Non-enveloped spherical viruses (NSVs) are characterized by their highly symmetrical capsids that serve to protect and encapsulate the genomes. The stability and functionality of the capsids determine their ability for survival and proliferation in harsh environments. Over four decades of structural studies using X-ray crystallography and NMR have provided static, high-resolution snapshots of several viruses. Recently, advances in cryo-electron microscopy, together with AI-based structure predictions and traditional methods, have aided in elucidating not only the structural details of complex NSVs but also the mechanistic processes underlying their assembly. The knowledge thus generated has been instrumental in critical understanding of the conformational changes and interactions associated with the coat proteins, the genome, and the auxiliary factors that regulate the capsid dynamics. This review seeks to summarize current literature regarding the structure and assembly of the NSVs and discusses how the data has facilitated a deeper understanding of their biology and phylogeny.

This work presents a novel application of additive manufacturing in the design of self-assembling helical viral capsids using 3D-printed components. Expanding on prior work with 3D-printed self-assembling spherical capsids, we developed helical models that integrate geometric parameters and magnetic interactions to mimic key features of the assembly process of helical viral capsids. Using dual-helix phyllotactic patterns and simplified electrostatic simulations, these models consistently self-assemble into a cylinder, providing unique insights into the structural organization and stability of helical capsids. This accessible 3D-printed approach demonstrates the potential of additive manufacturing for research in mesoscale self-assembling models and in the education of complex biological assembly processes, promoting hands-on exploration of viral architecture and self-assembly mechanisms.

Helices are one of the most frequently encountered symmetries in biological assemblies. Helical symmetry has been exploited in electron microscopic studies as a limited number of filament images, in principle, can provide all the information needed to do a three-dimensional reconstruction of a polymer. Over the past 25 years, three-dimensional reconstructions of helical polymers from cryo-EM images have shifted completely from Fourier-Bessel methods to single-particle approaches. The single-particle approaches have allowed people to surmount the problem that very few biological polymers are crystalline in order, and despite the flexibility and heterogeneity present in most of these polymers, reaching a resolution where accurate atomic models can be built has now become the standard. While determining the correct helical symmetry may be very simple for something like F-actin, for many other polymers, particularly those formed from small peptides, it can be much more challenging. This review discusses why symmetry determination can be problematic, and why trial-and-error methods are still the best approach. Studies of many macromolecular assemblies, such as icosahedral capsids, have usually found that not imposing symmetry leads to a great reduction in resolution while at the same time revealing possibly interesting asymmetric features. We show that for certain helical assemblies asymmetric reconstructions can sometimes lead to greatly improved resolution. Further, in the case of supercoiled flagellar filaments from bacteria and archaea, we show that the imposition of helical symmetry can not only be wrong, but is not necessary, and obscures the mechanisms whereby these filaments supercoil.

Since biochemists and biologists have progressed in understanding the mechanisms involved in living organisms, biological systems have become a source of inspiration for chemists. In this context, the concept of colloidal tectonics, describing the spontaneous formation of colloidal particles or supracolloidal structures in which the building blocks are called "tectons", has emerged. Therefore, a bottom-up edification of tectons towards (supra) colloidal structures is allowed. Each (supra) colloidal system has at least one of the following properties: amphiphilicity, predictability, versatility, commutability, and reversibility. However, for these systems to perform even more interesting functions, it is necessary for tectons to have very precise chemical and physical properties so that new properties emerge in (supra) colloidal systems. In this way, colloidal tectonics enables engineering at the nano- and micrometric level and contributes to the development of smart bioinspired systems with applications in catalysis, drug delivery, etc. In this review, an overview of the concept of colloidal tectonics is illustrated by some biotic systems. The design of abiotic (supra) colloidal systems and their applications in various fields are also addressed (notably Pickering emulsions for catalysis or drug delivery). Finally, theoretical directions for the design of novel self-assembled (supra) colloidal systems are discussed.

Spherical supramolecular dendrimers including helical, self-organize soft Frank-Kasper, other cubic such as body-centered cubic, and quasicrystal periodic and quasiperiodic arrays. When any of these periodic or quasiperiodic arrays forms immediately above a columnar phase, a supramolecular orientational memory effect was found to discriminate between mechanisms of self-organization of supramolecular spheres and generate unprecedented periodic arrays of helical columns which cannot be constructed by any other methodology. Here, we demonstrate that unwinding spherical helices, via their precursor nonhelical columns, increases the entropy and stability of their periodic and quasiperiodic spherical arrays and places the Frank-Kasper and other cubic phases immediately above the columnar phase. This process is not available in biology where spherical viruses self-organize body-centered cubic lattices. However, this concept reengineers, on increasing temperature, the originally expected position of the periodic and quasiperiodic array versus that of the columnar lattice. This process facilitates discrimination between different self-organization mechanisms of supramolecular spheres and also mediates the emergence of unprecedentedly complex and technologically important periodic arrays of nonhelical columns.

Cryo-electron microscopy and tomography have allowed us to unveil the remarkable structure of icosahedral viruses. However, in the past few years, the idea that these viruses must have perfectly symmetric virions, but in some cases, it might not be true. This has opened the door to challenging paradigms in structural virology and raised new questions about the biological implications of "unusual" or "defective" symmetries and structures. Also, the continual improvement of these technologies, coupled with more rigorous sample purification protocols, improvements in data processing, and the use of artificial intelligence, has allowed solving the structure of sub-viral particles in highly heterogeneous samples and finding novel symmetries or structural defects. In this review, I initially analyzed the case of the symmetry and composition of hepatitis B virus-produced spherical sub-viral particles. Then, I focused on Alphaviruses as an example of "imperfect" icosahedrons and analyzed how structural biology has changed our understanding of the Alphavirus assembly and some biological implications arising from these discoveries.

