as well as the major capsid protein (ORF25). Capsid immunoprecipitation assays revealed the incorporation of ORF19 as well as ORF32 into assembled capsids. Similar experiments also showed that the incorporation of each protein occurred independent of the other. These studies reveal biochemically how the KSHV CVSC interacts with the capsid shell.

Self-assembly of virus capsids decorated with block copolymers: a simulation study

Self-assembly of biocompatible nanoparticles is part of a promising field in drug delivery and biomaterials. Virus capsids are an example of nanoparticles capable of being tethered with functional groups for specific targeting. There have been experimental efforts on grafting polymers to virus capsids to synthesize tailored nanostructures. To provide insight at the nanoscale, we perform a highly coarse-grained molecular dynamics study, simulating the self-aggregation of cowpea mosaic virus (CPMV) capsids decorated with polyethylene glycol (PEG) and PEG polylactic acid (PLA) block polymers. We examined the effects of grafting architecture and volume fraction on equilibrated clusters. Characterization of the aggregation dynamics are summarized by the radius of gyration of the clusters, coordination number distributions, and average cluster size. When the system and methods are parameterized with respect to atomistic models or empirical results, the results can serve as the basis in broadly mapping the theoretical design space for controlled self-assembly of polymer-decorated virus capsids.

Coarse-grained simulation reveals key features of HIV-1 capsid self-assembly.

The maturation of HIV-1 viral particles is essential for viral infectivity. During maturation, many copies of the capsid protein (CA) self-assemble into a capsid shell to enclose the viral RNA. The mechanistic details of the initiation and early stages of capsid assembly remain to be delineated. We present coarse-grained simulations of capsid assembly under various conditions, considering not only capsid lattice self-assembly but also the potential disassembly of capsid upon delivery to the cytoplasm of a target cell. The effects of CA concentration, molecular crowding, and the conformational variability of CA are described, with results indicating that capsid nucleation and growth is a multi-stage process requiring well-defined metastable intermediates. Generation of the mature capsid lattice is sensitive to local conditions, with relatively subtle changes in CA concentration and molecular crowding influencing self-assembly and the ensemble of structural morphologies.

Assembly, Engineering and Applications of Virus-Based Protein Nanoparticles.

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.

Early stage P22 viral capsid self-assembly mediated by scaffolding protein: atom-resolved model and molecular dynamics simulation.

Molecular dynamics simulation of an atom-resolved bacteriophage P22 capsid model is used to delineate the underlying mechanism of early stage P22 self-assembly. A dimer formed by the C-terminal fragment of scaffolding protein with a new conformation is demonstrated to catalyze capsomer (hexamer and pentamer) aggregation efficiently. Effects of scaffolding protein/coat protein binding patterns and scaffolding protein concentration on efficiency, fidelity, and capsid curvature of P22 self-assembly are identified.

Role of dynamic capsomere supply for viral capsid self-assembly

Many viruses rely on the self-assembly of their capsids to protect and transport their genomic material. For many viral systems, in particular for human viruses like hepatitis B, adeno or human immunodeficiency virus, that lead to persistent infections, capsomeres are continuously produced in the cytoplasm of the host cell while completed capsids exit the cell for a new round of infection. Here we use coarse-grained Brownian dynamics simulations of a generic patchy particle model to elucidate the role of the dynamic supply of capsomeres for the reversible self-assembly of empty T1 icosahedral virus capsids. We find that for high rates of capsomere influx only a narrow range of bond strengths exists for which a steady state of continuous capsid production is possible. For bond strengths smaller and larger than this optimal value, the reaction volume becomes crowded by small and large intermediates, respectively. For lower rates of capsomere influx a broader range of bond strengths exists for which a steady state of continuous capsid production is established, although now the production rate of capsids is smaller. Thus our simulations suggest that the importance of an optimal bond strength for viral capsid assembly typical for in vitro conditions can be reduced by the dynamic influx of capsomeres in a cellular environment.