Plant Virus-Like Particles for RNA Delivery.

Plant viruses such as brome mosaic virus and cowpea chlorotic mottle virus are effectively purified through PEG precipitation and sucrose cushion ultracentrifugation. Increasing ionic strength and an alkaline pH cause the viruses to swell and disassemble into coat protein subunits. The coat proteins can be reassembled into stable virus-like particles (VLPs) that carry anionic molecules at low ionic strength and through two-step dialysis from neutral pH to acidic buffer. VLPs have been extensively studied due to their ability to protect and deliver cargo, particularly RNA, while avoiding degradation under physiological conditions. Furthermore, chemical functionalization of the surface of VLPs allows for the targeted drug delivery. VLPs derived from plants have demonstrated great potential in nanomedicine by offering a versatile platform for drug delivery, imaging, and therapeutic applications.

The Dynamics of Viruslike Capsid Assembly and Disassembly.

Cowpea chlorotic mottle virus (CCMV) is a widely used model for virus replication studies. A major challenge lies in distinguishing between the roles of the interaction between coat proteins and that between the coat proteins and the viral RNA in assembly and disassembly processes. Here, we report on the spontaneous and reversible size conversion of the empty capsids of a CCMV capsid protein functionalized with a hydrophobic elastin-like polypeptide which occurs following a pH jump. We monitor the concentrations of T = 3 and T = 1 capsids as a function of time and show that the time evolution of the conversion from one T number to another is not symmetric: The conversion from T = 1 to T = 3 is a factor of 10 slower than that of T = 3 to T = 1. We explain our experimental findings using a simple model based on classical nucleation theory applied to virus capsids, in which we account for the change in the free protein concentration, as the different types of shells assemble and disassemble by shedding or absorbing single protein subunits. As far as we are aware, this is the first study confirming that both the assembly and disassembly of viruslike shells can be explained through classical nucleation theory, reproducing quantitatively results from time-resolved experiments.

Controlling the surface charge of simple viruses.

The vast majority of plant viruses are unenveloped, i.e., they lack a lipid bilayer that is characteristic of most animal viruses. The interactions between plant viruses, and between viruses and surfaces, properties that are essential for understanding their infectivity and to their use as bionanomaterials, are largely controlled by their surface charge, which depends on pH and ionic strength. They may also depend on the charge of their contents, i.e., of their genes or-in the instance of virus-like particles-encapsidated cargo such as nucleic acid molecules, nanoparticles or drugs. In the case of enveloped viruses, the surface charge of the capsid is equally important for controlling its interaction with the lipid bilayer that it acquires and loses upon leaving and entering host cells. We have previously investigated the charge on the unenveloped plant virus Cowpea Chlorotic Mottle Virus (CCMV) by measurements of its electrophoretic mobility. Here we examine the electrophoretic properties of a structurally and genetically closely related bromovirus, Brome Mosaic Virus (BMV), of its capsid protein, and of its empty viral shells, as functions of pH and ionic strength, and compare them with those of CCMV. From measurements of both solution and gel electrophoretic mobilities (EMs) we find that the isoelectric point (pI) of BMV (5.2) is significantly higher than that of CCMV (3.7), that virion EMs are essentially the same as those of the corresponding empty capsids, and that the same is true for the pIs of the virions and of their cleaved protein subunits. We discuss these results in terms of current theories of charged colloidal particles and relate them to biological processes and the role of surface charge in the design of new classes of drug and gene delivery systems.

Fluorescent nanodiamonds encapsulated by Cowpea Chlorotic Mottle Virus (CCMV) proteins for intracellular 3D-trajectory analysis.

Long-term tracking of nanoparticles to resolve intracellular structures and motions is essential to elucidate fundamental parameters as well as transport processes within living cells. Fluorescent nanodiamond (ND) emitters provide cell compatibility and very high photostability. However, high stability, biocompatibility, and cellular uptake of these fluorescent NDs under physiological conditions are required for intracellular applications. Herein, highly stable NDs encapsulated with Cowpea chlorotic mottle virus capsid proteins (ND-CP) are prepared. A thin capsid protein layer is obtained around the NDs, which imparts reactive groups and high colloidal stability, while retaining the opto-magnetic properties of the coated NDs as well as the secondary structure of CPs adsorbed on the surface of NDs. In addition, the ND-CP shows excellent biocompatibility both in vitro and in vivo. Long-term 3D trajectories of the ND-CP with fine spatiotemporal resolutions are recorded; their intracellular motions are analyzed by different models, and the diffusion coefficients are calculated. The ND-CP with its brilliant optical properties and stability under physiological conditions provides us with a new tool to advance the understanding of cell biology, e.g., endocytosis, exocytosis, and active transport processes in living cells as well as intracellular dynamic parameters.

