Single-Molecule Analysis of Genome Uncoating from Individual Human Rhinovirus Particles, and Modulation by Antiviral Drugs.

Infection of humans by many viruses is typically initiated by the internalization of a single virion in each of a few susceptible cells. Thus, the outcome of the infection process may depend on stochastic single-molecule events. A crucial process for viral infection, and thus a target for developing antiviral drugs, is the uncoating of the viral genome. Here a force spectroscopy procedure using an atomic force microscope is implemented to study uncoating for individual human rhinovirus particles. Application of an increasing mechanical force on a virion led to a high force-induced structural transition that facilitated extrusion of the viral RNA molecule without loss of capsid integrity. Application of force to virions that h ad previously extruded the RNA, or to RNA-free capsids, led to a lower force-induced event associated with capsid disruption. The kinetic parameters are determined for each reaction. The high-force event is a stochastic process governed by a moderate free energy barrier (≈20 kcal mol-1 ), which results in a heterogeneous population of structurally weakened virions in which different fractions of the RNA molecule are externalized. The effects of antiviral compounds or capsid mutation on the kinetics of this reaction reveal a correlation between the reaction rate and virus infectivity.

Electrostatic Screening, Acidic pH and Macromolecular Crowding Increase the Self-Assembly Efficiency of the Minute Virus of Mice Capsid In Vitro.

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.

Equilibrium Dynamics of a Biomolecular Complex Analyzed at Single-amino Acid Resolution by Cryo-electron Microscopy.

The biological function of macromolecular complexes depends not only on large-scale transitions between conformations, but also on small-scale conformational fluctuations at equilibrium. Information on the equilibrium dynamics of biomolecular complexes could, in principle, be obtained from local resolution (LR) data in cryo-electron microscopy (cryo-EM) maps. However, this possibility had not been validated by comparing, for a same biomolecular complex, LR data with quantitative information on equilibrium dynamics obtained by an established solution technique. In this study we determined the cryo-EM structure of the minute virus of mice (MVM) capsid as a model biomolecular complex. The LR values obtained correlated with crystallographic B factors and with hydrogen/deuterium exchange (HDX) rates obtained by mass spectrometry (HDX-MS), a gold standard for determining equilibrium dynamics in solution. This result validated a LR-based cryo-EM approach to investigate, with high spatial resolution, the equilibrium dynamics of biomolecular complexes. As an application of this approach, we determined the cryo-EM structure of two mutant MVM capsids and compared their equilibrium dynamics with that of the wild-type MVM capsid. The results supported a previously suggested linkage between mechanical stiffening and impaired equilibrium dynamics of a virus particle. Cryo-EM is emerging as a powerful approach for simultaneously acquiring information on the atomic structure and local equilibrium dynamics of biomolecular complexes.

Molecular Determinants of Human Rhinovirus Infection, Assembly, and Conformational Stability at Capsid Protein Interfaces.

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. IMPORTANCE 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.

Antiviral compounds modulate elasticity, strength and material fatigue of a virus capsid framework.

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.

A Genetically Engineered, Chain Mail-Like Nanostructured Protein Material with Increased Fatigue Resistance and Enhanced Self-Healing.

Protein-based nanostructured materials are being developed for many biomedical and nanotechnological applications. Despite their many desirable features, protein materials are highly susceptible to disruption by mechanical stress and fatigue. This study is aimed to increase fatigue resistance and enhance self-healing of a natural protein-based supramolecular nanomaterial through permanent genetic modification. The authors envisage the conversion of a model nanosheet, formed by a regular array of noncovalently bound human immunodeficiency virus capsid protein molecules, into a supramolecular "chain mail." Rationally engineered mutations allow the formation of a regular network of disulfide bridges in the protein lattice. This network links each molecule in the lattice to each adjacent molecule through one covalent bond, analogous to the rivetting of interlinked iron rings in the chain mail of a medieval knight. The engineered protein nanosheet shows greatly increased thermostability and resistance to mechanical stress and fatigue in particular, as well as enhanced self-healing, without undesirable stiffening compared to the original material. The results provide proof of concept for a genetic design to permanently increase fatigue resistance and enhance self-healing of protein-based nanostructured materials. They also provide insights into the molecular basis for fatigue of protein materials.

Visualization of Single Molecules Building a Viral Capsid Protein Lattice through Stochastic Pathways.

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.