In a recent issue of Nature, Coshic et al. employ a computational multiscale approach to package the complete HK97 viral genome into its capsid. They find both good agreement with experimental observations and shed new light on the heterogeneity of genome structures and the mechanism by which they package.

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.

Methods in protein design have made it possible to create large and complex, self-assembling protein cages with diverse applications. These have largely been based on highly symmetric forms exemplified by the Platonic solids. Prospective applications of protein cages would be expanded by strategies for breaking the designed symmetry, for example, so that only one or a few (instead of many) copies of an exterior domain or motif might be displayed on their surfaces. Here we demonstrate a straightforward design approach for creating symmetry-broken protein cages able to display singular copies of outward-facing domains. We modify the subunit of an otherwise symmetric protein cage through fusion to a small inward-facing domain, only one copy of which can be accommodated in the cage interior. Using biochemical methods and native mass spectrometry, we show that co-expression of the original subunit and the modified subunit, which is further fused to an outward-facing anti-GFP DARPin domain, leads to self-assembly of a protein cage presenting just one copy of the DARPin protein on its exterior. This strategy of designed occlusion provides a facile route for creating new types of protein cages with unique properties.

The emergence of antibiotic resistant bacteria and the ineffectiveness of routine treatments inspired development of alternatives to biocides for antibacterial applications. Bacteriophages are natural predators of bacteria and are promising alternatives to antibiotics. This study presents fabrication of a Salmonella enterica bacteriophage containing ultra-thin multilayer film composed of chitosan and alginate and demonstrates its potential as an antibacterial coating for food packaging applications. Chitosan/alginate film was prepared through layer-by-layer (LbL) self-assembly technique. A bacteriophage, which belongs to Siphoviridae morphotype (MET P1-001_43) and infects Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis), was post-loaded into chitosan/alginate film. The LbL growth, stability, and surface morphology of chitosan/alginate film as well as phage deposition into multilayers were analysed through ellipsometry, QCM-D and AFM techniques. The bacteriophage containing multilayers showed antibacterial activity at pH 7.0. In contrast, anti-bacterial activity was not observed at acidic conditions. We showed that wrapping a Salmonella Enteritidis contaminated chicken piece with aluminium foil whose surface was modified with phage loaded chitosan/alginate multilayers decreased the number of colonies on the chicken meat, and it was as effective as treating the meat directly with phage solution.

Protein capsids are a widespread form of compartmentalisation in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximises 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 novel 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 cryo-EM, we determine the structures of a precedented 60-mer icosahedral assembly and an unprecedented 36-mer tetrahedron that features significant geometric rearrangements around a novel 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 the first example of tetrahedral geometry across all characterised 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 protein sequence.

Protein nanocages have diverse applications in medicine and biotechnology, including molecular delivery. However, although numerous studies have demonstrated the ability of protein nanocages to encapsulate various molecular species, limited methods are available for subsequently opening a nanocage for cargo release under specific conditions. A modular platform with a specific protein-target-based mechanism of nanocage opening is notably lacking. To address this important technology gap, we present a new class of designed protein cages, the Ligand-Operable Cage (LOC). LOCs primarily comprise a protein nanocage core and a fused surface binding adaptor. The geometry of the LOC is designed so that binding of a target protein ligand (or multiple copies thereof) to the surface binder is sterically incompatible with retention of the assembled state of the cage. Therefore, the tight binding of a target ligand drives cage disassembly by mass action, subsequently exposing the encapsulated cargo. LOCs are modular; direct substitution of the surface binder sequence can reprogram the nanocage to open in response to any target protein ligand of interest. We demonstrate these design principles using both a natural and a designed protein cage as the core, with different proteins acting as the triggering ligand and with different reporter readouts─fluorescence unquenching and luminescence─for cage disassembly. These developments advance the critical problem of targeted molecular delivery and detection.

Artificial protein cages have great potential in diverse fields including as vaccines and drug delivery vehicles. TRAP-cage is an artificial protein cage notable for the way in which the interface between its ring-shaped building blocks can be modified such that the conditions under which cages disassemble can be controlled. To date, TRAP-cages have been constructed from homo-11mer rings, i.e., hendecamers. This is interesting as convex polyhedra with identical regular faces cannot be formed from hendecamers. TRAP-cage overcomes this limitation due to intrinsic flexibility, allowing slight deformation to absorb any error. The resulting TRAP-cage made from 24 TRAP 11mer rings is very close to regular with only very small errors necessary to allow the cage to form. The question arises as to the limits of the error that can be absorbed by a protein structure in this way before the formation of an apparently regular convex polyhedral becomes impossible. Here we use a naturally occurring TRAP variant consisting of twelve identical monomers (i.e., a dodecamer) to probe these limits. We show that it is able to form an apparently regular protein cage consisting of twelve TRAP rings. Comparison of the cryo-EM structure of the new cage with theoretical models and related cages gives insight into the rules of cage formation and allows us to predict other cages that may be formed given TRAP-rings consisting of different numbers of monomers.

All matter must obey the general laws of physics and living matter is not an exception. Viruses have not only learnt how to cope with them but have managed to use them for their own survival. In this chapter, we will review some of the exciting physics that are behind viruses and discuss simple physical models that can shed some light on different aspects of the viral life cycle and viral properties. In particular, we will focus on how the structure and shape of the viral capsid, its assembly and stability, and the entry and exit of viral particles and their genomes can be explained using fundamental physics theories.