Virus-Like Particles as Positive Controls for COVID-19 RT-LAMP Diagnostic Assays.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid and inexpensive isothermal alternative to the current gold standard reverse transcription quantitative polymerase chain reaction (RT-qPCR) for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, unlike RT-qPCR, there are no consensus detection regions or optimal RT-LAMP methods, and most protocols do not include internal controls to ensure reliability. Naked RNAs, plasmids, or even RNA from infectious COVID-19 patients have been used as external positive controls for RT-LAMP assays, but such reagents lack the stability required for full-process control. To overcome the lack of proper internal and external positive controls and the instability of the detection RNA, we developed virus-like particles (VLPs) using bacteriophage Qß and plant virus cowpea chlorotic mottle virus (CCMV) for the encapsidation of target RNA, namely a so-called SARS-CoV-2 LAMP detection module (SLDM). The target RNA is a truncated segment of the SARS-CoV-2 nucleocapsid (N) gene and human RNase P gene (internal control) as positive controls for RT-qPCR and RT-LAMP. Target RNAs stably encapsidated in Qß and CCMV VLPs were previously shown to function as full-process controls in RT-qPCR assays, and here we show that SLDMs can fulfill the same function for RT-LAMP and swab-to-test (direct RT-LAMP with heat lysis) assays. The SLDM was validated in a clinical setting, highlighting the promise of VLPs as positive controls for molecular assays.

Optimizing fluorophore density for single virus counting: a photophysical approach.

In health and environmental research, it is often necessary to quantify the concentrations of single (bio) nanoparticles present at very low concentrations. Suitable quantification approaches that rely on counting and tracking of single fluorescently labelled (bio) nanoparticles are often challenging since fluorophore self-quenching limits the maximum particle brightness. Here we study how the number of labels per nanoparticle influences the total brightness of fluorescently labelled cowpea chlorotic mottle virus (CCMV). We analyze in detail the photophysical interplay between the fluorophores on the virus particles. We deduce that the formation of dark aggregates and energy transfer towards these aggregates limits the total particle brightness that can be achieved. We show that by carefully selecting the number of fluorescent labels per CCMV, and thus minimizing the negative effects on particle brightness, it is possible to quantify fluorescently labelled CCMV concentrations down to fM concentrations in single particle counting experiments.

Self-Assembly of Viral Capsid Proteins Driven by Compressible Nanobubbles.

Colloidal nanobubbles occur in gas-saturated aqueous solutions following high power water electrolysis. Here the influence of nanobubble solutions on the self-assembly properties of viral capsid proteins (CP) was investigated. Interestingly, we found that gas solutions were able to trigger the self-assembly of CP of cowpea chlorotic mottle virus (CCMV) in the absence of the viral genome, most likely by acting as a negatively charged template. The process was demonstrated by three distinct techniques, namely, dynamic light scattering (DLS), atomic force microscopy (AFM), and transmission electron microscopy (TEM). Furthermore, nanobubble-induced self-assembly of viral CP was found to depend on protein concentration. Low CP concentrations led to assembly of 18 nm virus-like particles (VLPs), comparable to T = 1 (Casper and Klug triangulation number) virus capsids, whereas high CP concentrations led to 28 nm VLPs (similar to T = 3 capsids). This paves a new route for self-assembly of VLPs.

How a Virus Circumvents Energy Barriers to Form Symmetric Shells.