Negatively charged amino acids at the foot-and-mouth disease virus capsid reduce the virion-destabilizing effect of viral RNA at acidic pH.

Elucidation of the molecular basis of the stability of foot-and-mouth disease virus (FMDV) particles is relevant to understand key aspects of the virus cycle. Residue N17D in VP1, located at the capsid inner surface, modulates the resistance of FMDV virion to dissociation and inactivation at acidic pH. Here we have studied whether the virion-stabilizing effect of amino acid substitution VP1 N17D may be mediated by the alteration of electrostatic charge at this position and/or the presence of the viral RNA. Substitutions that either introduced a positive charge (R,K) or preserved neutrality (A) at position VP1 17 led to increased sensitivity of virions to inactivation at acidic pH, while replacement by negatively charged residues (D,E) increased the resistance of virions to acidic pH. The role in virion stability of viral RNA was addressed using FMDV empty capsids that have a virtually unchanged structure compared to the capsid in the RNA-filled virion, but that are considerably more resistant to acidic pH than WT virions, supporting a virion-destabilizing effect of the RNA. Remarkably, no differences were observed in the resistance to dissociation at acidic pH between the WT empty capsids and those harboring replacement N17D. Thus, the virion-destabilizing effect of viral RNA at acidic pH can be partially restored by introducing negatively charged residues at position VP1 N17.

Structural determinants of mechanical resistance against breakage of a virus-based protein nanoparticle at a resolution of single amino acids.

Virus particles and other protein-based supramolecular complexes have a vast nanotechnological potential. However, protein nanostructures are "soft" materials prone to disruption by force. Whereas some non-biological nanoparticles (NPs) may be stronger, for certain applications protein- and virus-based NPs have potential advantages related to their structure, self-assembly, production, engineering, and/or inbuilt functions. Thus, it may be desirable to acquire the knowledge needed to engineer protein-based nanomaterials with a higher strength against mechanical breakage. Here we have used the capsid of the minute virus of mice to experimentally identify individual chemical groups that determine breakage-related properties of a virus particle. Individual amino acid side chains that establish interactions between building blocks in the viral particle were truncated using protein engineering. Indentation experiments using atomic force microscopy were carried out to investigate the role of each targeted side chain in determining capsid strength and brittleness, by comparing the maximum force and deformation each modified capsid withstood before breaking apart. Side chains with major roles in determining capsid strength against breakage included polar groups located in solvent-exposed positions, and did not generally correspond with those previously identified as determinants of mechanical stiffness. In contrast, apolar side chains buried along the intersubunit interfaces that generally determined capsid stiffness had, at most, a minor influence on strength against disruption. Whereas no correlated variations between strength and either stiffness or brittleness were found, brittleness and stiffness were quantitatively correlated. Implications for developing robust protein-based NPs and for acquiring a deeper physics-based perspective of viruses are discussed.

Thermostability of the Foot-and-Mouth Disease Virus Capsid Is Modulated by Lethal and Viability-Restoring Compensatory Amino Acid Substitutions.

Infection by viruses depends on a balance between capsid stability and dynamics. This study investigated biologically and biotechnologically relevant aspects of the relationship in foot-and-mouth disease virus (FMDV) between capsid structure and thermostability and between thermostability and infectivity. In the FMDV capsid, a substantial number of amino acid side chains at the interfaces between pentameric subunits are charged at neutral pH. Here a mutational analysis revealed that the essential role for virus infection of most of the 8 tested charged groups is not related to substantial changes in capsid protein expression or processing or in capsid assembly or stability against a thermally induced dissociation into pentamers. However, the positively charged side chains of R2018 and H3141, located at the interpentamer interfaces close to the capsid 2-fold symmetry axes, were found to be critical both for virus infectivity and for keeping the capsid in a state of weak thermostability. A charge-restoring substitution (N2019H) that was repeatedly fixed during amplification of viral genomes carrying deleterious mutations reverted both the lethal and capsid-stabilizing effects of the substitution H3141A, leading to a double mutant virus with close to normal infectivity and thermolability. H3141A and other thermostabilizing substitutions had no detectable effect on capsid resistance to acid-induced dissociation into pentamers. The results suggest that FMDV infectivity requires limited local stability around the 2-fold axes at the interpentamer interfaces of the capsid. The implications for the mechanism of genome uncoating in FMDV and the development of thermostabilized vaccines against foot-and-mouth disease are discussed.IMPORTANCE This study provides novel insights into the little-known structural determinants of the balance between thermal stability and instability in the capsid of foot-and-mouth disease virus and into the relationship between capsid stability and virus infectivity. The results provide new guidelines for the development of thermostabilized empty capsid-based recombinant vaccines against foot-and-mouth disease, one of the economically most important animal diseases worldwide.