Viruses are elegant macromolecular assemblies and constitute a paradigm of the economy of genomic resources; they must use simple general principles to complete their life cycles successfully. Viruses need only one or a few different capsid structural subunits to build an infectious particle, which is possible for two reasons: extensive use of symmetry and built-in conformational flexibility. Although viruses come in many shapes and sizes, two major symmetric assemblies are found: icosahedral and helical. The enormous diversity of virus structures appears to be derived from one or a limited number of basic schemes that became more complex by consecutive incorporation of additional structural elements. The intrinsic structural polymorphism of the viral proteins results in dynamic capsids. The study of virus structures is required to understand structure-function relationships, including those related to morphogenesis and antigenicity, among many others. These structural foundations can be extended to other macromolecular complexes that control many fundamental processes in biology.

AlphaFold can predict the structures of monomeric and multimeric proteins with high accuracy but has a limit on the number of chains and residues it can fold. Here we show that a combination of AlphaFold and all-atom symmetric docking simulations enables highly accurate prediction of the structure of complex symmetrical assemblies. We present a method to predict the structure of complexes with cubic - tetrahedral, octahedral and icosahedral - symmetry from sequence. Focusing on proteins where AlphaFold can make confident predictions on the subunit structure, 27 cubic systems were assembled with a median TM-score of 0.99 and a DockQ score of 0.72. 21 had TM-scores of above 0.9 and were categorized as acceptable- to high-quality according to DockQ. The resulting models are energetically optimized and can be used for detailed studies of intermolecular interactions in higher-order symmetrical assemblies. The results demonstrate how explicit treatment of structural symmetry can significantly expand the size and complexity of AlphaFold predictions.

Protein nanoparticles play pivotal roles in many areas of bionanotechnology, including drug delivery, vaccination, and diagnostics. These technologies require control over the distinct particle morphologies that protein nanocontainers can adopt during self-assembly from their constituent protein components. The geometric construction principle of virus-derived protein cages is by now fairly well understood by analogy to viral protein shells in terms of Caspar and Klug's quasi-equivalence principle. However, many artificial, or genetically modified, protein containers exhibit varying degrees of quasi-equivalence in the interactions between identical protein subunits. They can also contain a subset of protein subunits that do not participate in interactions with other assembly units, called capsomers, leading to gaps in the particle surface. We introduce a method that exploits information on the local interactions between the capsomers to infer the geometric construction principle of these nanoparticle architectures. The predictive power of this approach is demonstrated here for a prominent system in nanotechnology, the AaLS pentamer. Our method not only rationalises hitherto discovered cage structures but also predicts geometrically viable options that have not yet been observed. The classification of nanoparticle architecture based on the geometric properties of the interaction network closes a gap in our current understanding of protein container structure and can be widely applied in protein nanotechnology, paving the way to programmable control over particle polymorphism.

The ability to view structures of proteins at atomic resolution, facilitated by the rise of macromolecular crystallography, has had a tremendous impact in many areas of sciences, including molecular pharmacology, drug discovery and biotechnology. However, the teaching of macromolecular crystallography in universities across the globe has been less than optimal. This could be attributed to the interdisciplinary nature of this subject, making it appear esoteric and incomprehensible, at least at first glance, for students who have exclusive training in only one specific discipline. For the instructor, this problem is compounded further by the plethora of complex concepts and specialized terminologies that the science of macromolecular crystallography has accumulated over the course of its evolution. Moreover, the advent of robotics and several sophisticated software algorithms have reduced the incentive to understand the beautiful conceptual bedrock on which this subject is based. As a way of addressing some of the challenges delineated above, this Words of Advice article attempts to formulate the broad framework within which the teaching and learning of macromolecular crystallography should be approached. It advocates the acknowledgement that this is an interdisciplinary field, with substantial contributions from chemical, physical, biological and mathematical sciences, requiring the evolution of teaching approaches that acknowledge this reality. Moreover, it suggests the use of visual tools, use of computational resources and history to make the subject more relatable to students.

As nanotechnology continues to push the boundaries across disciplines, there is an increasing need for engineering nanomaterials with atomic-level precision for self-assembly across length scales, i.e., from the nanoscale to the macroscale. Although molecular self-assembly allows atomic precision, extending it beyond certain length scales presents a challenge. Therefore, the attention has turned to size and shape-controlled metal nanoparticles as building blocks for multifunctional colloidal self-assemblies. However, traditionally, metal nanoparticles suffer from polydispersity, uncontrolled aggregation, and inhomogeneous ligand distribution, resulting in heterogeneous end products. In this feature article, I will discuss how virus capsids provide clues for designing subunit-based, precise, efficient, and error-free self-assembly of colloidal molecules. The atomically precise nanoscale proteinic subunits of capsids display rigidity (conformational and structural) and patchy distribution of interacting sites. Recent experimental evidence suggests that atomically precise noble metal nanoclusters display an anisotropic distribution of ligands and patchy ligand bundles. This enables symmetry breaking, consequently offering a facile route for two-dimensional colloidal crystals, bilayers, and elastic monolayer membranes. Furthermore, inter-nanocluster interactions mediated via the ligand functional groups are versatile, offering routes for discrete supracolloidal capsids, composite cages, toroids, and macroscopic hierarchically porous frameworks. Therefore, engineered nanoparticles with atomically precise structures have the potential to overcome the limitations of molecular self-assembly and large colloidal particles. Self-assembly allows the emergence of new optical properties, mechanical strength, photothermal stability, catalytic efficiency, quantum yield, and biological properties. The self-assembled structures allow reproducible optoelectronic properties, mechanical performance, and accurate sensing. More importantly, the intrinsic properties of individual nanoclusters are retained across length scales. The atomically precise nanoparticles offer enormous potential for next-generation functional materials, optoelectronics, precision sensors, and photonic devices.

