Virus-Like Particles Produced Using the Brome Mosaic Virus Recombinant Capsid Protein Expressed in a Bacterial System.

Virus-like particles (VLPs), due to their nanoscale dimensions, presence of interior cavities, self-organization abilities and responsiveness to environmental changes, are of interest in the field of nanotechnology. Nevertheless, comprehensive knowledge of VLP self-assembly principles is incomplete. VLP formation is governed by two types of interactions: protein-cargo and protein-protein. These interactions can be modulated by the physicochemical properties of the surroundings. Here, we used brome mosaic virus (BMV) capsid protein produced in an E. coli expression system to study the impact of ionic strength, pH and encapsulated cargo on the assembly of VLPs and their features. We showed that empty VLP assembly strongly depends on pH whereas ionic strength of the buffer plays secondary but significant role. Comparison of VLPs containing tRNA and polystyrene sulfonic acid (PSS) revealed that the structured tRNA profoundly increases VLPs stability. We also designed and produced mutated BMV capsid proteins that formed VLPs showing altered diameters and stability compared to VLPs composed of unmodified proteins. We also observed that VLPs containing unstructured polyelectrolyte (PSS) adopt compact but not necessarily more stable structures. Thus, our methodology of VLP production allows for obtaining different VLP variants and their adjustment to the incorporated cargo.

Genome organization and interaction with capsid protein in a multipartite RNA virus.

We report the asymmetric reconstruction of the single-stranded RNA (ssRNA) content in one of the three otherwise identical virions of a multipartite RNA virus, brome mosaic virus (BMV). We exploit a sample consisting exclusively of particles with the same RNA content-specifically, RNAs 3 and 4-assembled in planta by agrobacterium-mediated transient expression. We find that the interior of the particle is nearly empty, with most of the RNA genome situated at the capsid shell. However, this density is disordered in the sense that the RNA is not associated with any particular structure but rather, with an ensemble of secondary/tertiary structures that interact with the capsid protein. Our results illustrate a fundamental difference between the ssRNA organization in the multipartite BMV viral capsid and the monopartite bacteriophages MS2 and Qß for which a dominant RNA conformation is found inside the assembled viral capsids, with RNA density conserved even at the center of the particle. This can be understood in the context of the differing demands on their respective lifecycles: BMV must package separately each of several different RNA molecules and has been shown to replicate and package them in isolated, membrane-bound, cytoplasmic complexes, whereas the bacteriophages exploit sequence-specific "packaging signals" throughout the viral RNA to package their monopartite genomes.

Intermolecular RNA Recombination Occurs at Different Frequencies in Alternate Forms of Brome Mosaic Virus RNA Replication Compartments.

Positive-strand RNA viruses replicate their genomes in membrane-bound replication compartments. Brome mosaic virus (BMV) replicates in vesicular invaginations of the endoplasmic reticulum membrane. BMV has served as a productive model system to study processes like virus-host interactions, RNA replication and recombination. Here we present multiple lines of evidence showing that the structure of the viral RNA replication compartments plays a fundamental role and that recruitment of parental RNAs to a common replication compartment is a limiting step in intermolecular RNA recombination. We show that a previously defined requirement for an RNA recruitment element on both parental RNAs is not to function as a preferred crossover site, but in order for individual RNAs to be recruited into the replication compartments. Moreover, modulating the form of the replication compartments from spherular vesicles (spherules) to more expansive membrane layers increased intermolecular RNA recombination frequency by 200- to 1000-fold. We propose that intermolecular RNA recombination requires parental RNAs to be recruited into replication compartments as monomers, and that recruitment of multiple RNAs into a contiguous space is much more common for layers than for spherules. These results could explain differences in recombination frequencies between viruses that replicate in association with smaller spherules versus larger double-membrane vesicles and convoluted membranes.

The Effect of RNA Secondary Structure on the Self-Assembly of Viral Capsids.