Previous self-assembly experiments on a model icosahedral plant virus have shown that, under physiological conditions, capsid proteins initially bind to the genome through an en masse mechanism and form nucleoprotein complexes in a disordered state, which raises the question as to how virions are assembled into a highly ordered structure in the host cell. Using small-angle X-ray scattering, we find out that a disorder-order transition occurs under physiological conditions upon an increase in capsid protein concentrations. Our cryo-transmission electron microscopy reveals closed spherical shells containing in vitro transcribed viral RNA even at pH 7.5, in marked contrast with the previous observations. We use Monte Carlo simulations to explain this disorder-order transition and find that, as the shell grows, the structures of disordered intermediates in which the distribution of pentamers does not belong to the icosahedral subgroups become energetically so unfavorable that the caps can easily dissociate and reassemble, overcoming the energy barriers for the formation of perfect icosahedral shells. In addition, we monitor the growth of capsids under the condition that the nucleation and growth is the dominant pathway and show that the key for the disorder-order transition in both en masse and nucleation and growth pathways lies in the strength of elastic energy compared to the other forces in the system including protein-protein interactions and the chemical potential of free subunits. Our findings explain, at least in part, why perfect virions with icosahedral order form under different conditions including physiological ones.

Versatile Reversible Cross-Linking Strategy to Stabilize CCMV Virus Like Particles for Efficient siRNA Delivery.

Virus like particles obtained from the Cowpea Chlorotic Mottle Virus (CCMV) represent an innovative platform for drug delivery applications. Their unique reversible self-assembly properties as well as their suitability for both cargo loading and functionalization make them a versatile scaffold for numerous purposes. One of the main drawbacks of this platform is however its limited stability at physiological conditions. Herein, we report the development of a general reversible cross-linking strategy involving the homobifunctional cross-linker DTSSP (3,3'-dithiobis (sulfosuccinimidylpropionate)) which is suitable for particle stabilization. This methodology is adaptable to different CCMV variants in the presence or absence of a stabilizing cargo without varying neither particle shape nor size thus extending the potential use of these protein cages in nanomedical applications. Cross-linked particles are stable at neutral pH and 37 °C and they are capable of protecting loaded cargo against enzymatic digestion. Furthermore, the reversible nature of the cross-linking ensures particle disassembly when they are taken up by cells. This was demonstrated via the highly effective delivery of active siRNA into cells.

RNA Homopolymers Form Higher-Curvature Virus-like Particles Than Do Normal-Composition RNAs.

Unlike double-stranded DNA, single-stranded RNA can be spontaneously packaged into spherical capsids by viral capsid protein (CP) because it is a more compact and flexible polymer. Many systematic investigations of this self-assembly process have been carried out using CP from cowpea chlorotic mottle virus, with a wide range of sequences and lengths of single-stranded RNA. Among these studies are measurements of the relative packaging efficiencies of these RNAs into spherical capsids. In this work, we address a fundamental issue that has received very little attention, namely the question of the preferred curvature of the capsid formed around different RNA molecules. We show in particular that homopolymers of RNA-polyribouridylic acid and polyriboadenylic acid-form exclusively T = 2-sized (∼22-nm diameter) virus-like particles (VLPs) when mixed with cowpea chlorotic mottle virus CP, independent of their length, ranging from 500 to more than 4000 nucleotides. This is in contrast to "normal-composition" RNAs (i.e., molecules with comparable numbers of each of the four nucleotides and hence capable of developing a large amount of secondary structure because of intramolecular complementarity/basepairing); a curvature corresponding to T = 3-size (∼28 nm in diameter) is preferred for the VLPs formed with such RNAs. Our work is consistent with the preferred curvature of VLPs being a consequence of interaction of CP with RNA-in particular, the presence or absence of short RNA duplexes-and suggests that the equilibrium size of the capsid results from a trade-off between this optimum size and the cost of confinement.

Intermittency of Deformation and the Elastic Limit of an Icosahedral Virus under Compression.

Viruses undergo mesoscopic morphological changes as they interact with host interfaces and in response to chemical cues. The dynamics of these changes, over the entire temporal range relevant to virus processes, are unclear. Here, we report on creep compliance experiments on a small icosahedral virus under uniaxial constant stress. We find that even at small stresses, well below the yielding point and generally thought to induce a Hookean response, strain continues to develop in time via sparse, randomly distributed, relatively rapid plastic events. The intermittent character of mechanical compliance only appears above a loading threshold, similar to situations encountered in granular flows and the plastic deformation of crystalline solids. The threshold load is much smaller for the empty capsids of the brome mosaic virus than for the wild-type virions. The difference highlights the involvement of RNA in stabilizing the assembly interface. Numerical simulations of spherical crystal deformation suggest intermittency is mediated by lattice defect dynamics and identify the type of compression-induced defect that nucleates the transition to plasticity.

Interactions between the Molecular Components of the Cowpea Chlorotic Mottle Virus Investigated by Molecular Dynamics Simulations.