Understanding the principles governing protein arrangement in viral capsids and structurally similar protein shells can enable the development of new antiviral strategies and the design of artificial protein cages for various applications. We study these principles within the context of the close packing problem, by analyzing dozens of small spherical shells assembled from a single type of protein. First, we use icosahedral spherical close packings containing 60T identical disks, where T ≤ 4, to rationalize the protein arrangement in twenty real icosahedral shells both satisfying and violating the paradigmatic Caspar-Klug model. We uncover a striking correspondence between the protein mass centers in the considered shells and the centers of disks in the close packings. To generalize the packing model, we consider proteins with a weak shape anisotropy and propose an interaction energy, minimization of which allows us to obtain spherical dense packings of slightly anisotropic structural units. In the case of strong anisotropy, we model the proteins as sequences of overlapping discs of different sizes, with minimum energy configuration not only resulting in packings, accurately reproducing locations and orientations of individual proteins, but also revealing that icosahedral packings that display the handedness of real capsids are energetically more favorable. Finally, by introducing effective disc charges, we rationalize the formation of inter-protein bonds in protein shells.

Methods in protein design have made it possible to create large and complex, self-assembling protein cages with diverse applications. These have largely been based on highly symmetric forms exemplified by the Platonic solids. Prospective applications of protein cages would be expanded by strategies for breaking the designed symmetry, e.g., so that only one or a few (instead of many) copies of an exterior domain or motif might be displayed on their surfaces. Here we demonstrate a straightforward design approach for creating symmetry-broken protein cages able to display singular copies of outward-facing domains. We modify the subunit of an otherwise symmetric protein cage through fusion to a small inward-facing domain, only one copy of which can be accommodated in the cage interior. Using biochemical methods and native mass spectrometry, we show that co-expression of the original subunit and the modified subunit, which is further fused to an outward-facing anti-GFP DARPin domain, leads to self-assembly of a protein cage presenting just one copy of the DARPin protein on its exterior. This strategy of designed occlusion provides a facile route for creating new types of protein cages with unique properties.

Artificial protein cages made from multiple copies of a single protein can be produced such that they only assemble upon addition of a metal ion. Consequently, the ability to remove the metal ion triggers protein-cage disassembly. Controlling assembly and disassembly has many potential uses including cargo loading/unloading and hence drug delivery. TRAP-cage is an example of such a protein cage which assembles due to linear coordination bond formation with Au(I) which acts to bridge constituent proteins. Here we describe the method for production and purification of TRAP-cage.

The capsid-like structure of the virus-based protein nanoparticles (NPs) can serve as bionanomaterials, with applications in biomedicines and nanotechnology. Release of packaged material from these nanocontainers is associated with subtle conformational changes of the NP structure, which in vitro, is readily accomplished by heating. Characterizing the structural changes as a function of temperature may provide fresh insights into nanomaterial/antiviral strategies. Here, we have calculated heat induced changes in the properties of an empty minute virus of mice particle using large-scale ≈ 3.0 × 10⁶ all-atom molecular dynamics simulations. We focus on two heat induced structural changes of the NP, namely, dynamical transition (DT) and breathing transition (BT), both characterized by sudden and sharp change of measured parameters at temperatures, T_DT and T_BT, respectively. While DT is assessed by mean-square fluctuation of hydrogen atoms of the NP, BT is monitored through internal volume and permeation rate of water molecules through the NP. Both the transitions, resulting primarily from collective atomistic motion, are found to occur at temperatures widely separated from one another (T_BT>T_DT). The breathing motions, responsible for the translocation events of the packaged materials through the NP to kick off, are further probed by computing atomic resolution stresses from NVE simulations. Distribution of equilibrium atomistic stresses on the NP reveals a largely asymmetric nature and suggests structural breathing may actually represent large dynamic changes in the hotspot regions, far from the NP pores, which is in remarkable resemblance with recently conducted hydrogen-deuterium exchange coupled to mass spectrometry experiment. Communicated by Ramaswamy H. Sarma.

Viruses are the most abundant biological entities on Earth, and yet, they have not received enough consideration in astrobiology. Viruses are also extraordinarily diverse, which is evident in the types of relationships they establish with their host, their strategies to store and replicate their genetic information and the enormous diversity of genes they contain. A viral population, especially if it corresponds to a virus with an RNA genome, can contain an array of sequence variants that greatly exceeds what is present in most cell populations. The fact that viruses always need cellular resources to multiply means that they establish very close interactions with cells. Although in the short term these relationships may appear to be negative for life, it is evident that they can be beneficial in the long term. Viruses are one of the most powerful selective pressures that exist, accelerating the evolution of defense mechanisms in the cellular world. They can also exchange genetic material with the host during the infection process, providing organisms with capacities that favor the colonization of new ecological niches or confer an advantage over competitors, just to cite a few examples. In addition, viruses have a relevant participation in the biogeochemical cycles of our planet, contributing to the recycling of the matter necessary for the maintenance of life. Therefore, although viruses have traditionally been excluded from the tree of life, the structure of this tree is largely the result of the interactions that have been established throughout the intertwined history of the cellular and the viral worlds. We do not know how other possible biospheres outside our planet could be, but it is clear that viruses play an essential role in the terrestrial one. Therefore, they must be taken into account both to improve our understanding of life that we know, and to understand other possible lives that might exist in the cosmos.