Previous work has shown that purified capsid protein (CP) of cowpea chlorotic mottle virus (CCMV) is capable of packaging both purified single-stranded RNA molecules of normal composition (comparable numbers of A, U, G, and C nucleobases) and of varying length and sequence, and anionic synthetic polymers such as polystyrene sulfonate. We find that CCMV CP is also capable of packaging polyU RNAs, which-unlike normal-composition RNAs-do not form secondary structures and which act as essentially structureless linear polymers. Following our canonical two-step assembly protocol, polyU RNAs ranging in length from 1000 to 9000 nucleotides (nt) are completely packaged. Surprisingly, negative-stain electron microscopy shows that all lengths of polyU are packaged into 22-nm-diameter particles despite the fact that CCMV CP prefers to form 28-nm-diameter (T = 3) particles when packaging normal-composition RNAs. PolyU RNAs >5000 nt in length are packaged into multiplet capsids, in which a single RNA molecule is shared between two or more 22-nm-diameter capsids, in analogy with the multiplets of 28-nm-diameter particles formed with normal-composition RNAs >5000 nt long. Experiments in which viral RNA competes for viral CP with polyUs of equal length show that polyU, despite its lack of secondary structure, is packaged more efficiently than viral RNA. These findings illustrate that the secondary structure of the RNA molecule-and its absence-plays an essential role in determining capsid structure during the self-assembly of CCMV-like particles.

An unrecognized function for COPII components in recruiting the viral replication protein BMV 1a to the perinuclear ER.

Positive-strand RNA viruses invariably assemble their viral replication complexes (VRCs) by remodeling host intracellular membranes. How viral replication proteins are targeted to specific organelle membranes to initiate VRC assembly remains elusive. Brome mosaic virus (BMV), whose replication can be recapitulated in Saccharomyces cerevisiae, assembles its VRCs by invaginating the outer perinuclear endoplasmic reticulum (ER) membrane. Remarkably, BMV replication protein 1a (BMV 1a) is the only viral protein required for such membrane remodeling. We show that ER-vesicle protein of 14 kD (Erv14), a cargo receptor of coat protein complex II (COPII), interacts with BMV 1a. Moreover, the perinuclear ER localization of BMV 1a is disrupted in cells lacking ERV14 or expressing dysfunctional COPII coat components (Sec13, Sec24 or Sec31). The requirement of Erv14 for the localization of BMV 1a is bypassed by addition of a Sec24-recognizable sorting signal to BMV 1a or by overexpressing Sec24, suggesting a coordinated effort by both Erv14 and Sec24 for the proper localization of BMV 1a. The COPII pathway is well known for being involved in protein secretion; our data suggest that a subset of COPII coat proteins have an unrecognized role in targeting proteins to the perinuclear ER membrane.

Tools for Model Building and Optimization into Near-Atomic Resolution Electron Cryo-Microscopy Density Maps.

Electron cryo-microscopy (cryoEM) has advanced dramatically to become a viable tool for high-resolution structural biology research. The ultimate outcome of a cryoEM study is an atomic model of a macromolecule or its complex with interacting partners. This chapter describes a variety of algorithms and software to build a de novo model based on the cryoEM 3D density map, to optimize the model with the best stereochemistry restraints and finally to validate the model with proper protocols. The full process of atomic structure determination from a cryoEM map is described. The tools outlined in this chapter should prove extremely valuable in revealing atomic interactions guided by cryoEM data.

Phosphorylation of the Brome Mosaic Virus Capsid Regulates the Timing of Viral Infection.

UNLABELLED: The four brome mosaic virus (BMV) RNAs (RNA1 to RNA4) are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for the differential release of BMV RNAs from virions is unknown, since 180 copies of the same coat protein (CP) encapsidate each of the BMV genomic RNAs. Using mass spectrometry, we found that the BMV CP contains a complex pattern of posttranslational modifications. Treatment with phosphatase was found to not significantly affect the stability of the virions containing RNA1 but significantly impacted the stability of the virions that encapsidated BMV RNA2 and RNA3/4. Cryo-electron microscopy reconstruction revealed dramatic structural changes in the capsid and the encapsidated RNA. A phosphomimetic mutation in the flexible N-terminal arm of the CP increased BMV RNA replication and virion production. The degree of phosphorylation modulated the interaction of CP with the encapsidated RNA and the release of three of the BMV RNAs. UV cross-linking and immunoprecipitation methods coupled to high-throughput sequencing experiments showed that phosphorylation of the BMV CP can impact binding to RNAs in the virions, including sequences that contain regulatory motifs for BMV RNA gene expression and replication. Phosphatase-treated virions affected the timing of CP expression and viral RNA replication in plants. The degree of phosphorylation decreased when the plant hosts were grown at an elevated temperature. These results show that phosphorylation of the capsid modulates BMV infection. IMPORTANCE: How icosahedral viruses regulate the release of viral RNA into the host is not well understood. The selective release of viral RNA can regulate the timing of replication and gene expression. Brome mosaic virus (BMV) is an RNA virus, and its three genomic RNAs are encapsidated in separate virions. Through proteomic, structural, and biochemical analyses, this work shows that posttranslational modifications, specifically, phosphorylation, on the capsid protein regulate the capsid-RNA interaction and the stability of the virions and affect viral gene expression. Mutational analysis confirmed that changes in modification affected virion stability and the timing of viral infection. The mechanism for modification of the virion has striking parallels to the mechanism of regulation of chromatin packaging by nucleosomes.