The formation of a viral particle generally involves hundreds of proteins, making the assembly process intricate. Despite its intrinsic complexity, the production of a viral particle begins through the interaction between the basic assembly components. For the cowpea chlorotic mottle virus (CCMV), the first steps of the assembly process involve dimers of the capsid protein. Here, we carried out atomistic molecular dynamics simulations to investigate the initial assembly process of CCMV to get insight into the interactions at the molecular level. We found that salinity not only affects the electrostatic interactions between dimers but also changes the conformation of the positively charged N-terminal tails and can cause a serious steric hindrance for other dimers binding to the hydrophobic domains. An RNA rod was used to mimic a long segment of a viral genome and to study its interaction with dimers. We observed that the dimer with tails prefers to bind on the RNA rod with its positively charged inner side. The dimer-RNA interaction was found to be as strong as the dimer-dimer interaction, whereas the association energies between a dimer and a pentamer or a hexamer of dimers were high but strongly depended on the presence of the tails. Upon heating, the capsid experienced a shrinkage accompanied by a loss of order in the icosahedral crystal structure.

Defects and Chirality in the Nanoparticle-Directed Assembly of Spherocylindrical Shells of Virus Coat Proteins.

Virus coat proteins of small isometric plant viruses readily assemble into symmetric, icosahedral cages encapsulating noncognate cargo, provided the cargo meets a minimal set of chemical and physical requirements. While this capability has been intensely explored for certain virus-enabled nanotechnologies, additional applications require lower symmetry than that of an icosahedron. Here, we show that the coat proteins of an icosahedral virus can efficiently assemble around metal nanorods into spherocylindrical closed shells with hexagonally close-packed bodies and icosahedral caps. Comparison of chiral angles and packing defects observed by in situ atomic force microscopy with those obtained from molecular dynamics models offers insight into the mechanism of growth, and the influence of stresses associated with intrinsic curvature and assembly pathways.

Oligonucleotide Length-Dependent Formation of Virus-Like Particles.

Understanding the assembly pathway of viruses can contribute to creating monodisperse virus-based materials. In this study, the cowpea chlorotic mottle virus (CCMV) is used to determine the interactions between the capsid proteins of viruses and their cargo. The assembly of the capsid proteins in the presence of different lengths of short, single-stranded (ss) DNA is studied at neutral pH, at which the protein-protein interactions are weak. Chromatography, electrophoresis, microscopy, and light scattering data show that the assembly efficiency and speed of the particles increase with increasing length of oligonucleotides. The minimal length required for assembly under the conditions used herein is 14 nucleotides. Assembly of particles containing such short strands of ssDNA can take almost a month. This slow assembly process enabled the study of intermediate states, which confirmed a low cooperative assembly for CCMV and allowed for further expansion of current assembly theories.

Modular, Bioorthogonal Strategy for the Controlled Loading of Cargo into a Protein Nanocage.

Virus capsids, i.e., viruses devoid of their genetic material, are suitable nanocarriers for biomedical applications such as drug delivery and diagnostic imaging. For this purpose, the reliable encapsulation of cargo in such a protein nanocage is crucial, which can be accomplished by the covalent attachment of the compounds of interest to the protein domains positioned at the interior of the cage. This approach is particularly valid for the capsid proteins of the cowpea chlorotic mottle virus (CCMV), which have their N-termini located at the inside of the capsid structure. Here, we examined several site-selective modification methods for covalent attachment and encapsulation of cargo at the N-terminus of the CCMV protein. Initially, we explored approaches to introduce an N-terminal azide functionality, which would allow the subsequent bioorthogonal modification with a strained alkyne to attach the desired cargo. As these methods showed compatibility issues with the CCMV capsid proteins, a strategy based on 2-pyridinecarboxaldehydes for site-specific N-terminal protein modification was employed. This method allowed the successful modification of the proteins, and was applied for the introduction of a bioorthogonal vinylboronic acid moiety. In a subsequent reaction, the proteins could be modified further with a fluorophore using the tetrazine ligation. The application of capsid assembly conditions on the functionalized proteins led to successful particle formation, showing the potential of this covalent encapsulation strategy.

Ultra-high mass multimer analysis of protein-1a capping domains by a silicon nanomembrane detector.