Understanding the principles of protein packing and the mechanisms driving morphological transformations in virus shells (capsids) during their maturation can be pivotal for the development of new antiviral strategies. Here, we study how these principles and mechanisms manifest themselves in icosahedral viral capsids assembled from identical symmetric structural units (capsomeres). To rationalize such shells, we model capsomers as symmetrical groups of identical particles interacting with a short-range potential typical of the classic Tammes problem. The capsomere particles are assumed to retain their relative positions on the vertices of planar polygons placed on the spherical shell and to interact only with the particles from other capsomeres. Minimization of the interaction energy enforces equal distances between the nearest particles belonging to neighboring capsomeres and minimizes the number of different local environments. Thus, our model implements the Caspar and Klug quasi-equivalence principle and leads to packings strikingly similar to real capsids. We then study a reconstruction of protein trimers into dimers in a Flavivirus shell during its maturation, connecting the relevant structural changes with the modifications of the electrostatic charges of proteins, wrought by the oxidative switch in the bathing solution that is essential for the process. We highlight the key role of pr peptides in the shell reconstruction and show that the highly ordered arrangement of these subunits in the dimeric state is energetically favored at a low pH level. We also discuss the electrostatic mechanisms controlling the release of pr peptides in the last irreversible step of the maturation process.

Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA-protein interactions are thought to play a critical role in the assembly, our understanding of their effects is limited because the assembly process is difficult to observe directly. We address this problem by using interferometric scattering microscopy, a sensitive optical technique with high dynamic range, to follow the in vitro assembly kinetics of more than 500 individual particles of brome mosaic virus (BMV)-for which RNA-protein interactions can be controlled by varying the ionic strength of the buffer. We find that when RNA-protein interactions are weak, BMV assembles by a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before additional proteins can bind. As the strength of RNA-protein interactions increases, the nucleation time becomes shorter and more narrowly distributed, but the time to grow a capsid after nucleation is largely unaffected. These results suggest that the nucleation rate is controlled by RNA-protein interactions, while the growth process is driven less by RNA-protein interactions and more by protein-protein interactions and intraprotein forces. The nucleated pathway observed with the plant virus BMV is strikingly similar to that previously observed with bacteriophage MS2, a phylogenetically distinct virus with a different host kingdom. These results raise the possibility that nucleated assembly pathways might be common to other RNA viruses.

Tile-based DNA self-assembly is a powerful approach for nano-constructions. In this approach, individual DNA single strands first assemble into well-defined structural tiles, which, then, further associate with each other into final nanostructures. It is a general assumption that the lower-level structures (tiles) determine the higher-level, final structures. In this study, we present concrete experimental data to show that higher-level structures could, at least in the current example, also impact on the formation of lower-level structures. This study prompts questions such as: how general is this phenomenon in programmed DNA self-assembly and can we turn it into a useful tool for fine tuning DNA self-assembly?

While the application of cryogenic electron microscopy (cryo-EM) to helical polymers in biology has a long history, due to the huge number of helical macromolecular assemblies in viruses, bacteria, archaea, and eukaryotes, the use of cryo-EM to study synthetic soft matter noncovalent polymers has been much more limited. This has mainly been due to the lack of familiarity with cryo-EM in the materials science and chemistry communities, in contrast to the fact that cryo-EM was developed as a biological technique. Nevertheless, the relatively few structures of self-assembled peptide nanotubes and ribbons solved at near-atomic resolution by cryo-EM have demonstrated that cryo-EM should be the method of choice for a structural analysis of synthetic helical filaments. In addition, cryo-EM has also demonstrated that the self-assembly of soft matter polymers has enormous potential for polymorphism, something that may be obscured by techniques such as scattering and spectroscopy. These cryo-EM structures have revealed how far we currently are from being able to predict the structure of these polymers due to their chaotic self-assembly behavior.

Gene therapy provides the exciting opportunity of a curative single treatment for devastating diseases, eradicating the need for chronic medication. Adeno-associated viruses (AAVs) are among the most attractive vector carriers for gene replacement in vivo. Yet, despite the success of recent AAV-based clinical trials, the clinical use of these vectors has been limited. For instance, the AAV packaging capacity is restricted to ~4.7 kb, making it a substantial challenge to deliver large gene products.

In this review, we explore established and emerging strategies that circumvent the packaging limit of AAVs to make them effective vehicles for gene replacement therapy of monogenic disorders, with a particular focus on diseases affecting the nervous system. We report historical references, design remarks, as well as strengths and weaknesses of these approaches. We additionally discuss examples of neurological disorders for which such strategies have been attempted.

The field of AAV-gene therapy has experienced enormous advancements in the last decade. However, there is still ample space for improvement aimed at overcoming existing challenges that are slowing down the progressive trajectory of this field.

Goldberg polyhedra have been widely studied across multiple fields, as their distinctive pattern can lead to many useful applications. Their topology can be determined using Goldberg's method through generating topologically equivalent structures, named cages. However, the geometry of Goldberg polyhedra remains underexplored. This study extends Goldberg's framework to a new method that can systematically determine the topology and effectively control the geometry of Goldberg polyhedra based on the initial shapes of cages. In detail, we first parametrize the cage's geometry under specified topology and polyhedral symmetry; then, we manipulate the predefined independent variables through optimization to achieve the user-defined geometric properties. The benchmark problem of finding equilateral Goldberg polyhedra is solved to demonstrate the effectiveness of the proposed method. Using this method, we have successfully achieved nearly exact spherical Goldberg polyhedra, with all vertices on a sphere and all faces being planar under extremely low numerical errors. Such results serve as strong numerical evidence for the existence of this new type of Goldberg polyhedra. Furthermore, we iteratively perform k-means clustering and optimization to significantly reduce the number of different edge lengths to benefit the cost reduction for architectural and engineering applications.