Comparative Study of Non-Enveloped Icosahedral Viruses Size.

Now, as before, transmission electron microscopy (TEM) is a widely used technique for the determination of virions size. In some studies, dynamic light scattering (DLS) has also been applied for this purpose. Data obtained by different authors and using different methods could vary significantly. The process of TEM sample preparation involves drying on the substrate, which can cause virions to undergo morphology changes. Therefore, other techniques should be used for measurements of virions size in liquid, (i.e. under conditions closer to native). DLS and nanoparticle tracking analysis (NTA) provide supplementary data about the virions hydrodynamic diameter and aggregation state in liquid. In contrast to DLS, NTA data have a higher resolution and also are less sensitive to minor admixtures. In the present work, the size of non-enveloped icosahedral viruses of different nature was analyzed by TEM, DLS and NTA: the viruses used were the encephalomyocarditis virus (animal virus), and cauliflower mosaic virus, brome mosaic virus and bean mild mosaic virus (plant viruses). The same, freshly purified, samples of each virus were used for analysis using the different techniques. The results were compared with earlier published data and description databases. DLS data about the hydrodynamic diameter of bean mild mosaic virus, and NTA data for all examined viruses, were obtained for the first time. For all virus samples, the values of size obtained by TEM were less than virions sizes determined by DLS and NTA. The contribution of the electrical double layer (EDL) in virions hydrodynamic diameter was evaluated. DLS and NTA data adjusted for EDL thickness were in better agreement with TEM results.

Approximation of virus structure by icosahedral tilings.

Viruses are remarkable examples of order at the nanoscale, exhibiting protein containers that in the vast majority of cases are organized with icosahedral symmetry. Janner used lattice theory to provide blueprints for the organization of material in viruses. An alternative approach is provided here in terms of icosahedral tilings, motivated by the fact that icosahedral symmetry is non-crystallographic in three dimensions. In particular, a numerical procedure is developed to approximate the capsid of icosahedral viruses by icosahedral tiles via projection of high-dimensional tiles based on the cut-and-project scheme for the construction of three-dimensional quasicrystals. The goodness of fit of our approximation is assessed using techniques related to the theory of polygonal approximation of curves. The approach is applied to a number of viral capsids and it is shown that detailed features of the capsid surface can indeed be satisfactorily described by icosahedral tilings. This work complements previous studies in which the geometry of the capsid is described by point sets generated as orbits of extensions of the icosahedral group, as such point sets are by construction related to the vertex sets of icosahedral tilings. The approximations of virus geometry derived here can serve as coarse-grained models of viral capsids as a basis for the study of virus assembly and structural transitions of viral capsids, and also provide a new perspective on the design of protein containers for nanotechnology applications.

An atomic model of brome mosaic virus using direct electron detection and real-space optimization.

Advances in electron cryo-microscopy have enabled structure determination of macromolecules at near-atomic resolution. However, structure determination, even using de novo methods, remains susceptible to model bias and overfitting. Here we describe a complete workflow for data acquisition, image processing, all-atom modelling and validation of brome mosaic virus, an RNA virus. Data were collected with a direct electron detector in integrating mode and an exposure beyond the traditional radiation damage limit. The final density map has a resolution of 3.8 Å as assessed by two independent data sets and maps. We used the map to derive an all-atom model with a newly implemented real-space optimization protocol. The validity of the model was verified by its match with the density map and a previous model from X-ray crystallography, as well as the internal consistency of models from independent maps. This study demonstrates a practical approach to obtain a rigorously validated atomic resolution electron cryo-microscopy structure.

Self-assembly and modular functionalization of three-dimensional crystals from oppositely charged proteins.

Multicomponent crystals and nanoparticle superlattices are a powerful approach to integrate different materials into ordered nanostructures. Well-developed, especially DNA-based, methods for their preparation exist, yet most techniques concentrate on molecular and synthetic nanoparticle systems in non-biocompatible environment. Here we describe the self-assembly and characterization of binary solids that consist of crystalline arrays of native biomacromolecules. We electrostatically assembled cowpea chlorotic mottle virus particles and avidin proteins into heterogeneous crystals, where the virus particles adopt a non-close-packed body-centred cubic arrangement held together by avidin. Importantly, the whole preparation process takes place at room temperature in a mild aqueous medium allowing the processing of delicate biological building blocks into ordered structures with lattice constants in the nanometre range. Furthermore, the use of avidin-biotin interaction allows highly selective pre- or post-functionalization of the protein crystals in a modular way with different types of functional units, such as fluorescent dyes, enzymes and plasmonic nanoparticles.