Systematic analysis of biological roles of charged amino acid residues located throughout the structured inner wall of a virus capsid.

Structure-based mutational analysis of viruses is providing many insights into the relationship between structure and biological function of macromolecular complexes. We have systematically investigated the individual biological roles of charged residues located throughout the structured capsid inner wall (outside disordered peptide segments) of a model spherical virus, the minute virus of mice (MVM). The functional effects of point mutations that altered the electrical charge at 16 different positions at the capsid inner wall were analyzed. The results revealed that MVM capsid self-assembly is rather tolerant to point mutations that alter the number and distribution of charged residues at the capsid inner wall. However, mutations that either increased or decreased the number of positive charges around capsid-bound DNA segments reduced the thermal resistance of the virion. Moreover, mutations that either removed or changed the positions of negatively charged carboxylates in rings of acidic residues around capsid pores were deleterious by precluding a capsid conformational transition associated to through-pore translocation events. The results suggest that number, distribution and specific position of electrically charged residues across the inner wall of a spherical virus may have been selected through evolution as a compromise between several different biological requirements.

Mechanical stiffening of human rhinovirus by cavity-filling antiviral drugs.

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.

Structural basis for biologically relevant mechanical stiffening of a virus capsid by cavity-creating or spacefilling mutations.

Recent studies reveal that the mechanical properties of virus particles may have been shaped by evolution to facilitate virus survival. Manipulation of the mechanical behavior of virus capsids is leading to a better understanding of viral infection, and to the development of virus-based nanoparticles with improved mechanical properties for nanotechnological applications. In the minute virus of mice (MVM), deleterious mutations around capsid pores involved in infection-related translocation events invariably increased local mechanical stiffness and interfered with pore-associated dynamics. To provide atomic-resolution insights into biologically relevant changes in virus capsid mechanics, we have determined by X-ray crystallography the structural effects of deleterious, mechanically stiffening mutations around the capsid pores. Data show that the cavity-creating N170A mutation at the pore wall does not induce any dramatic structural change around the pores, but instead generates subtle rearrangements that propagate throughout the capsid, resulting in a more compact, less flexible structure. Analysis of the spacefilling L172W mutation revealed the same relationship between increased stiffness and compacted capsid structure. Implications for understanding connections between virus mechanics, structure, dynamics and infectivity, and for engineering modified virus-based nanoparticles, are discussed.

Kinetics of Surface-Driven Self-Assembly and Fatigue-Induced Disassembly of a Virus-Based Nanocoating.

Self-assembling protein layers provide a "bottom-up" approach for precisely organizing functional elements at the nanoscale over a large solid surface area. The design of protein sheets with architecture and physical properties suitable for nanotechnological applications may be greatly facilitated by a thorough understanding of the principles that underlie their self-assembly and disassembly. In a previous study, the hexagonal lattice formed by the capsid protein (CA) of human immunodeficiency virus (HIV) was self-assembled as a monomolecular layer directly onto a solid substrate, and its mechanical properties and dynamics at equilibrium were analyzed by atomic force microscopy. Here, we use atomic force microscopy to analyze the kinetics of self-assembly of the planar CA lattice on a substrate and of its disassembly, either spontaneous or induced by materials fatigue. Both self-assembly and disassembly of the CA layer are cooperative reactions that proceed until a phase equilibrium is reached. Self-assembly requires a critical protein concentration and is initiated by formation of nucleation points on the substrate, followed by lattice growth and eventual merging of CA patches into a continuous monolayer. Disassembly of the CA layer showed hysteresis and appears to proceed only after large enough defects (nucleation points) are formed in the lattice, whose number is largely increased by inducing materials fatigue that depends on mechanical load and its frequency. Implications of the kinetic results obtained for a better understanding of self-assembly and disassembly of the HIV capsid and protein-based two-dimensional nanomaterials and the design of anti-HIV drugs targeting (dis)assembly and biocompatible nanocoatings are discussed.