The symmetry of biological molecules has fascinated structural biologists ever since the structure of hemoglobin was determined. The Protein Data Bank (PDB) archive is the central global archive of three-dimensional (3D), atomic-level structures of biomolecules, providing open access to the results of structural biology research with no limitations on usage. Roughly 40% of the structures in the archive exhibit some type of symmetry, including formal global symmetry, local symmetry, or pseudosymmetry. The Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (founding member of the Worldwide Protein Data Bank partnership that jointly manages, curates, and disseminates the archive) provides a variety of tools to assist users interested in exploring the symmetry of biological macromolecules. These tools include multiple modalities for searching and browsing the archive, turnkey methods for biomolecular visualization, documentation, and outreach materials for exploring functional biomolecular symmetry.

The history of tobacco mosaic virus (TMV) includes many firsts in science, beginning with its being the first virus identified. This review offers an overview of a history of research on TMV, with an emphasis on its close connections to the emergence and development of molecular biology. Expected final online publication date for the Annual Review of Virology, Volume 9 is September 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Spindle- or lemon-shaped viruses infect archaea in diverse environments. Due to the highly pleomorphic nature of these virions, which can be found with cylindrical tails emanating from the spindle-shaped body, structural studies of these capsids have been challenging. We have determined the atomic structure of the capsid of Sulfolobus monocaudavirus 1, a virus that infects hosts living in nearly boiling acid. A highly hydrophobic protein, likely integrated into the host membrane before the virions assemble, forms 7 strands that slide past each other in both the tails and the spindle body. We observe the discrete steps that occur as the tail tubes expand, and these are due to highly conserved quasiequivalent interactions with neighboring subunits maintained despite significant diameter changes. Our results show how helical assemblies can vary their diameters, becoming nearly spherical to package a larger genome and suggest how all spindle-shaped viruses have evolved from archaeal rod-like viruses.

Proteins that self-assemble into polyhedral shell-like structures are useful molecular containers both in nature and in the laboratory. Here we review efforts to repurpose diverse protein cages, including viral capsids, ferritins, bacterial microcompartments, and designed capsules, as vaccines, drug delivery vehicles, targeted imaging agents, nanoreactors, templates for controlled materials synthesis, building blocks for higher-order architectures, and more. A deep understanding of the principles underlying the construction, function, and evolution of natural systems has been key to tailoring selective cargo encapsulation and interactions with both biological systems and synthetic materials through protein engineering and directed evolution. The ability to adapt and design increasingly sophisticated capsid structures and functions stands to benefit the fields of catalysis, materials science, and medicine.

Lattice self-assemblies (LSAs), which mimic protein assemblies, were studied using a new nonlinear vibrational imaging technique called vibrational sum-frequency generation (VSFG) microscopy. This technique successfully mapped out the mesoscopic morphology, microscopic geometry, symmetry, and ultrafast dynamics of an LSA formed by β-cyclodextrin (β-CD) and sodium dodecyl sulfate (SDS). The spatial imaging also revealed correlations between these different physical properties. Such knowledge shed light on the functions and mechanical properties of LSAs. In this Feature Article, we briefly introduce the fundamental principles of the VSFG microscope and then discuss the in-depth molecular physics of the LSAs revealed by this imaging technique. The application of the VSFG microscope to the artificial LSAs also paved the way for an alternative approach to studying the structure-dynamic-function relationships of protein assemblies, which were essential for life and difficult to study because of their various and complicated interactions. We expect that the hyperspectral VSFG microscope could be broadly applied to many noncentrosymmetric soft materials.

Biological systems display a broad palette of hierarchically ordered designs spanning over many orders of magnitude in size. Remarkably enough, periodic order, which profusely shows up in non-living ordered compounds, plays a quite subsidiary role in most biological structures, which can be appropriately described in terms of the more general aperiodic crystal notion instead. In this topical review I shall illustrate this issue by considering several representative examples, including botanical phyllotaxis, the geometry of cell patterns in tissues, the morphology of sea urchins, or the symmetry principles underlying virus architectures. In doing so, we will realize that albeit the currently adopted quasicrystal notion is not general enough to properly account for the rich structural features one usually finds in biological arrangements of matter, several mathematical tools and fundamental notions belonging to the aperiodic crystals science toolkit can provide a useful modeling framework to this end.

Hepatitis B virus (HBV) is a small enveloped DNA virus which replicates its tiny 3.2 kb genome by reverse transcription inside an icosahedral nucleocapsid, formed by a single ~180 amino acid capsid, or core, protein (Cp). HBV causes chronic hepatitis B (CHB), a severe liver disease responsible for nearly a million deaths each year. Most of HBV's only seven primary gene products are multifunctional. Though less obvious than for the multi-domain polymerase, P protein, this is equally crucial for Cp with its multiple roles in the viral life-cycle. Cp provides a stable genome container during extracellular phases, allows for directed intracellular genome transport and timely release from the capsid, and subsequent assembly of new nucleocapsids around P protein and the pregenomic (pg) RNA, forming a distinct compartment for reverse transcription. These opposing features are enabled by dynamic post-transcriptional modifications of Cp which result in dynamic structural alterations. Their perturbation by capsid assembly modulators (CAMs) is a promising new antiviral concept. CAMs inappropriately accelerate assembly and/or distort the capsid shell. We summarize the functional, biochemical, and structural dynamics of Cp, and discuss the therapeutic potential of CAMs based on clinical data. Presently, CAMs appear as a valuable addition but not a substitute for existing therapies. However, as part of rational combination therapies CAMs may bring the ambitious goal of a cure for CHB closer to reality.