Conventional time of flight ion detectors are based on secondary electron multipliers encountering a significant loss in detection efficiency, sensitivity and resolution with protein mass above 50kDa. In this work we employ a silicon nanomembrane detector in a Matrix-Assisted Laser Desorption/Ionization coupled to time of flight (MALDI-TOF) mass spectrometer. The operating principle relies on phonon-assisted field emission with excellent performance in the high mass range from 0.001-2MDa. In addition to the analysis of standard proteins the nanomembrane detector (NMD) has the potential for the detection and structural investigation of complex macromolecular assemblies through non-covalent interactions. In order to investigate this hypothesis, the N-terminal capping/methyltransferase domain (CAP) of the Brome Mosaic Virus (BMV) 1a replication protein by MALDI-TOF-NMD is analyzed. The signals detected at the high m/z-ratios of 912.6/982.7 (×103) and 1333.3 (×103) could be modified species of CAP-tricta/tetractamer and the octadecamer. For the first time, the NMD is applied to detect biologically complex macromolecular protein assemblies. Hence, this technology overcomes the limitations of conventional TOF-detectors and increases the analytical range of MALDI-TOF. This technology will be a future alternative for the structural analysis of intact virus capsids that will complement other MS-based techniques such as native mass spectrometry.

Biophysical analysis of BMV virions purified using a novel method.

Brome mosaic virus (BMV) has been successfully loaded with different types of nanoparticles. However, studies concerning its application as a nanoparticle carrier demand high-purity virions in large amounts. Existing BMV purification protocols rely on multiple differential ultracentrifugation runs of the initially purified viral preparation. Herein, we describe an alternative method for BMV purification based on ion-exchange chromatography and size-exclusion chromatography (SEC) yielding 0.2mg of virus from 1g of plant tissue. Our method is of similar efficiency to previously described protocols and can easily be scaled up. The method results in high-quality BMV preparations as confirmed by biophysical analyses, including cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), static light scattering (SLS), and circular dichroism (CD) measurements and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy. Our results revealed that purified BMV capsids are stable and monodisperse and can be used for further downstream applications. In this work, we also characterize secondary structure and size fluctuations of the BMV virion at different pH values.

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.

Stabilization of a Virus-Like Particle and Its Application as a Nanoreactor at Physiological Conditions.

Virus-like particles are very interesting tools for application in bionanotechnology, due to their monodisperse features and biocompatibility. In particular, the cowpea chlorotic mottle virus (CCMV) capsid has been studied extensively as it can be assembled and disassembled reversibly, facilitating cargo encapsulation. CCMV is, however, only stable at physiological conditions when its endogenous nucleic acid cargo is present. To gain more flexibility in the type of cargo encapsulated and to broaden the window of operation, it is interesting to improve the stability of the empty virus-like particles. Here, a method is described to utilize the CCMV capsid at close to physiological conditions as a stable, enzyme-filled nanoreactor. As a proof-of-principle, the encapsulation of T4 lysozyme (T4L) was chosen; this enzyme is a promising antibiotic, but its clinical application is hampered by, for example, its cationic character. It was shown that four T4L molecules can successfully be encapsulated inside CCMV capsids, while remaining catalytically active, which could thus improve the enzyme's application potential.

Predicting the Initial Steps of Salt-Stable Cowpea Chlorotic Mottle Virus Capsid Assembly with Atomistic Force Fields.

Computational prediction of native protein-protein interfaces still remains a challenging task. In virus capsids, each protein unit is in contact with copies of itself through several interfaces. The relative strengths of the different contacts affect the dynamics of the assembly, especially if the process is hierarchical. We investigate the dimerization of the salt-stable cowpea chlorotic mottle virus (CCMV) capsid protein using a combination of different computational tools. The best predictions of dimer configurations provided by blind docking with ZDOCK are rescored using geometry optimization with the Amber and Rosetta force fields. We also evaluate the relative stabilities of the three main interfaces present in the icosahedral capsid using locally restricted docking with Rosetta. Both the rescoring and locally restricted docking results report a particularly stable protein-protein interface, which is the most likely intermediate during the first stage of the hierarchical capsid assembly. The blind docking results rescored with both Amber and Rosetta yield docking funnels, i.e., three or more near-native structures among the top five predictions. The results support experimental observations on in vitro assembly of CCMV capsids. The cross-validation of the results suggests that energy-landscape-based methods with biomolecular force fields have the potential to improve existing docking procedures.