Role of electrostatics in the assembly pathway of a single-stranded RNA virus.

UNLABELLED: We have recently discovered (R. D. Cadena-Nava et al., J. Virol. 86:3318-3326, 2012, doi:10.1128/JVI.06566-11) that the in vitro packaging of RNA by the capsid protein (CP) of cowpea chlorotic mottle virus is optimal when there is a significant excess of CP, specifically that complete packaging of all of the RNA in solution requires sufficient CP to provide charge matching of the N-terminal positively charged arginine-rich motifs (ARMS) of the CPs with the negatively charged phosphate backbone of the RNA. We show here that packaging results from the initial formation of a charge-matched protocapsid consisting of RNA decorated by a disordered arrangement of CPs. This protocapsid reorganizes into the final, icosahedrally symmetric nucleocapsid by displacing the excess CPs from the RNA to the exterior surface of the emerging capsid through electrostatic attraction between the ARMs of the excess CP and the negative charge density of the capsid exterior. As a test of this scenario, we prepare CP mutants with extra and missing (relative to the wild type) cationic residues and show that a correspondingly smaller and larger excess, respectively, of CP is needed for complete packaging of RNA. IMPORTANCE: Cowpea chlorotic mottle virus (CCMV) has long been studied as a model system for the assembly of single-stranded RNA viruses. While much is known about the electrostatic interactions within the CCMV virion, relatively little is known about these interactions during assembly, i.e., within intermediate states preceding the final nucleocapsid structure. Theoretical models and coarse-grained molecular dynamics simulations suggest that viruses like CCMV assemble by the bulk adsorption of CPs onto the RNA driven by electrostatic attraction, followed by structural reorganization into the final capsid. Such a mechanism facilitates assembly by condensing the RNA for packaging while simultaneously concentrating the local density of CP for capsid nucleation. We provide experimental evidence of such a mechanism by demonstrating that efficient assembly is initiated by the formation of a disordered protocapsid complex whose stoichiometry is governed by electrostatics (charge matching of the anionic RNA and the cationic N termini of the CP).

Versatile post-functionalization of the external shell of cowpea chlorotic mottle virus by using click chemistry.

We present the modification of the outer protein shell of cowpea chlorotic mottle virus (CCMV) with linear and strained alkyne groups. These functionalized protein capsids constitute valuable platforms for post-functionalization via click chemistry. After modification, the integrity of the capsid and the reversible disassembly behavior are preserved.

Chemotherapy pro-drug activation by biocatalytic virus-like nanoparticles containing cytochrome P450.

This work shows, for the first time, the encapsulation of a highly relevant protein in the biomedical field into virus-like particles (VLPs). A bacterial CYP variant was effectively encapsulated in VLPs constituted of coat protein from cowpea chlorotic mottle virus (CCMV). The catalytic VLPs are able to transform the chemotherapeutic pro-drug, tamoxifen, and the emerging pro-drug resveratrol. The chemical nature of the products was identified, confirming similar active products than those obtained with human CYP. The enzymatic VLPs remain stable after the catalytic reaction. The potential use of these biocatalytic nanoparticles as targeted CYP carriers for the activation of chemotherapy drugs is discussed.

Probing the impact of loading rate on the mechanical properties of viral nanoparticles.

The effects of changes in the loading rate during the forced dissociation of single bonds have been studied for a wide variety of interactions. Less is known on the loading rate dependent behaviour of more complex systems that consist of multiple bonds. Here we focus on viral nanoparticles, in particular the protein shell (capsid) that protects the viral genome. As model systems we use the well-studied capsids of the plant virus Cowpea Chlorotic Mottle Virus (CCMV) and of the bacteriophages φ29 and HK97. By applying an atomic force microscopy (AFM) nanoindentation approach we study the loading rate dependency of their mechanical properties. Our AFM results show very diverse behaviour for the different systems. In particular, we find that not only the breaking force, but also the spring constant of some capsids depend on the loading rate. We describe and compare the measured data with simulation results from the literature. The unexpected complex loading rate dependencies that we report present a challenge for the current theoretical considerations aimed at understanding the molecular level interactions of highly ordered protein assemblies.