Five libraries of natural and synthetic phenolic acids containing five AB₃, ten constitutional isomeric AB₂, one AB₄, and one AB₅ were previously synthesized and reported by our laboratory in 5 to 11 steps. They were employed to construct seven libraries of self-assembling dendrons, by divergent generational, deconstruction, and combined approaches, enabling the discovery of a diversity of supramolecular assemblies including Frank-Kasper phases, soft quasicrystals, and complex helical organizations, some undergoing deracemization in the crystal state. However, higher substitution patterns within a single dendron were not accessible. Here we report three libraries consisting of 30 symmetric and nonsymmetric constitutional isomeric phenolic acids with unprecedented sequenced patterns, including two AB₂, three AB₃, eight AB₄, five AB₅, six AB₆, three AB₇, two AB₈, and one AB₉ synthesized by accelerated modular-orthogonal Ni-catalyzed borylation and cross-coupling. A single etherification step with 4-(n-dodecyloxy)benzyl chloride transformed all these phenolic acids, of interest also for other applications, into self-assembling dendrons. Despite this synthetic simplicity, they led to a diversity of unprecedented self-organizing principles: lamellar structures of interest for biological membrane mimics, helical columnar assemblies from rigid-solid angle dendrons forming Tobacco Mosaic Virus-like assemblies, columnar organizations from adaptable-solid angle dendrons forming disordered micellar-like nonhelical columns, columns from supramolecular spheres, five body-centered cubic phases displaying supramolecular orientational memory, rarely encountered in previous libraries forming predominantly Frank-Kasper phases, and two Frank-Kasper phases. Lessons from these self-organizing principles, discovered within a single generation of self-assembling dendrons, may help elaborate design principles for complex helical and nonhelical organizations of synthetic and biological matter.

The geometrical structures of single- and multiple-shell icosahedral virus capsids are reproduced as the targets that minimize the cost corresponding to relatively simple design functions. Capsid subunits are first identified as building blocks at a given coarse-grained scale and then represented in these functions as point particles located on an appropriate number of concentric spherical surfaces. Minimal design cost is assigned to optimal spherical packings of the particles. The cost functions are inspired by the packings favored for the Thomson problem, which minimize the electrostatic potential energy between identical charged particles. In some cases, icosahedral symmetry constraints are incorporated as external fields acting on the particles. The simplest cost functions can be obtained by separating particles in disjoint nonequivalent sets with distinct interactions, or by introducing interacting holes (the absence of particles). These functions can be adapted to reproduce any capsid structure found in real viruses. Structures absent in Nature require significantly more complex designs. Measures of information content and complexity are assigned to both the cost functions and the capsid geometries. In terms of these measures, icosahedral structures and the corresponding cost functions are the simplest solutions.

Broad-spectrum antiviral platforms that can decrease or inhibit viral infection would alleviate many threats to global public health. Nonetheless, effective technologies of this kind are still not available. Here, we describe a programmable icosahedral canvas for the self-assembly of icosahedral shells that have viral trapping and antiviral properties. Programmable triangular building blocks constructed from DNA assemble with high yield into various shell objects with user-defined geometries and apertures. We have created shells with molecular masses ranging from 43 to 925 MDa (8 to 180 subunits) and with internal cavity diameters of up to 280 nm. The shell interior can be functionalized with virus-specific moieties in a modular fashion. We demonstrate this virus-trapping concept by engulfing hepatitis B virus core particles and adeno-associated viruses. We demonstrate the inhibition of hepatitis B virus core interactions with surfaces in vitro and the neutralization of infectious adeno-associated viruses exposed to human cells.

The viral protein containers that encapsulate a virus' genetic material are repurposed as virus-like particles in a host of nanotechnology applications, including cargo delivery, storage, catalysis, and vaccination. These viral architectures have evolved to sit on the knife's edge between stability, to provide adequate protection for their genetic cargoes, and instability, to enable their efficient and timely release in the host cell environment upon environmental cues. By introducing a percolation theory for viral capsids, we demonstrate that the geometric characteristics of a viral capsid in terms of its subunit layout and intersubunit interaction network are key for its disassembly behavior. A comparative analysis of all alternative homogeneously tiled capsid structures of the same stoichiometry identifies evolutionary drivers favoring specific viral geometries in nature and offers a guide for virus-like particle design in nanotechnology.

Molecular self-assembly has forged a new era in the development of advanced biomaterials for local drug delivery and tissue engineering applications. Given their innate biocompatibility and biodegradability, self-assembling peptides (SAPs) have come in the spotlight of such applications. Short and water-soluble SAP biomaterials associated with enhanced pharmacokinetic (PK) and pharmacodynamic (PD) responses after the topical administration of the therapeutic systems, or improved regenerative potential in tissue engineering applications will be the focus of the current review. SAPs are capable of generating supramolecular structures using a boundless array of building blocks, while peptide engineering is an approach commonly pursued to encompass the desired traits to the end composite biomaterials. These two elements combined, expand the spectrum of SAPs multi-functionality, constituting them potent biomaterials for use in various biomedical applications.

It is intriguing to think that over millions of years, groups of nucleic acids got the chance to hold together with groups of proteins to build up what today is called a virus. Their only goal is to guarantee a successful replication inside a host. If their genome information is preserved, the task is accomplished. Viruses have evolved to infect organisms and propagate with high degree of adaptation, as it is the case of the SARS-CoV-2, agent of the 2020 world pandemic. The technological progress observed in the field of structural biology, especially in cryo-EM, has offered scientists the possibility of a better understanding of virus origins, behavior, and structural organization. In this minireview we summarize few perspectives about the origins and organization of viruses and the advances of cryo-EM to aid structural virologists to sample the virosphere.