Structural Analysis of a Temperature-Induced Transition in a Viral Capsid Probed by HDX-MS.

Icosahedral viral capsids are made of a large number of symmetrically organized protein subunits whose local movements can be essential for infection. In the capsid of the minute virus of mice, events required for infection that involve translocation of peptides through capsid pores are associated with a subtle conformational change. In vitro, this change can be reversibly induced by overcoming the energy barrier through mild heating of the capsid, but little is known about the capsid regions involved in the process. Here, we use hydrogen-deuterium exchange coupled to mass spectrometry to analyze the dynamics of the minute virus of mice capsid at increasing temperatures. Our results indicate that the transition associated with peptide translocation involves the structural rearrangement of regions distant from the capsid pores. These alterations are reflected in an increased dynamics of some secondary-structure elements in the capsid shell from which spikes protrude, and a decreased dynamics in the long intertwined loops that form the large capsid spikes. Thus, the translocation events through capsid pores involve a global conformational rearrangement of the capsid and a complex alteration of its equilibrium dynamics. This study additionally demonstrates the potential of hydrogen-deuterium exchange coupled to mass spectrometry to explore in detail temperature-dependent structural dynamics in large macromolecular protein assemblies. Most importantly, it paves the way for undertaking novel studies of the relationship between structure, dynamics, and biological function in virus particles and other large protein cages.

Amino Acid Side Chains Buried along Intersubunit Interfaces in a Viral Capsid Preserve Low Mechanical Stiffness Associated with Virus Infectivity.

Single-molecule experimental techniques and theoretical approaches reveal that important aspects of virus biology can be understood in biomechanical terms at the nanoscale. A detailed knowledge of the relationship in virus capsids between small structural changes caused by single-point mutations and changes in mechanical properties may provide further physics-based insights into virus function; it may also facilitate the engineering of viral nanoparticles with improved mechanical behavior. Here, we used the minute virus of mice to undertake a systematic experimental study on the contribution to capsid stiffness of amino acid side chains at interprotein interfaces and the specific noncovalent interactions they establish. Selected side chains were individually truncated by introducing point mutations to alanine, and the effects on local and global capsid stiffness were determined using atomic force microscopy. The results revealed that, in the natural virus capsid, multiple, mostly hydrophobic, side chains buried along the interfaces between subunits preserve a comparatively low stiffness of most (S2 and S3) regions. Virtually no point mutation tested substantially reduced stiffness, whereas most mutations increased stiffness of the S2/S3 regions. This stiffening was invariably associated with reduced virus yields during cell infection. The experimental evidence suggests that a comparatively low stiffness at S3/S2 capsid regions may have been biologically selected because it facilitates capsid assembly, increasing infectious virus yields. This study demonstrated also that knowledge of individual amino acid side chains and biological pressures that determine the physical behavior of a protein nanoparticle may be used for engineering its mechanical properties.

Imaging and Quantitation of a Succession of Transient Intermediates Reveal the Reversible Self-Assembly Pathway of a Simple Icosahedral Virus Capsid.

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.

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.

Quantification and modification of the equilibrium dynamics and mechanics of a viral capsid lattice self-assembled as a protein nanocoating.

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.

Different functional sensitivity to mutation at intersubunit interfaces involved in consecutive stages of foot-and-mouth disease virus assembly.

Small spherical viruses are paradigms of supramolecular self-assembly. Identifying the specific structural determinants for virus assembly provides guidelines to develop new antiviral drugs or engineer modified viral particles for medical or technological applications. However, very few systematic studies have been carried out so far to identify those chemical groups at interfaces between virus capsid subunits that are important for viral assembly and function. Foot-and-mouth disease virus (FMDV) and other picornaviruses are assembled in a stepwise process in which different protein-protein interfaces are formed: 5 protomeric subunits oligomerize to form a pentameric intermediate, and 12 of these stable pentameric building blocks associate to form a labile capsid. In this study, a systematic mutational analysis revealed that very few amino acid side chains involved in substantial interactions between protomers within each pentamer are individually required for virus infectivity. This result contrasts sharply with the previous finding that most amino acid side chains involved in interactions between pentamers during the next assembly step are individually required for infectivity. The dramatic difference in sensitivity to single mutations between the two types of protein-protein interfaces in FMDV is discussed in terms of possible structural strategies for achieving self-assembly and genome uncoating in the face of diverse selective constraints.