Magnetic virus-like nanoparticles in N. benthamiana plants: a new paradigm for environmental and agronomic biotechnological research.

This article demonstrates the encapsulation of cubic iron oxide nanoparticles (NPs) by Brome mosaic virus capsid shells and the formation, for the first time, of virus-based nanoparticles (VNPs) with cubic cores. Cubic iron oxide NPs functionalized with phospholipids containing poly(ethylene glycol) tails and terminal carboxyl groups exhibited exceptional relaxivity in magnetic resonance imaging experiments, which opens the way for in vivo MRI studies of systemic virus movement in plants. Preliminary data on cell-to-cell and long-distance transit behavior of cubic iron oxide NPs and VNPs in Nicotiana benthamiana leaves indicate that VNPs have specific transit properties, i.e., penetration into tissue and long-distance transfer through the vasculature in N. benthamiana plants, even at low temperature (6 °C), while NPs devoid of virus protein coats exhibit limited transport by comparison. These particles potentially open new opportunities for high-contrast functional imaging in plants and for the delivery of therapeutic antimicrobial cores into plants.

All-atom multiscale simulation of cowpea chlorotic mottle virus capsid swelling.

An all-atom multiscale computational modeling approach, molecular dynamics/order parameter extrapolation (MD/OPX), has recently been developed for simulating large bionanosystems. It accelerates MD simulations and addresses rapid atomistic fluctuations and slowly varying nanoscale dynamics of bionanosystems simultaneously. With modules added to account for water molecules and ions, MD/OPX is applied to simulate the swelling of cowpea chlorotic mottle virus (CCMV) capsid solvated in a host medium in this study. Simulation results show that the N-terminal arms of capsid proteins undergo large deviations from the initial configurations with their length extended quickly during the early stage of capsid swelling. The capsid swelling is a symmetry-breaking process involving local initiation and front propagation. The capsid swelling rate is approximately 0.25 nm/ns (npn) during the early stage of the simulation, and propagation of the structural transition across the capsid is roughly 0.6 npn. The system conditions that affect swelling of the capsid are analyzed. Prospects for creating a phase diagram for CCMV capsid swelling and using predictions to guide experiments are discussed.

Self-assembly and optically triggered disassembly of hierarchical dendron-virus complexes.

Nature offers a vast array of biological building blocks that can be combined with synthetic materials to generate a variety of hierarchical architectures. Viruses are particularly interesting in this respect because of their structure and the possibility of them functioning as scaffolds for the preparation of new biohybrid materials. We report here that cowpea chlorotic mottle virus particles can be assembled into well-defined micrometre-sized objects and then reconverted into individual viruses by application of a short optical stimulus. Assembly is achieved using photosensitive dendrons that bind on the virus surface through multivalent interactions and then act as a molecular glue between the virus particles. Optical triggering induces the controlled decomposition and charge switching of dendrons, which results in the loss of multivalent interactions and the release of virus particles. We demonstrate that the method is not limited to the virus particles alone, but can also be applied to other functional protein cages such as magnetoferritin.

An amphipathic alpha-helix controls multiple roles of brome mosaic virus protein 1a in RNA replication complex assembly and function.

Brome mosaic virus (BMV) protein 1a has multiple key roles in viral RNA replication. 1a localizes to perinuclear endoplasmic reticulum (ER) membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA replication complexes form, and recruits and stabilizes BMV 2a polymerase (2a(Pol)) and RNA replication templates at these sites to establish active replication complexes. During replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic alpha-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a-ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a-membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and replication, but doubles the 1a-mediated increase in 2a(Pol) accumulation. The second class of helix A mutations not only maintains efficient 1a-membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2a(Pol) accumulation. The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action.

Synergistic effects of mutations and nanoparticle templating in the self-assembly of cowpea chlorotic mottle virus capsids.

A study of the in vitro nanoparticle-templated assembly of a mutant of cowpea chlorotic mottle virus lacking most of the N-terminal domain (residues 4-37), NDelta34, is presented. Mutant empty proteins assemble into empty capsids with a much broader distribution of sizes than the wild-type virus. This increased flexibility in the assembly outcomes is known to be detrimental for the assembly process in the presence of molecular polyanions. However, when rigid polyanionic cores are used, such as nanoparticles, the assembly process is restored and virus-like particles form. Moreover, the breadth of the nanoparticle-templated capsid size distribution becomes comparable with the wild-type virus size distribution.