Self-assembly is a ubiquitous process in synthetic and biological systems, broadly defined as the spontaneous organization of multiple subunits (e.g. macromolecules, particles) into ordered multi-unit structures. The vast majority of equilibrium assembly processes give rise to two states: one consisting of dispersed disassociated subunits, and the other, a bulk-condensed state of unlimited size. This review focuses on the more specialized class of self-limiting assembly, which describes equilibrium assembly processes resulting in finite-size structures. These systems pose a generic and basic question, how do thermodynamic processes involving non-covalent interactions between identical subunits "measure" and select the size of assembled structures? In this review, we begin with an introduction to the basic statistical mechanical framework for assembly thermodynamics, and use this to highlight the key physical ingredients that ensure equilibrium assembly will terminate at finite dimensions. Then, we introduce examples of self-limiting assembly systems, and classify them within this framework based on two broad categories: self-closing assemblies and open-boundary assemblies. These include well-known cases in biology and synthetic soft matter - micellization of amphiphiles and shell/tubule formation of tapered subunits - as well as less widely known classes of assemblies, such as short-range attractive/long-range repulsive systems and geometrically-frustrated assemblies. For each of these self-limiting mechanisms, we describe the physical mechanisms that select equilibrium assembly size, as well as potential limitations of finite-size selection. Finally, we discuss alternative mechanisms for finite-size assemblies, and draw contrasts with the size-control that these can achieve relative to self-limitation in equilibrium, single-species assemblies.

The lack of targeting efficacy has frequently led functionalized nanoparticles to accumulate in unwanted cells and tissues while boosting toxicity-related effects. Conversely, viruses are natural nanoparticles that precisely and responsively interact with the biological machinery through an effective-driven fashion. This interaction is enhanced by a meticulous spatial arrangement which results in a quasi-crystalline distribution of proteins on the viruses' surface. Amidst the COVID-19 pandemic, we propose to look at the SARS-CoV-2 nanoscale viral scaffold as an example of a highly-ordered architecture that must inspire and tailor the production of targeted synthetic nanoparticles.

What is the optimal distribution of two types of crystalline phases on the surface of icosahedral shells, such as of many viral capsids? We here investigate the distribution of a thin layer of soft material on a crystalline convex icosahedral shell. We demonstrate how the shapes of spherical viruses can be understood from the perspective of elasticity theory of thin two-component shells. We develop a theory of shape transformations of an icosahedral shell upon addition of a softer, but still crystalline, material onto its surface. We show how the soft component "invades" the regions with the highest elastic energy and stress imposed by the 12 topological defects on the surface. We explore the phase diagram as a function of the surface fraction of the soft material, the shell size, and the incommensurability of the elastic moduli of the rigid and soft phases. We find that, as expected, progressive filling of the rigid shell by the soft phase starts from the most deformed regions of the icosahedron. With a progressively increasing soft-phase coverage, the spherical segments of domes are filled first (12 vertices of the shell), then the cylindrical segments connecting the domes (30 edges) are invaded, and, ultimately, the 20 flat faces of the icosahedral shell tend to be occupied by the soft material. We present a detailed theoretical investigation of the first two stages of this invasion process and develop a model of morphological changes of the cone structure that permits noncircular cross sections. In conclusion, we discuss the biological relevance of some structures predicted from our calculations, in particular for the shape of viral capsids.

Vaccine-induced immune response can be greatly enhanced by mimicking pathogen properties. The size and the repetitive geometric shape of virus-like particles (VLPs) influence their immunogenicity by facilitating drainage to secondary lymphoid organs and enhancing interaction with and activation of B cells and innate humoral immune components. VLPs derived from the plant Bromovirus genus, specifically cowpea chlorotic mottle virus (CCMV), are T = 3 icosahedral particles. (T) is the triangulation number that refers to the number and arrangements of the subunits (pentamers and hexamers) of the VLPs. CCMV-VLPs can be easily expressed in an E. coli host system and package ssRNA during the expression process. Recently, we have engineered CCMV-VLPs by incorporating the universal tetanus toxin (TT) epitope at the N-terminus. The modified CCMV_TT-VLPs successfully form icosahedral particles T = 3, with a diameter of ~30 nm analogous to the parental VLPs. Interestingly, incorporating TT epitope at the C-terminus of CCMV_TT-VLPs results in the formation of Rod-shaped VLPs, ~1 μm in length and ~ 30 nm in width. In this study, we have investigated the draining kinetics and immunogenicity of both engineered forms (termed as Round-shaped CCMV_TT-VLPs and Rod-shaped CCMV_TT-VLPs) as potential B cell immunogens using different in vitro and in vivo assays. Our results reveal that Round-shaped CCMV_TT-VLPs are more efficient in draining to secondary lymphoid organs to charge professional antigen-presenting cells as well as B cells. Furthermore, compared to Rod-shaped CCMV_TT-VLPs, Round-shaped CCMV_TT-VLPs led to more than 100-fold increased systemic IgG and IgA responses accompanied by prominent formation of splenic germinal centers. Round-shaped CCMV_TT-VLPs could also polarize the induced T cell response toward Th1. To our knowledge, this is the first study investigating and comparing the draining kinetics and immunogenicity of one and the same VLP monomer forming nano-sized icosahedra or rods in the micrometer size.

This minireview provides an overview on the current knowledge of protein-protein interactions, common characterisation methods to characterise them, and their role in protein complex formation with some examples. A deep understanding of protein-protein interactions and their molecular interactions is important for a number of applications, including drug design. Protein-protein interactions and their discovery are thus an interesting avenue for understanding how protein complexes, which make up the majority of proteins, work.